SEM and TEM results demonstrate that the micro-sized LiFeBO3/C hollow spheres consist of LiFeBO3@C particles and the average size of LiFeBO3@C particles is around 50–100 nm. The thickness of the amorphous carbon layer which is coated on the surface of LiFeBO3 nanoparticles is about 2.5 nm. LiFeBO3@C particles are connected by carbon layers and formed conductive network in the LiFeBO3/C hollow spheres, leading to improved electrical conductivity. Meanwhile, the hollow structure boosts the Li+ diffusion and the carbon layers of LiFeBO3@C particles protect LiFeBO3 from moisture corrosion. Consequently, synthesized LiFeBO3/C sample exhibits good electrochemical properties and storage performance.LiFeBO For instance, the theoretical capacity of LiMnBO3 is 222 mAh g−1, whereas the phosphate counterpart, LiMnPO4, is limited to 170 mAh g−1  pathway for Li+ diffusing in the LiFeBO3 crystal, which leading to low electronic and ionic conductivity  play an increasingly important role in modern society 3·9H2O, and 1.24g H3BO3 were weighed and dissolved gradually in deionized water under stirring, and the mixed solution was named solution A. Meanwhile, 1.22 g PEG-6000 was dropped and dissolved in deionized water under stirring to form solution B. Then the solution B was added into the solution A and stirred carefully. A homogeneous sol was obtained when the mixed solution has been stirred at 80°C for 1 h. The precursor of LiFeBO3/C was synthesized using the obtained sol as raw material via a spray drying process. The inlet and outlet air temperatures are 200 and 100°C during the spray drying process with the air pressure of 0.25 MPa. The as-obtained precursor was calcined at 350°C (3 h), followed by crystallized at 450°C for 10 h in an argon atmosphere, and the final LiFeBO3/C was obtained. For contrast, the LiFeBO3 sample was also prepared by the similar synthetic process but without PEG-6000. All the chemical regents employed in this work were of analytic grade.Firstly, LiNO3/C and LiFeBO3 samples was investigated by XRD (Rigaku/Ultima-IV) and Raman micro-spectroscopy. And the range of XRD and Raman spectroscopic analysis is 2θ = 10 ~ 80° and 600–2,000 cm−1, respectively. Morphology and microstructure of LiFeBO3/C and LiFeBO3 samples were investigated by SEM (JSM/6380LV) and TEM (TecnaiG220). C-S analysis (Eltar) was used to determine the carbon content of samples.The structure of LiFeBO3/C or LiFeBO3 powder, acetylene black  and polyvinylidene fluoride  in N-methylpyrolline onto an Al foil and dried at 120°C for 4 h in vacuum oven. Then the coin-type cells were assembled in a glove box filled with high purity argon. 1M LiPF6 solution in a mixture of ethylene carbonate and dimethyl carbonate with 1:1 volumetric ratio was used as electrolyte . The cells were tested in the voltage range of 1.5–4.5 V under various charge/discharge current densities from 5 to 100 mA g−1 at room temperature. The electrochemical impedance spectroscopy (EIS) and the cyclic voltammetry (CV) was both measured by a CHI 660D workstation.The electrochemical measurements were performed in coin-type cells (CR2025) with lithium metal as the negative electrode. The positive electrodes were prepared by mixing as-prepared LiFeBO3/C and LiFeBO3 samples are shown in 3 with monoclinic structure is well-crystallized. XRD patterns of two samples are similar, indicating the combination of LiFeBO3 and carbon has little influence on the crystal structure. Although the amount of carbon in the LiFeBO3/C composites is 4.9 wt. % , the diffraction peak of carbon is not detected from the XRD patterns, implying the carbon in the LiFeBO3/C composite is amorphous.XRD patterns of the synthesized LiFeBO3/C, XRD data was refined by Rietveld method  band of carbon while the broad peak located at 1,590 cm−1 agreeing well with G (graphitized) band of sp2 type carbon  range of 1.5–4.5 V, the cells were charged/discharged at 10 mA g−1, and the curves of the as-synthesized LiFeBO3/C and LiFeBO3 samples are exhibited in 3 and LiCoBO3  can be recovered for LiFeBO3/C but only 75.1% was recovered for LiFeBO3 electrode (149.6 mAh g−1), implying the LiFeBO3/C sample has a better capacity recovery ability than LiFeBO3. The improved electrochemical performances can be attributed to the special hollow sphere structure and network of amorphous carbon layer. The conductivity is improved by the network of amorphous carbon layer and the volume expansion during the charge and discharge progresses is againsted due to the special hollow sphere architecture provides a flexible structure, so that the electrochemical performance of the synthesized cathode is improved  is lower than that of LiFeBO3 (354.1 Ω), implying the charge transfer speed during the charge and discharge processes of electrode is significantly improved. The exchange current density (j0) of synthesized LiFeBO3/C and LiFeBO3 is calculated by the following equation, respectively.In order to analyze the conductivity of the as-prepared LiFeBOIS tests  are meast insert  represenT and n refer to the temperature and the electrons number, respectively. The exchange current density (j0) of LiFeBO3/C (10.78 × 10−4 mA cm−2) is higher than that of LiFeBO3 (7.26 × 10−5 mA cm−2), which indicates that LiFeBO3/C has a better electrode reaction reversibility than LiFeBO3.Where 3/C and LiFeBO3 samples were stored in air exposure for 6 months. XRD patterns of the LiFeBO3/C and LiFeBO3 samples before and after storage are shown in 3/C sample also remains its original crystal structure, verifying high storage stability of LiFeBO3/C, as shown in 3 changed after storing 6 months. As can be seen from 3 from monoclinic to orthorhombic structure with air corrosion (Bo et al., To evaluate the storage stability, LiFeBO3/C and LiFeBO3 samples are investigated, and the results are shown in 3/C is as high as 198.9 mAh g−1 and there is 76.9% capacity retention after 50 cycles. However, the stored LiFeBO3 delivers a capacity of 191.7 mAh g−1 and only 56.1% capacity retention after 50 cycles. Obviously, the stored LiFeBO3/C shows higher stability and cycling performance than the stored LiFeBO3. In summary, carbon coating and structural modification improve the conductivity of LiFeBO3, shorten the Li+ diffusion/conduction path and protect LiFeBO3 from moisture corrosion, thus leading to good electrochemical performance.After 6 months of storage, the cycling performances of LiFeBO3/C hollow sphere is successfully synthesized by a facile spray-drying method. Primary LiFeBO3@C particles are connected by carbon layers and formed LiFeBO3/C hollow spheres with improved electrical conductivity. Meanwhile, the hollow porous structure boosts the Li+ diffusion and the carbon layers of LiFeBO3@C particles protect LiFeBO3 from moisture corrosion. Therefore, the synthesized LiFeBO3/C sample shows good electrochemical performances and improved storage performance. This work is conducive to obtaining promising and high-performance cathode materials for LIBs.LiFeBOThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.YS, ST, and WC did the main experiment and write the manuscript. LW and BW envolved the discussion of the experiment and revised the manuscript. HL and WL assisted the material synthesis. LW and SZ made the research plan. SZ and LW also provided the financial support.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PRNP gene, have been associated with autosomal dominant neurodegenerative disorders, such as Creutzfeldt–Jacob disease (CJD), Gerstmann–Sträussler–Scheinker syndrome (GSS), and Fatal Familial Insomnia (FFI). Notably, PRNP mutations have also been described in clinical pictures resembling other neurodegenerative diseases, such as frontotemporal dementia. Regarding the pathogenesis, it has been observed that these point mutations are located in the C-terminal region of the PRNP gene and, currently, the potential significance of the N-terminal domain has largely been underestimated. The purpose of this report is to review and provide current insights into the pathogenic mechanisms of PRNP mutations, emphasizing the differences between the C- and N-terminal regions and focusing, in particular, on the lesser-known flexible N-terminal, for which recent biophysical evidence has revealed a physical interaction with the globular C-terminal domain of the cellular prion protein (PrPC).Inherited mutations in the Prion protein (PrP), encoded by the  Sc) [PRNP) gene, such as in the case of familial CJD, genetic CJD, GSS, and FFI , with an autosomal dominant inheritance pattern [Prion diseases, also known as transmissible spongiform encephalopathies, are progressive, fatal, neurodegenerative disorders based on the misfolding of the prion protein . They caSc) ,3. Accor pattern . HoweverC, into the typically protease-resistant-sheet rich isoform, defined as the scrapie prion protein (PrPSc), by a conformational rearrangement. The PrPSc constitutes the transmissible agent (“prion”), able to recruit and convert natively folded PrPC into de novo PrPSc via an autocatalytic process [A key event in the pathogenesis of infectious, sporadic, and IPD is the misfolding of the normal form of the prion protein, PrP process ,7.C undergoes conformational changes into the self-propagating, misfolded PrPSc conformer. Conversely, in the inherited form of the disease, an alteration in the conformation of PrPC may be induced by a genetic mutation in the PRNP gene, but, despite the misfolding of the prion protein (PrP) also playing a central pathogenic role, the process by which PRNP mutations promote the development of self-propagating conformations has not been completely elucidated [C is largely unknown; however, it appears to be concentrated primarily at pre- and postsynaptic neuronal membranes [C to coordinate Cu2+ and Zn2+ suggests it plays a role in metal ion homeostasis [C protein (residues 23–231) is composed of an independent and flexible N-terminal region (residues 23–120) and a C-terminal globular domain (residues 121–231), which physically interact with each other [PRNP pathogenic mutations have been identified in the C-terminal domain. Recently, a missense P39L mutation in N-terminal domain of the prion protein was reported by several authors, in patients affected by frontotemporal lobar degeneration (FTLD) syndrome, which were negative for mutations in genes causative of dementia [PRNP mutations, emphasizing the differences between the C- and N-terminal domains and focusing in particular on the lesser-known flexible N-terminal, for which recent biophysical evidence has revealed a physical interaction with globular C-terminal domains of PrPC [In the case of infectious and sporadic prion diseases, the normal prion protein PrPucidated . The preembranes  and the eostasis ,10. In sch other . To datech other . Indeed,dementia ,14,15. GC into a protease-resistant β-sheet, PrPSc [C, which is required and rate-limiting [To date, the exact physiological role of PrP has not yet been definitively clarified. The expression of the wild type PrP is diffuse in neurons, neuroendocrine cells, and stromal cells of the lymphoreticular system. The highest levels have been observed in the central nervous system in the synaptic membrane. The determinant step in prion infection is the conversion of the conformation of PrPt, PrPSc , with colimiting .2+ and Zn2+ ions coordinate to the N-terminal PrP differently—Cu2+ interacts with the octarepeat domain, residues 60–91 with the sequence (PHGGGWGQ) [2+ binds to the octarepeat domain, in which all four histidine residues coordinate a single Zn2+ ion [The PrP is bound to the outer membrane of the cell surface, in specific “rafts” (cholesterol- and glycosphingolipid-rich lipid sites) , by a glHGGGWGQ) ,23, and HGGGWGQ) , whilst Zn2+ ion .C is a copper-binding protein showing superoxide dismutase activity, appearing to protect against oxidative damage [C protein is extremely versatile, involved in proliferation, differentiation, cell adhesion, and synaptic plasticity [C protein depend on its interaction with extra- and intra-cellular signaling partners (ligands). Among these ligands, those found to be advantageous to the cell [PrPe damage  and actie damage . Severalasticity . Severalthe cell  are lamithe cell , and neuthe cell .PRNP (NC_000020.11), located on chromosome 20 (4686151-4701588), is a 16 Kb long gene, containing two exons. Exon 1 has the role of a transcriptional initiation site, whereas the open reading frame (ORF) encoding the PrP protein, composed of 253 amino acids, is located in the exon 2 [PRNP are the only known causes of IPD [PRNP mutations consist of point mutations leading to an amino acid substitution or a premature stop codon, and insertions/deletions of additional  octapeptide repeats (OPRI/OPRD) in the region between codons 51–91 of the PrP that encodes a 5-mer repeat region consisting of a nonapeptide followed by four identical octapeptides. The frequency and distribution of these mutations differ between Europeans and East Asians [PRNP mutations are typically associated with particular clinical categories of prion disease [PRNP gene mutations have been suggested to play a potential role in clinical pictures mimicking other neurodegenerative diseases, such as Frontotemporal dementia (FTD) [PRNP mutation was described in a young GSS patient, presenting a particular clinical picture with status epilepticus at the age of 34, prefaced by night terrors at age 26, memory problems, behavioral changes and parasomnias subsided after a six-year period, emerged at this age [C that prefigures the PrP mutated fragment pathognomonic for end-stage GSS with multicentric amyloid plaques [e exon 2 . Differee exon 2 . Pathoges of IPD . PRNP mut Asians . Some pa disease , conferr disease , often wia (FTD) ,36,37,38 20 468611-4701588ia (FTD) , familiaia (FTD) , and Hunia (FTD) ; whereasia (FTD) . Of noteia (FTD)  and succia (FTD) , in whicthis age . This muthis age , involvi plaques  that migPRNP gene [PRNP Met129Val SNP is responsible for the diagnosis of FFI or GSS  have also been detected in the ORF of the RNP gene . The mosI or GSS  when assd in CJD . Furtherly127Val  and the ly127Val , completly127Val . The Gluadic CJD , and theadic CJD . It is ptype PrP .+- and Zn2+-binding octarepeat domain, OR) domain, as well as two polybasic charged clusters, and a hydrophobic linker domain.As for the structure of the PrP, the mature protein (residues 23–231) is composed of two independent structures, the N-terminal (23–120) and the C-terminal domains (residues 121–231). The N-terminal region is a flexible, random coil presenting with a disordered amino acid sequence, whereas the C-terminal region forms a rigid globular domain ; it contPRNP pathogenic mutations are involved in generating a misfolded PrP remains not clarified and how pathogenic mutations in PRNP cause prion disease has yet to be solved. However, study efforts about the mechanism involved in this conformational rearrangement of this protein have indicated that the variation of the PRNP sequence by pathological mutations is sufficient to generate prions [PRNP gene were found mostly in the β2–α2-loop region and in the α2–α3 inter-helical interfaces, which are assembled against each other in the hydrophobic core. Experimental data suggested that the conformation of the β2–α2-loop plays a role in the transmission of prion disease and its susceptibility. Mammals carrying a flexible β2–α2 loop are easily infected by prions, while in animals carrying a rigid loop, prions are poorly infected [Despite important advances in the last decade, how e prions . Geneticinfected . Notablyinfected ,53,54,55PRNP mutations, the mutant structures in aqueous solution have been investigated [Sc. Indeed, the regions involved in the pathogenic conversion of PrPC to the scrapie form of the protein appear to be the same as affected by disease-linked mutations in terms of structure and flexibility [PrP structures are characterized by a rigid β2–α2 loop and by a closer contact between the loop and the α3 helix . Thus, istigated . Structuxibility . In factxibility . The flexibility . Moleculxibility ,53,54,55xibility . Using ixibility . The importance of the N-terminal region has largely been underestimated because it does not appear involved in prion replication. Nevertheless, it has been shown that this domain is involved in fibrillation and the determination of the physical properties of disease-related forms of PrP . The N-tC protein in the interaction with multiple extra- and intra-cellular signaling partners (ligands) is due to the structural disorder of the N-terminal domain, which depends on its specific conserved, and not random, amino acid sequence [C internalization [C, and may also regulate the acquisition of the strain-specific conformations in the disease [Many functional advantages to intrinsically disordered proteins/regions (IDPs/IPRs) are due to the lack of stable tertiary and secondary structures. These advantages are represented by the disorder-to-order transition, increased binding rate malleability of interaction with different partners (binding promiscuity), and specific low-affinity binding. These characteristics of disordered, unstructured proteins, which constitute the basis of modulation of post-translational modifications, such as phosphorylation, acetylation, acylation, carboxylation, glycosylation, methylation, hydroxylation, etc. Post-translational modifications, involving low affinity and high-specificity interactions between a protein and a specific ligand and associated with IDPs and IPRs are especially important for signaling and regulation of the cell  . The capsequence . The suslization , for whilization . Moreovelization . The pol disease . The reg disease .C with several advantages. The extended linear protein region may allow interaction with many ligands ranging from small molecules  [C molecule are represented by lipids, nucleic acids, and glycosaminoglycans, which confer to the protein diverse, and sometimes contrasting, activities [Natural ligands promote structural rearrangements and play a significant role in the modulation and stabilization of the structure of proteins. These proteins undergo, during the course of their biological function, several types of conformational changes, which are responsible for interactions between proteins and low molecular-weight ligands or larger macromolecules. The structural transformations induced by a ligand in a protein can vary, ranging from a negligible decrease in the conformational stability to complete protein unfolding ,63. The +, Zn2+) ,64 to mativities ,68,69,702+ and Zn2+), which bind to the amino acid residues 59–90, demonstrate the involvement of this region in copper endocytosis and metabolism [C conversion into PrPSc-like species [Different studies have shown that the N-terminus of PrP can interact with a broad range of ligands: (1) Metal ions  Aβ tabolism ; (3) Tubtabolism ; (4) Acetabolism ; (5) Meltabolism ; and (6) species .C consists of two functionally distinct modules, with the globular domain and the flexible tail-exerting regulatory and executive functions, respectively [C N-Terminal domain in prion species barriers examined the role of amino acids 23–90 in cross-species conversion using real-time, quaking-induced conversion (RT-QuIC) to model the central molecular event in prion disease, i.e., the template misfolding of the PrPC to the pathogenic isoform. By comparing the conversion efficiency of various prion seeds in either full-length (amino acids 23–231) or truncated (amino acids 90–231) PrPC, it was observed that, in addition to the primary sequence, prion species barriers are controlled by interactions of the N-Terminal domain with PrPC [To date, the N-terminal and C-terminal domains have often been considered as independent and non-interacting units. Cellular and biophysical studies have demonstrated that this scheme cannot be correct, and rather, the PrPectively . In factectively . An inteith PrPC .PRNP gene was tolerated both in cells and in transgenic animals. Similarly, the deletion of the N-terminal domain (residues 23–124) resulted to be benign [C N-terminal domain have been reported to induce varying degrees of neurotoxicity in transgenic mice, and the severity of the neurotoxic phenotype depended on the length of the amino acid deletion. Deletions in residues spanning 105–125 at the end of the N-terminus domain produce spontaneous neurodegeneration similar to that of natural prion diseases but without accumulation of PrPSc [C, the N-terminal domain may act as a neurotoxic effector whose activity is regulated by its C-terminal domain. Thus, this interaction involving the N- and C-domain may regulate the physiological activity of PrPC, and the disruption of this interaction could play a role in the pathophysiology of neurodegenerative disorders [In another study, it was observed that deletion of the e benign . Insteadof PrPSc . These pof PrPSc . Recentlof PrPSc  discoverisorders .C physiology and in prion disease pathogenesis. To date, growing evidence supports the concept that the physiological function of PrPC is associated with its metal-binding properties [2+ and Zn2+ promote an interdomain interaction in cis, in terms of an association of the PrPC N- and C-terminal domains [cis interaction leads to sequestration of the N-terminal domain and regulation of cell surface receptor interactions via an autoinhibitory-like mechanism, with the direct involvement of metal ions [2+ or Zn2+ ions but is attributed to the long-range tertiary interactions involving the N-terminal domain, given that the interdomain cis interaction is stabilized by the coordination of Cu2+ and Zn2+ ions [cis interactions between the OR region, while disruptions of the site 5 region influenced pathogenic outcomes by impacting on the PrP globular C-terminus domain [Among the known ligands of the N-terminal domain, there has been substantial effort to better understand the role of metal ions in normal PrPoperties . The oct domains . This dotal ions . Howevern2+ ions . In a ren2+ ions  establisn2+ ions . The auts domain .Proline is a nonpolar, non-essential, cyclic amino acid, and imparts a degree of the structure onto proteins due to the steric constraints of the rigid pyrrolidine ring . ProlineC, or in the linker region that separates the C-terminal from the N-terminal domain [cis interaction with the N-terminal domain, suggests that the human mutations responsible for familial CJD  and either CJD or FFI based on the codon at residue 129), respectively, showed weakened cis interactions. Otherwise, the GSS-associated Pro102Leu mutation in humans resulted in a mild weakening of the strength of the cis interaction, indicating that the conformational rigidity of the proline at this codon may be involved in correctly orienting the two PrP domains for suitable cis interactions. A similar pathogenic mechanism could be shared from all mutations involving proline, including the recently published Pro39Leu  or assoutations , should utations  to inves


Bacillus subtilis prophages have been characterized and it was found that only XepA establishes cytotoxic activity in plaque assays. The crystal structures of both proteins show a unique pentameric assembly, in which YomS adopts a very similar fold to the C-terminal domain of the XepA dumbbell pentamer. The overall architecture of XepA, with the N-terminal domain subunits resembling cytoplasmic membrane-binding C2-domain folds, suggests that any lytic functionality could be based on disruption of the proton motive force of the cytoplasmic membrane, which induces cell lysis.The lytic cassette proteins XepA and YomS from  Bacillus subtilis prophage PBSX and YomS from prophage SPβ were cloned and the proteins were subsequently produced and functionally characterized. In order to elucidate the role and the molecular mechanism of XepA and YomS, the crystal structures of these proteins were solved at resolutions of 1.9 and 1.3 Å, respectively. XepA consists of two antiparallel β-sandwich domains connected by a 30-amino-acid linker region. A pentamer of this protein adopts a unique dumbbell-shaped architecture consisting of two discs and a central tunnel. YomS (12.9 kDa per monomer), which is less than half the size of XepA (30.3 kDa), shows homology to the C-terminal part of XepA and exhibits a similar pentameric disc arrangement. Each β-sandwich entity resembles the fold of typical cytoplasmic membrane-binding C2 domains. Only XepA exhibits distinct cytotoxic activity in vivo, suggesting that the N-terminal pentameric domain is essential for this biological activity. The biological and structural data presented here suggest that XepA disrupts the proton motive force of the cytoplasmatic membrane, thus supporting cell lysis.As part of the Virus-X Consortium that aims to identify and characterize novel proteins and enzymes from bacteriophages and archaeal viruses, the genes of the putative lytic proteins XepA from  The respective PCR fragments were cloned in-frame into the rhamnose-inducible vector pJOE5751  and S12646 (gcctgtacaTTAACGCACATCCATCTCACCCG) and yielded a 441 bp fragment. The N-terminal xepA domain with the tunnel region was amplified with the primers S11669 (see above) and S12647 (gcctgtacaTTAAGCCTCGACTTTCAGCCGTC), yielding a 520 bp fragment, while for amplification of the C-domain of xepA the primers S12648 (gcggatccGTCAATGAAAAAACGCCTTTACA) and S11670 (see above) were used and resulted in a 325 bp fragment. The respective PCR fragments, which contained additional BamHI and BsrGI restriction sites, respectively, were cloned in-frame into pJOE5751 as mentioned above and resulted in pHWG1319 for His6-xepA without the tunnel region (XepA_N), pHWG1320 for His6-xepA with the tunnel region (XepA_NL) and pHWG1321 for His6-xepA-C-domain (XepA_C). After transformation of the plasmids in E. coli JM109 cells, 15 ml precultures were grown overnight in LB–ampicillin at 37°C. After inoculating a 1 l LB–ampicillin culture with the preculture, the bacteria were grown for 4 h to an OD600 nm of 0.6–0.8. Protein production was induced by the addition of rhamnose to a final concentration of 0.2% at 30°C. The culture was incubated overnight and was subsequently centrifuged at 1300g at 4°C. The pellet was resuspended in lysis buffer (20 mM Tris–HCl pH 7.5) with added protease inhibitor. After sonication  the supernatant was filtered and was subsequently loaded onto a HisTrap HP affinity column, which was subjected to an imidazole-gradient FPLC separation. The protein was dialyzed into 10 mM ammonium bicarbonate buffer pH 7.5 and the molecular weight was verified by ESI-MS and SDS–PAGE. For YomS, selenomethionine (SeMet) needed to be incorporated in the protein sequence for crystallographic structure solution. Accordingly, the YomS expression construct was transformed into the methionine-auxotrophic E. coli strain B834(DE3). SeMet incorporation was performed following the general recommendations  and 10 µl was pipetted into a 96-well PCR plate. 10 µl of the screens were added to the wells and the plate was sealed with thermostable film. The plate was centrifuged at 160g at 4°C for 2 min and was subsequently placed in a Real-Time PCR machine for melting-temperature experiments. Data from the thermal shift assay screen were analysed using in-house Microsoft Excel scripts and NAMI  and the fluorescence was measured after each increment. In addition, thermal shift assays with all target proteins and a number of glycosides were performed using the same protocol to assess ligand binding.Thermal shift assays were performed to identify stabilizing conditions for protein storage and crystallization  PEG 4000. Co-crystals with a terbium compound  were obtained by adding 100 µl protein solution (as above) to 0.6 mg of a Tb cluster compound  PEG 4000. Crystallization experiments were set up in sitting drops in MRC 3-well plates using a Mosquito robot (TTP Labtech). Long needles  were grown using 0.1 M magnesium acetate, 0.1 M potassium chloride, 12% PEG Smear High (Molecular Dimensions), 0.1 M MES pH 5.5 and 4 mg ml−1 XepA in Tris buffer and were crystallized manually in vapour-diffusion sitting drops. YomS crystals (∼0.03 × 0.2 × 0.3 mm) were obtained at 20°C using 13.4 mg ml−1 YomS in 20 mM MES pH 6.0, 150 mM sodium chloride mixed with an equal volume of 0.1 M cacodylate pH 5.5, 0.2 M ammonium nitrate, 18%(w/v) PEG Smear Low (Molecular Dimensions). Selenomethionine-containing YomS crystals (∼0.03 × 0.1 × 0.2 mm) were grown from 6 mg ml−1 SeMet-YomS under the same conditions by self-seeding techniques.Initial crystallization experiments were performed using a range of commercially available crystallization screens. XepA crystallized in several crystal forms. Crystal form I  was obtained at 4°C using a protein solution consisting of 15 mg ml2.4.M sodium acetate pH 5.0, 9%(w/v) PEG 4000, 50 mM sodium chloride, 30%(v/v) glycerol. Tb-derivatized XepA form II crystals were soaked in a solution with 0.6 mg terbium cluster added to 10–100 µl cryosolution for approximately 1 min. Native YomS crystals were cryoprotected in 0.1 M sodium cacodylate pH 5.5, 0.2 M ammonium nitrate, 22%(w/v) PEG Smear Low, 25% glycerol, 30 mM sodium chloride. SeMet-YomS crystals were transferred into 0.1 M sodium cacodylate pH 5.5, 0.2 M ammonium nitrate, 20%(w/v) PEG Smear Low, 25% PEG 400. Data were collected using a PILATUS pixel-array detector , 6ia5 (XepA form II) and 6i5o (YomS).All crystals were transferred into cryosolution and flash-cooled in liquid nitrogen before data collection  agar] that contained 500 µl of concentrated bacterial cells. Eight bacterial strains were tested: Bacillus megaterium ATCC 14581, B. subtilis subsp. spizizenii ATCC 6633, B. pumilus KPD 181, B. thuringiensis KPD 114, B. mycoides KPD 15, Micrococcus luteus ATCC 4698, E. coli MG1655 and B. subtilis 168 DSM 23778. Cells were prepared by growing them to an OD600 nm of 0.3 in 25 ml LB. Bacterial cultures were centrifuged at 5000g for 30 min at 4°C and the pellet was washed in 20 mM HEPES pH 7.4. The washed pellet was resuspended in 500 µl HEPES buffer added to the 50°C semi-solid LB–agar. The plates were left for 20 min at room temperature to set before spotting the protein solutions and were subsequently incubated overnight at 37°C. Hen egg-white lysozyme (HEWL) was applied as a positive control, whereas the negative control was HEPES buffer . A summary of the results of the in vivo assays can be found in Table 2XepA, YomS, XlyA and XlyB were dialysed after purification into 20 m3.3.1.Supplementary Fig. S1). However, neither XepA nor YomS shows significant sequence similarity to the lysins XlyA and XlyB, indicating that these two enzymes have different functionalities and/or mechanisms. In database searches no assigned enzymatic domains could be found for the XepA and YomS sequences. However, YomS corresponds to the C-terminal half of XepA. A sequence search for the N-terminal XepA domain in the SPβ prophage genome in the B. subtilis database SubtiWiki , whereas the identity for YomS is 33% . The high sequence identity to B. subtilis YqxG suggests that this protein might have a very similar three-dimensional assembly to the XepA structure described below. The XepA and YomS protein sequences were also screened for transmembrane regions but none were identified, thus indicating that both proteins are soluble components of the lytic system.The putative lysins XepA and YomS align very well, with a sequence identity of 38% (54% similarity) covering 85% of the overall sequence , which as an N-acetylmuramide glycanhydrolase attacks the peptidoglycan at the glucose 3 (16 mM) show an increase in the melting temperature, Tm, from 68 to 77°C. The same experiments with d-GlcNAc (N-acetyl-d-glucosamine) did not show a stabilization effect. All four proteins were tested for (GlcNAc)3 and d-GlcNAc affinity. A similar stabilization (ΔTm = 2°C) was observed for XlyA and XlyB in TSAs with (GlcNAc)3. For XepA and YomS, however, no stabilizing effect was observed in the presence of these compounds, which suggests that any XepA/YomS lytic action is likely to be based on a different mechanism to the HEWL muramidase activity.In addition to XepA and YomS, the lytic enzymes XlyA and XlyB were subjected to thermal shift assays (TSAs). XepA, XlyA and XlyB are significantly more thermostable, with 3.3.a). Superpositions of the N-terminal (residues 35–142) and the C-terminal (residues 174–279) β-sandwiches show that they are similar in fold but have major differences . In the crystal, XepA forms a highly symmetric pentamer , whereas the full pentamers superimpose with an r.m.s.d. of 1.2 Å . Although the sequence identity between XepA and YomS is only 38%, the three-dimensional structures of YomS and the C-terminal XepA domain are highly conserved, supporting the notion that the domains in each protein serve similar functionalities.The three-dimensional structure of YomS was determined to a resolution of 1.3 Å by SeMet SAD phasing. YomS adopts an antiparallel β-sandwich fold, which arranges as a pentameric disc similar to the C-terminal domain of XepA Fig. 6. In this Å Fig. 7b. Altho3.5.2, respectively. Rather than being based on one or two key residues, the interfaces are mainly held together by a large number of hydrogen bonds (over 40 for XepA and approximately 20 for YomS) and salt bridges.The individual interfaces within the pentamers of both proteins show extensive electrostatic interactions. The buried surfaces between two XepA chains and two YomS chains amount to approximately 3300 and 1100 Å3.6.B. megaterium, B. pumilus, B. subtilis subsp. spizizenii, B. thuringiensis, B. mycoides, M. luteus, E. coli and B. subtilis 168) were tested and the results are summarized in Table 2B. megaterium, B. pumilus, B. subtilis subsp. spizizenii, M. luteus and B. subtilis 168, whereas XepA and XlyB show plaque formation with the same samples and in addition with B. thuringiensis and B. mycoides. The N-acetylmuramoyl-l-alanine amidase XlyA only shows lysis with B. megaterium and B. thuringiensis. YomS does not exhibit any effects on the tested bacteria. As the crystal structures revealed a high degree of structural similarity of the C-terminal XepA domain and YomS, truncated mutants of XepA were designed and the variants were produced. They included (i) only the N-terminal protein (XepA_N), (ii) the N-terminal domain including the linker region (XepA_NL) and (iii) only the C-terminal domain (XepA_C). In vivo plate assays with these variants and B. megaterium  displayed no activity for XepA_N and XepA_C. XepA_NL shows minimal activity. This clearly indicates that the full-length XepA protein is required for cytotoxic activity.As the target proteins had been described to play a role in the lytic systems of their prophages, they were tested for cytotoxic activity in bacterial plate assays Fig. 8. Purifie4.4.1.Bacillus species and M. luteus, and the Gram-negative E. coli to four proteins with putative lytic activity. As a positive control, the known lytic enzyme HEWL was tested as an N-acetylmuramide glycanhydrolase. The Gram-negative E. coli did not show any disruption of the bacterial cells, which means that none of the proteins were able to attack the bacterial outer membrane. Interestingly, XepA showed lytic activity against all Bacillus species tested : a C-terminal cell-wall-binding domain (CBD) and an N-terminal catalytic domain with a Zn2+-dependent active site  could be involved in similar interactions. As XepA displays two domains with very similar folds, a tandem C2 domain is also possible.XepA forms a remarkable pentameric structure in which two disc-shaped domains are connected by a linker region. As shown above, the C-terminal disc of XepA is very similar to the structure of YomS, and the full-length protein including both domains is essential for cytotoxic activity. Both the XepA and YomS monomeric β-sandwiches adopt a fold resembling the C2 domain, which usually contains about 110 amino acids and 2–3 Caet al., 2015et al., 2018Looking at the three-dimensional structure of XepA with the linker region connecting the N-terminal and C-terminal pentameric discs, it appears possible that the structure may form a tunnel involved in DNA release. However, the absence of a transmembrane domain and the relatively small size of the formed pore discourage this notion. Comparisons with viral portal proteins  and helping the virus particle to escape (XepA). Many phages have additional domains lying on the capsid surface, which can be seen in cryo-EM reconstructions as protrusions. In the tailed bacteriophage φ29, for example, head fibres decorate the phage head  may be able to bind to the negatively charged phosphate moieties of the phospholipid bilayer. Furthermore, the crystal structure of XepA reveals acetate ions from the crystallization solutions that bind on the electropositive surface in the centre of the dumbbell-shaped protein, showing potential phosphate-binding sites. In contrast, the XepA C-terminal surface is almost exclusively electronegatively charged , which suggests that the same interaction is not feasible between the C-terminal surface and the cytoplasmic membrane.DNA phages can adopt multiple strategies to accomplish host-cell lysis, including the holin-dependent export of lysins, the Sec-mediated export of lysins with signal peptides (as observed in fOg44) and the holin-independent export of lysins with SAR  as observed in coliphage P1  and thus cannot be integrated into the cytoplasmic membrane.Like all members of the Caudovirales, PBSX and SPβ form an icosahedral head made of hexamers and pentamers of its major coat protein (MCP). Considering the conserved fivefold symmetry in the protein structures described here, it is feasible that the C-terminal pentamer of XepA as well as the structurally very similar YomS may bind at least transiently to the prophage capsid proteins. The XepA pentamer might dock onto the phospholipid membrane with its N-terminal positively charged surface and hence disrupt the proton motive force (PMF) in a similar way to pinholins (Catalão We hypothesize that XepA may be located on the phage capsid, binding to the cytoplasmic membrane and dissipating the proton motive force of the membrane. XepA could thereby interfere with the host-cell secretion machinery and/or subvert the bacterial lytic system to induce host-cell lysis, as shown previously (Fernandes & São-José, 20185.et al. (2007The following references are cited in the supporting information for this article: Larkin  al. 2007 and Robe al. 2007.YomS, 6i50PDB reference: XepA, 6i56PDB reference: 6ia5PDB reference: 10.1107/S2059798319013330/gm5068sup1.pdfSupplementary Tables and Figures. DOI:


We further extend this class of nets by including a ditopic linker to form [4 + 3 + 2] COFs. The results open up possibilities towards a new class of sub-valent COFs with unique structural, topological and compositional complexities for diverse applications.Covalent organic frameworks (COFs) are typically designed by breaking down the desired network into feasible building blocks - either simple and highly symmetric, or more convoluted and thus less symmetric. The linkers are chosen complementary to each other such that an extended, fully condensed network structure can form. We show not only an exception, but a design principle that allows breaking free of such design rules. We show that tri- and tetratopic linkers can be combined to form imine-linked [4 + 3] sub-stoichiometric 2D COFs featuring an unexpected  bex topology by combining non-complementary triangular and rectangular linkers.Using complementary linkers as building blocks in the design of 2D covalent organic frameworks (COFs) limits the formation of compositionally and structurally complex networks. Here, the authors demonstrate a COF with a  Such materials are synthesized by topologically directed condensation reactions, based on matching the geometry of the building blocks to form the desired net composed of the respective vertex and edge linker units9. The shapes and dimensions of the vertex and the edge linkers determine the pore shape and size, respectively. Most of the 2D COF structures reported till date are based on four simple topologies for which the vertices are connected by only one kind of edge—the honeycomb, square, kagome, and hexagonal lattice complexes3. In order to make the COFs, these nets are typically deconstructed into highly symmetric building blocks  represent an intriguing class of crystalline porous materials, where appropriately functionalized organic molecules are linked with an atomic level precision to give extended periodic structuresbex topology with two distinct vertex molecules catalysis29, ion conduction31, and optoelectronics33. Such diverse applicability demands diversified structural characteristics and importantly, appropriate functionality, which is often difficult to achieve in a single-step and requires post-synthetic modification of the framework. With examples limited to only a few35, it is still challenging to tune the structure and the physical properties of the COF post-synthetically while maintaining its crystallinity and porosity. This is because generating functional groups in the framework by post-synthetic modification requires orthogonal reaction chemistry, which also limits probable options considerably. In this regard, the unconventional combination of building blocks to produce periodic, uncondensed amine functional groups in the fully extended, crystalline and porous imine-linked COFs, is a new approach toward parallel and simultaneous functionalization of 2D COFs. Also, this method of introducing free functional groups by generating defined sub-valent COFs is fundamentally different from previous examples that rely on the introduction of defects37, or the introduction of periodic linker vacancies into a saturated host lattice to introduce dangling functional groups in the framework as reported by Loh and co-workers38. We further show that the free amine functional groups enhance CO2 adsorption of the framework, can be transformed in a subsequent reaction, and can also act as catalysts in organic transformations.Contrary to these typical design principles, in this manuscript, we show that triangular tritopic and rectangular tetratopic linkers can actually be combined to form a crystalline, dual pore 2D COF belonging to a previously unidentified 39. In this report, we also demonstrate the combination of tritopic, tetratopic, and linear ditopic linkers to form COFs, which have crystallinity and surface area comparable to the state-of-the art examples.All three-component COFs reported thus far use building blocks of only two different symmetriesThis class of sub-stoichiometric COFs with a frustrated bonding network prompts us to think outside the set rules of topological deconstruction and paves way for a new class of structures with unique structural, topological, and compositional complexities. Such COFs are inherently linked to free functional groups, and can be exploited in a diverse, application-specific manner.Triazine tribenzaldehyde (T) and benzene tripicolinaldehyde (Y) were chosen as the triangular tritopic linkers for reaction with pyrene tetraaniline (P) as the tetratopic linker. A fully condensed 2D framework cannot be envisaged from a combination of molecules with such symmetries, and thus periodic, crystalline frameworks are typically not expected to form. Nevertheless, when these linkers are reacted in an equimolar ratio in 1:1 mesitylene/dioxane under solvothermal conditions, crystalline PT-, and PY-COFs result  at ~1700 cm−1 corresponding to the aldehyde groups in the starting materials, T and Y  at 1625 cm−1 corresponding to the imine bonds formed in the framework by condensation of the aldehyde and the amine groups in the starting materials. Interestingly, similar to that in P  in the region 3430 cm−1–3190 cm−1 is observed for both PT- and PY-COFs. 13C cross-polarization magic angle spinning solid-state NMR (ssNMR) spectroscopy  for PT-COF and at 159 ppm (1′) for PY-COF proving the condensation of the starting materials into the framework. Retention of the molecular structure of the building blocks in the framework is also evident from an analysis of the 13C NMR spectrum. For PT-COF, the signals for carbon 2, 3, and 4 can be identified at 170 ppm, 150 ppm, and 116 ppm, respectively42. Likewise, for PY-COF, carbon 3′, 4′, 5′, and 6′ appear at 150 ppm, 116 ppm, 155 ppm, and 146 ppm, respectively. This analysis is further corroborated with 15N-ssNMR measurements  and the pyridine (10′) nitrogen appear between −40 ppm and −78 ppm. The signal at −262 ppm in the 15N ssNMR spectrum of PY-COF probably arises from oxidation of a fraction of the imine-linkages to amides in the COF  at −323 ppm to −330 ppm  measurements Fig.  of PT- aopy Fig.  shows thpyridine ′ nitrogeppm Fig. , inset.Fθ. In addition, a broad stacking reflection can be seen at 2θ = 24° for both COFs hinting towards the formation of 2D rather than a 3D-COF (vide infra). The PXRD patterns could be simulated by a structure model (Supplementary Methods) as shown in Fig. 2 groups result per unit cell, the presence of which were confirmed with 15N-ssNMR and IR experiments. The unit cell parameters for this structure model with P1 space group and bex crystallographic network topology were obtained by force-field optimizations and were then Pawley refined against the experimental PXRD pattern  possibly increases the predilection towards stacking and thus the consequent formation of such structures over stoichiometric 3D-COFs.PY- and PT-COFs are crystalline materials  linker. In the resulting structure, the ribbons should now be linked by B molecules instead of P, and thus the COF should not have any free-amine functional groups. Thus, P, T/Y, and B were reacted in a 1:2:1 molar ratio in the same solvent mixture as before under the same conditions to get highly crystalline PTνC=O(stretch) signal corresponding to the aldehyde groups in the starting materials and a simultaneous appearance of νC=N(stretch) at 1625 cm−1, validating the formation of the framework. Indeed, as expected, the νN-H(stretch) signal, as observed for PT- and PY-COFs, is absent in PT2B- and PY2B-COFs. 13C ssNMR spectrum  for PT2B-COF and at 159 ppm (11′) for PY2B-COF proving the formation of the framework. Retention of the molecular structure of the building blocks in the framework is again evident. 15N ssNMR is identical to PT- and PY-COFs , 6.9, 7.3, 9.4, 10.6, 12.3, 14.7 (for PY2B-COF), 15.9, 24.2, 25.8–26.5° 2θ. The Pawley refined unit cell parameters . Making COFs by combining three different linkers has been reported, albeit using intuitive combinations of either two differently sized linear linkers of the same geometry22, or using two molecules of similar geometry but having different linking groups39. PT2B- and PY2B-COFs, on the other hand, are the first example of COFs made from a combination of three linkers with different topicity.PTrns Fig.  have 12 d-spacings can be observed in the same sample—and often in the same crystallite—strongly suggesting a structural model based on a unit cell with significantly different a and b parameters describing the in-plane structure. The observed crystallites, in many cases bent, feature a large repeat unit of 3.9–4.1 nm for PT2B- and PY2B-COFs  surface area as evaluated by measuring Argon sorption isotherms at 87 K 53, 4PE-1P-COF (2140 m2 g−1)54 and TPB-DMTP-COF (2015 m2 g−1)55. The absence of free amine functional groups in PT2B- and PY2B-COFs also reduces the possibility of chemisorbed molecules clogging the pores and possibly contributes to the larger surface area. A pore size distribution (PSD) was calculated from the Argon sorption isotherms using a Quenched Solid DFT adsorption kernel for Argon at 87 K on carbon with cylindrical pores. For the PT- and PY-COFs, only one type of pore centered at 1.77–1.83 nm is observed  as the base for trapping the HI generated, the emission intensity first drops and then increases beyond the initial value on addition of increasing amounts of methyl iodide (MeI) . On the other hand, with PT- and PY-COFs, the reaction yields of 22% and 8%, respectively, with excellent regioselectivity  in the region 3430 cm−1–3190 cm−1 corresponding to the free amines in PY-COF is found to be almost absent in PY-NCS-COF. This is accompanied with the appearance of νN=C=S(stretch) signal at 2050 cm−1. 15N NMR spectrum , T/Y , 1,4-dioxane (3 ml), mesitylene (3 ml), and aqueous acetic acid  were added to a Biotage© precision glass vial, sealed, and heated under autogenous pressure at 120 °C for 72 h. After the reaction was allowed to cool down, the reaction mixture was filtered and washed thoroughly with water, tetrahydrofuran, chloroform, and acetone, and then dried in high dynamic vacuum overnight to get PT- and PY-COF in 72% and 68% yield, respectively.2B- and PY2B-COF in 75% and 72% yield, respectively.P , T/Y , B , 1,4-dioxane (5 ml), mesitylene (5 ml), and aqueous acetic acid  were added to a Biotage© precision glass vial, sealed, and heated under autogenous pressure at 120 °C for 72 h. After the reaction was allowed to cool down, the reaction mixture was filtered and washed thoroughly with water, tetrahydrofuran, chloroform, and acetone and then dried in high dynamic vacuum overnight overnight to get PT2CO3  in water (10 ml), 50 mg of PY-COF was added. To the resulting suspension, CS2  was added dropwise. After the addition was complete, the reaction mixture was stirred overnight at room temperature. The reaction mixture was then cooled to 0 oC and a solution of cyanuric chloride  in 5 ml dichloromethane was added dropwise. After complete addition, the reaction mixture was stirred for 3 h and then filtered. The residue was basified with 6 N NaOH solution and then washed with water until the washings were pH neutral followed by washing with acetone, chloroform, and tetrahydrofuran and then dried in high dynamic vacuum overnight to get PY-NCS-COF in 62% yield.To a solution of KSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5


Staphylococcal pathogens adhere to their human targets with exceptional resilience to mechanical stress, some propagating force to the bacterium via small, Ig-like folds called B domains. We examine the mechanical stability of these folds using atomic force microscopy-based single-molecule force spectroscopy. The force required to unfold a single B domain is larger than 2 nN – the highest mechanostability of a protein to date by a large margin. B domains coordinate three calcium ions, which we identify as crucial for their extreme mechanical strength. When calcium is removed through chelation, unfolding forces drop by a factor of four. Through systematic mutations in the calcium coordination sites we can tune the unfolding forces from over 2 nN to 0.15 nN, and dissect the contribution of each ion to B domain mechanostability. Their extraordinary strength, rapid refolding and calcium-tunable force response make B domains interesting protein design targets. Staphylococcal pathogens adhere to their human targets using adhesins, which can withstand extremely high forces. Here, authors use single-molecule force spectroscopy to determine the similarly high unfolding forces of B domains that link the adhesin to the bacterium. Covalently bound to the Gram-positive peptidoglycan and extruding into the extracellular space, these adhesins target sequences on the order of 15 amino acids in human proteins, notably all chains of fibrinogen, but also other members of the extracellular and adhesive matrix, such as fibronectin3 and keratin4. Initial adhesion is crucial to begin infection, especially under hydrodynamic forces. The ligand binding region A at the N-terminus of these adhesins employs the “Dock, Lock and Latch” (DLL) mechanism6. In DLL, the peptide target is tightly confined between the N2 and N3 domain of region A, achieving mechanically hyperstable adhesion to host proteins. These adhesins, such as SdrG from S. epidermidis, and its homologs from S. aureus  can withstand forces in the 2 nN force regime12, approaching the strength of a covalent bond13. This extreme strength is achieved through a recently identified molecular mechanism9. Along their stalks are so-called B domains, which appear in adhesins such as S.aureus SdrD and Bbp, S. epidermidis SdrG, and S. saprophyticus UafA14. B domains are small domains (~ 13 kDa) that link the ligand binding region to a sortase motif, which mediates covalent anchoring of the adhesin to the bacterial peptidoglycan15. Thus, it is to be expected that these folds must propagate the extreme mechanical force withstood by the DLL adhesin.Pathogenic bacteria have evolved to strongly and persistently adhere to their hosts using a variety of mechanisms. Among them are microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), which adhere to target proteins of their human hosts19 we investigate the mechanical strength of B domains from S. epidermidis SdrG20 and S. aureus SdrD22. We found their mechanostability to be exceptional, far exceeding all other proteins investigated to date. B domains unfold at forces larger than 2 nN – a strength reminiscent of breaking a covalent bond. In comparison, pili domains of FimA from Actinomyces oris have been shown to unfold at ~ 0.7 nN23 and cohesin domains from cellulosomal bacteria unfold at ~ 0.6 nN25, both at similar force loading rates. Through site-directed mutations, we demonstrate that this stability rests on the coordination of calcium (Ca2+) ions. Each B domain coordinates three Ca2+ ions in different positions. When these are chelated from the domain their mechanostability drastically decreases by a factor of four – yet forces are still in the vicinity of 0.6–0.8 nN. Systematically incapacitating the Ca2+ coordination sites revealed which Ca2+ ion is most important to the mechanostability. Furthermore, there are subtle differences between B domains from related organisms – even the same gene – opening multiple scenarios for their role in pathogen adhesion.Using atomic force microscopy-based (AFM) single-molecule force spectroscopy (SMFS)S. aureus as a handle, which binds a 12 amino-acid C-terminal peptide of dermokine (DK) and conveniently has no B domains, as well as a higher unbinding force than SdrG  can only present a limited number of molecules. Yet, its unfolding appeared in almost every trace for more than 12 h (N = 3712 events), thus we conclude that it refolds. A dynamic force spectrum was acquired for the high-force unfolding of B1  B1 domain structure -like fold containing exclusively ß-strands , which binds divalent ions. When probed in 10 mM EDTA, the stability of SdrG B1 dramatically decreased, and the previously described weak unfolding event ~ 600 pN appeared exclusively , reflected in similar distances to the transition state ∆x (within ~ 17% of each other), whereas the large difference in unfolding force is given through the zero force off-rates koff0, which differ by >14 orders of magnitude.The presence of calcium is relevant for both folding and stability of many protein domains2+-free buffers  and found mostly in the strong state. This puzzling contradiction of Ca2+-dependent folding could be explained by trace amounts of contaminating Ca2+ in the buffer, estimated to be in the nM range . Thus, the affinity of SdrG B1 for Ca2+ must be extremely high. Moving to higher purity Ca2+-free reagents (see Methods), a trace Ca2+ concentration low enough to keep the domains in the weak state was achieved. Previously, the Ca2+-dependent folding and thus Ca2+ affinity of homologous B domains from S. aureus SdrD had been measured31. As our AFM experiments could clearly discern between the strong Ca2+ saturated and weak, Ca2+-depleted state, we titrated the amount of Ca2+ to estimate the affinity of the weak state SdrG B1 domain for Ca2+ by plotting the fraction of strong to weak state events against Ca2+ concentration. The inflection point was below 1 nM, within our conservative estimate of Ca2+ concentration uncertainty, hinting at a sub-nM KD for Ca2+ , shown in Figs. 2+ sites deleted, leaving only a single Ca2+ bound, were created , hinting at a complete unfolding of the domains, a marked difference from to the mere weakening of SdrG B1. The Ca2+-EDTA switching, for SdrD B1 , whose sequence is 45% identical to B1  adhesins attach to their human targets through a covalent isopeptide bond35. Covalent bonds are mechanically stronger and irreversible compared with the non-covalent, spontaneously reversible, DLL attachment (KD for SdrG N2N3:Fgß ~ 400 nM)5. Isopeptide bonds are covalent amide bonds between amino-acid side chains that stabilize a fold, or connect two proteins. TEDs and collagen-binding MSCRAMMs are linked by Ig-like and Ca2+ coordinating folds such the Spy0128 pilus of S. pyogenes, which contain intramolecular isopeptide bonds37. These block the mechanical extension of Spy012838. Notably, another Ig-like domain from S. pyogenens fibronectin binding protein (fba2) follows a remarkably similar fold to the B domains here  at least SdrG B1 would be found almost exclusively in its strong state. However, the mechanical stabilities of the strongest state of SdrG B1 and B2 are on the order of the interaction of the SdrG N2N3 domains with fibrinogen. Sometimes the interaction is not sufficient to unfold the B domain at force loading rates around 104 pN s−1 – in clear conflict with the proposed shock dissipater by unfolding hypothesis. The BE fits . Previous work suggested that these have different individual Ca2+. The coordination of Ca3, connecting the parallel very N and C-terminal, closing ß-strands  or Sigma-Aldrich  if not specified explicitly.Dictyostelium discoideum 4th filamin fingerprint domain , the Staphylococcus epidermidis SdrG N2N3, B1, and B2 domain genes (UniProt: Q9KI13), the Staphylococcus aureus ClfB N2 and N3 domains (UniProt: Q7A382); the SdrD B1 domain  were synthesized codon-optimized for expression in Escherichia Coli as linear DNA fragments  including suitable overhangs for Gibson assembly. Genes were inserted into pET28a Vectors with a hexahistidine-, ybbr-tag and in some cases a sortase motif via Gibson assembly 47 . All point mutations, deletions, or additions of amino acids in all systems were created through polymerase chain reactions  with appropriate primers and finally blunt end ligation cloning using T4 Ligase . Resulting open reading frames of all constructs were verified by DNA sequencing . All protein sequences of constructs used in this study are listed in the Supplementary Information.The www.addgene.org) and are available through the following AddgeneIDs:Important plasmids were deposited with Addgene (E. Coli NiCo21(DE3) . Bacterial starter cultures of 5 mL Lysogeny broth (LB) medium containing 50 μg mL−1 Kanamycin, were inoculated and grown overnight at 37 °C. These were added into in 100–200 mL of ZYM-5052 autoinduction media48 containing 100 μg mL−1 Kanamycin and grown for 6 h at 37 °C and cooled down, continuing overnight at 18 °C. For small-scale protein production, 8 mL cultures in ZYM-5052 autoinduction media were grown at 37 °C overnight. Bacteria were harvested by centrifugation at 8000 g, the supernatant was discarded, pellets were stored at −80 °C until purification.All proteins were expressed in 2, 0.1% (v/v) Tween-20, 10% (v/v) glycerol, pH 8.0) with 100 µg mL−1 lysozyme . Cells were lysed by sonication . Insoluble parts were separated by centrifugation at > 40,000 g for at least 30 min. The supernatant was sterile filtered , adjusted to contain 20 mM imidazole, and then loaded onto a Ni-NTA column  for HIS-Tag purification and washed extensively  Tween-20, 10% (v/v) glycerol, pH 7.4). The protein was eluted in the same buffer, only different in containing 200 mM imidazole and being at pH 7.8. Protein containing fractions were concentrated in centrifugal filters , exchanged into measurement buffer  by desalting columns , adjusted to 10% (v/v) glycerol, and frozen in aliquots in liquid nitrogen to be stored at −80 °C until thawed for experiments. Protein concentrations were determined by spectrophotometry at 280 nm with typical final concentrations of 30–1000 μM .All purification steps were performed at 4–8 °C. The bacterial pellet was resuspended in lysis buffer  and 24 mM diameter cover glass surfaces  were modified with Aminosilane.More detailed AFM-SMFS protocol have been published previously20 for 10 min, rinsed with ultrapure H20, and further cleaned and oxidized in 50% (v/v) H202 and 50% (v/v) of 30% (v/v) sulfuric acid for 20 min. Surfaces were washed in ultrapure H20, then ethanol. Surfaces were silanized by soaking in a solution of: 2% (v/v) (3-aminopropyl) dimethylethoxysilane , 88% (v/v) ethanol, and 10% (v/v) ultrapure H20 under gentle shaking for 1 h. Again, followed by two rinsing steps in ethanol, then rinsed in ultrapure H20, and afterwards dried in a gentle stream of nitrogen. Finally, surfaces were baked at 80 °C for 45 min. Glass surfaces were stored under Argon and typically used within 1 month.Glass surfaces: Glass surfaces were cleaned by sonication in 50% (v/v) 2-propanol in ultrapure H20 for 5 min. Each cantilever was rinsed in ethanol and subsequently in ultrapure H20. Finally, cantilevers were baked at 80 °C for 1 h to be stored overnight under Argon and used in the following steps the next day.Cantilevers: Following 15 min of UV-Ozone cleaning , cantilevers were silanized, submerged in 1 mL (3-aminopropyl)-dimethylethoxysilane  mixed with 1 mL ethanol and 5 µL ultrapure H−1) for 30 min. After rinsing surfaces and cantilevers in ultrapure water, 1 mM coenzyme A  was applied to both for at least 1 h. CoA functionalized surfaces and cantilevers stored in coupling buffer  at 4 °C were stable for >4 weeks.Both glass surfaces and cantilevers were covered with 5 kDa heterobifunctional ɑ-Maleinimidohexanoic-PEG-NHS  dissolved in 50 mM HEPES (pH 7.5) at 25 mM . Pegylation and CoA coupling in individual wells was achieved following identically to the protocol described above25.When different protein constructs were compared with a single cantilever, up to 10 spatially separated spots were created using a silicone mask , cleaned by sonication in isopropanol and ultrapure H2. Cantilevers were typically incubated with 50 μM of protein of interest and 3 μM Sfp phosphopantetheinyl transferase (SFP) for at least 1 h. The glass surfaces were incubated with 5–15 μM of protein construct of interest and 1–2 μM SFP for 30–60 min, depending on the desired surface density. Both samples were rinsed extensively with at least 60 mL measurement buffer  buffer before experiments.These steps yielded cantilevers and surfaces covalently coated in PEG-CoA. Cantilevers and surfaces were rinsed in ultrapure water. Protein functionalization was achieved by covalently pulling down proteins via their ybbr-tag to CoA by the SFP enzyme coupling. The proteins of interest were diluted into TBS150  supplemented with 10 mM MgCl−1 for a dynamic force spectrum, otherwise and for titration experiments a standard velocity of 1.6 µm s−1 was used. After each curve acquired, the glass surface was moved horizontally by at least 100 nm to expose an unused, fresh surface spot. Typically, 50,000 – 100,000 curves were recorded per experiment. When quantitative comparisons of absolute forces were required, a single cantilever was used to move between multiple spatially separated spots to be probed on the same surface (created using the protocol described above). To calibrate cantilevers, the inverse optical cantilever sensitivity (InvOLS) was determined as the linear slope of the most probable value of typically 40 hard (>2000 pN) indentation curves. Cantilevers spring constants were calculated using the equipartition theorem method with typical spring constants between 70–160 pN nm−151. A full list of calibrated spring constants from experiments presented in this work is provided in the supplementary methods, as the stiffness of the pulling handle, i.e., the cantilever, may influence the complex rupture and domain unfolding forces measured.AFM-SMFS data were acquired on a custom-built AFM operated in closed loop by a MFP3D controller  programmed in Igor Pro 6 . Experiments were conducted at room temperature (approximately 25 ˚C). Cantilevers were briefly (<200 ms) and gently (<200 pN) brought in contact with the functionalized surface and then retracted at constant velocities ranging from 0.4, 0.8, 1.6, 3.2 to 6.4 µm s2+ to reliably measure Ca2+ binding . Instead, water  containing ≤10 ppt Ca2+  according to the manufacturer was used. Buffering was achieved by dissolving a Ca2+-free PBS tablet . B domains immobilized on the surface were Ca2+ depleted with at least 10 mM EDTA and after repeated rinsing in Ultra Quality Ca2+-free PBS, the titration was started. For each concentration, thousands of curves were acquired, before the surface was rinsed with the next Ca2+ concentration. As the discerning between strong and week state should not depend on cantilever stiffness, multiple experiment data using identical buffers were pooled to build statistics.Buffer made from ultrapure water , TRIS and NaCl  contained too much Ca54. Laser spot drift on the cantilever relative to the calibration curve was corrected via the baseline noise (determined as the last 5% of datapoints in each curve) for all curves and smoothed with a moving median (windowsize 300 curves). The InvOLS for each curve was corrected relative to the InvOLS value of the calibration curve.Data analysis was carried out in Python 2.7 (Python Software Foundation)Raw data were transformed from photodiode and piezo voltages into physical units with the cantilever calibration values: The piezo sensitivity, the InvOLS  and the cantilever spring constant (k).x) by correcting with their corresponding force datapoint (F) as xcorr = x – F/k.The last rupture peak of every curve was coarsely detected and the subsequent 15 nm of the baseline force signal were averaged and used to determine the curve baseline, which was then set to zero force. The origin of molecule extension was then set as the first and closest point to zero force. A correction for cantilever bending, to convert extension data in the position of the cantilever tip was applied. Bending was determined through the forces measured and was used on all extension datapoints . A quantum mechanical correction was used to account for peptide bond stretching at high forces 59. Especially at forces larger than 1 nN, this correction was essential to be able to fit the data to polymer elasticity models accurately. Peaks were assigned their contour length in diagrams assembled through Kernel Density Estimates (KDE) of the contour length transformed force-extension data. The KDE bandwidth was chosen as 1 nm. The loading rate was fitted as the linear slope of force vs. time of the last 4 nm preceding a peak.To detect unfolding or unbinding peaks, data were denoised with total variation denoising off,0, the probability density p to unfold at a given force F was fit to a normalized force histogram. For a superposition of two BE fits as in Fig. For single BE model at a given force loading rate r  with the parameters Δx and kr1 and r2 were assigned at a force fcritical at the minimum value of ptotal between the maxima of both BE fits and then assigned to BE1 and BE2, as force loading rate and unfolding force correlate in a constant velocity experiment. The relative weight of each distribution was q for BE1 and (1 – q) for BE2 with 0 < q < 1.Force loading rates 60. This value was used to fit the unfolding or rupture force histograms with the BE model for each pulling velocity62. Errors in all diagrams are given as the asymmetric full-width at half maximum (FWHM) of each probability distribution. A final fit was performed through the most probable rupture forces and loading rates for each pulling velocity to determine the distance to the transition state Δx0 and natural off-rate at zero force koff0.For dynamic force, spectra rupture force histograms for the respective peaks and dynamic force spectra were assembled from all curves showing B domain unfolding, or (if applicable) a specific fingerprint domain, and/or a clean complex rupture event. The most probable loading rate of all complex rupture or domain unfolding events was determined with a KDE, bandwidth chosen through the Silverman estimator. Template file for the B domains was PDB 4JDZ (SdrD B1 domain) and for the SdrG N2N3-B1-B2 construct PDB 1R17 (SdrG N2N3) was added.Homology models for the SdrG B1, SdrG B2 domain, and SdrG N2N3-B1-B2 construct were created using Modeller 9.1928 molecular dynamics package with setups created by VMD65 plugin QwikMD27. The CHARMM3666 force field and TIP367 water model were used in all simulations. Structures were centered in a water box 15 Angstrom larger than the protein’s longest dimension, NaCl was added to 150 mM. Minimization (2000 steps), then Annealing , then equilibration , then MD simulation  were performed in the NpT ensemble. Final simulations were run for at least 100 ns for individual B domains and at least 30 ns for SdrG N2N3-B1-B2.Model structures were equilibrated in water using the NAMD68 method. The pressure was maintained at 1 atm using Nosé-Hoover Langevin piston70. The equations of motion were integrated using the reversible reference system propagator algorithm (r-RESPA) multiple time step scheme to update the short-range interactions every 1 steps and long-range electrostatics interactions every 2 steps. The time step of integration was chosen to be 2 fs for all simulations. The temperature was maintained at 300 K using Langevin dynamics.Simulation parameters were: a distance cut-off of 12.0 Å was applied to short-range, non-bonded interactions, and 10.0 Å for the smothering functions. Long-range electrostatic interactions were treated using the particle-mesh EwaldSupplementary InformationPeer Review File


Here we discuss the formation of phyllotactic patterns in the shoot apical meristem (SAM) of plants, where the spatial distribution of the phytohormone auxin determines phyllotaxis in a domain that is growing and changing in time. We assume that the concentration of auxin modifies the mechanical properties of the domain and that the mechanical stress field in the SAM orients the flux of auxin. To study this problem we propose a mechanism for pattern formation in growing domains with variable curvature. The dynamics of chemicals is modeled by a reaction-diffusion system that produces a three dimensional pattern of chemical concentrations that changes the stress field in the domain while growing. The growth process is modeled by a phase-field order parameter which determines the location of the boundaries of the domain. This field is coupled to the chemical concentration through a curvature term that affects the local mechanical stress in the domain. The local stress changes in turn modify the chemical patterns. Our model constitutes a useful and novel approach in theoretical biology, as many developmental processes in organisms seem to be affected by the changes of curvature, size, mechanical stress and other physical aspects. Several patterns seen in many plants are reproduced under certain conditions by our model. Phyllotaxis is the arrangement of repeated plant organs such as leaves, floral structures, ribs in cacti or scales in a pine. This geometrical patterning has fascinated minds for centuries. The scientific importance of studying the form of living organisms was pertinently pointed out by Darcy Thompson . Phyllotmorphogenesis was a term coined by Alan Turing in his seminal article =-ϕf(ϕ) = −ϕ + ϕ3 is homogeneous and bistable, which is appropriate as ϕ must have two stable phases.Note that the function μ[ϕ] associated with the Cahn-Hilliard problem  can be obtained as the functional derivative of the free energy [The chemical potential e energy :μ[ϕ]=δFα = 1, 2, 3, and the subscripts on the gradient operator refer to differentiation with respect to the corresponding coordinate.Here we use spatial (Cartesian) coordinates so ϕ] = 0, which can be solved by adding the boundary conditions at the bulk phase ϕ(x) → ±1 as x → ±∞. In this way one obtains the equilibrium profile of the interface, that turns out to be a smooth tanh-like function with an interface width ϵ. The chemical potential is important because, as we will show, it governs the time evolution of the order parameter.The minimum of the free energy u. This can be done by adding an extra term in the free energy density so the new free energy functional will beWe now add a spontaneous curvature term in order to incorporate the influence of the chemical C0 is related to the spontaneous curvature of ϕ, and the term (1 − ϕ2) acts as a δ function centered at the interface, so it confines this effect to the surface ϕ = 0 and has no effect in the bulk phases. The interface is thus forced to accommodate its surface to the spontaneous curvature C0. It is important to mention that this phase-field model can be obtained from the formulations of the Canham-Helfrich problem  = mu2 is a quadratic functional that adds mass to ϕ, and m is the amount of added mass per unit time. The introduction of α allows for a plausible modelling of growth and not only deformation of a volume under stress.Now, since growth and deformation occur, and since cell proliferation and elongation take place during phyllotaxis, the amount of material is necessarily variable. We then need to introduce an additional term which adds mass to the domain. As the auxin promotes the tissue growth in plants, we add a term to the dynamic equation of ϕ is initialized as an axisymmetric dome and we introduce such a Gaussian distribution G[u], with a variable height that changes according to the displacement of the summit of the dome. This distribution is included in the dynamic Eqs  will provide almost stationary distributions of the chemical while the surface is changing (see the next Section for details).u is thus allowed to interact with another morphogen v and the two morphogens diffuse with different rates. For the non-linear kinetics, we adopt the so-called BVAM model  Eqs  and 13, de novo and contain almost a ten times more auxin concentration than any other tissue  of width R/2. The first whorl emerges at the basis of the dome, which is the region where the curvature of the domain changes more abruptly , located at the center of the dome near the tip, contains the stem cells which provide new cells to keep the plant growing. Daughter cells are displaced into the peripheral zone (PZ) at the flanks of the dome, where the new primordia emerge  (width of R/2), κ = 2, c = 0.57, η = 0.2014. The remaining parameters are indicated in the main text.Parameter values are (MP4)Click here for additional data file.S7 Videoβ = 0.5, γ = 0.2, G[u] (width of R/2), κ = 2, c = 0.33, η = 0.6504 and the rest of parameters as indicated in the main text.Parameter values are (MP4)Click here for additional data file.S8 Videoβ = 0.5, γ = 0.2, G[u] (width of R/2), κ = 2, c = 0.33, η = 0.8919 and the rest of parameters as indicated in the main text.Parameter values are (MP4)Click here for additional data file.S9 Videou concentration.In the top we plotted front and top views of the principal directions of the stress tensor (black arrows) in the volume. In the bottom, the principal directions of stress (black arrows) are plotted only in the surface. The domain was plotted in diffused color to identify the regions that are growing. The color scale indicates (MP4)Click here for additional data file.


Protein phosphorylation is the best characterized post-translational modification that regulates almost all cellular processes through diverse mechanisms such as changing protein conformations, interactions, and localization. While the inventory for phosphorylation sites across different species has rapidly expanded, their functional role remains poorly investigated. Here, we combine 537,321 phosphosites from 40 eukaryotic species to identify highly conserved phosphorylation hotspot regions within domain families. Mapping these regions onto structural data reveals that they are often found at interfaces, near catalytic residues and tend to harbor functionally important phosphosites. Notably, functional studies of a phospho-deficient mutant in the C-terminal hotspot region within the ribosomal S11 domain in the yeast ribosomal protein uS11 shows impaired growth and defective cytoplasmic 20S pre-rRNA processing at 16 °C and 20 °C. Altogether, our study identifies phosphorylation hotspots for 162 protein domains suggestive of an ancient role for the control of diverse eukaryotic domain families. Protein phosphorylation has various regulatory functions. Here, the authors map 241 phosphorylation hotspot regions across 40 eukaryotic species, showing that they are enriched at interfaces and near catalytic residues, and enable the discovery of functionally important phospho-sites. Mass spectrometry and biochemical enrichment methods allow for the study of PTM regulation at very large scale2 and such approaches have been extensively applied to study protein phosphorylation3. On the most comprehensive single study to date on the order of 75% of the detected proteome was found to be phosphorylated4 and approximately 160,000 non-redundant human phosphosites are listed in public repositories5. Although the regulation of protein functions by phosphorylation has been under study for over 60 years6 the recent wealth of knowledge regarding protein phosphorylation generated by mass-spectrometry remains mostly uncharacterized. Only around 5% of human phosphosites have an annotated regulatory role5. Evolutionary studies have suggested that some degree of protein phosphorylation, and other PTM sites, may have little to no biological function8. Given that some phosphosites may have no relevance to fitness, devising ways to rank sites according to functional importance is a crucial research question. Functionally important phosphosites have been shown to be more likely conserved across species and across protein domains of the same type9. For protein domains, conserved phosphorylation within a specific region—termed phosphorylation hotspot—tends to identify regions with regulatory potential8. Since each domain family is represented by multiple copies within each species, these domain-centric analyses have the advantage of increased statistical power when compared to the study of conservation of orthologous genes. Domain-centric analyses have also been used with success to study recurrence of mutations in cancer samples10.Protein post-translational regulation is a reversible and highly effective way to regulate protein functions within seconds to minutes time scales. Over 300 different types of protein post-translational modifications (PTMs) are known, ranging from single atom modifications (oxide) to small protein modifiers (ubiquitin)8. To perform a systematic analysis we compile here an expanded set of over 500,000 phosphosites across 40 eukaryotic species. These phosphosites are mapped to protein domain regions allowing us to identify phosphorylation hotspots within 162 domain families. We show that the identified regions are functionally relevant as they are enriched in previously known regulatory phosphosites, interaction residues, and positions that are close to catalytic residues. We identify a putative regulatory region in the ribosomal S11 domain and generate a phosphorylation-deficient mutant in two Saccharomyces cerevisiae phosphosites found within this region of the yeast ribosomal protein Rps14A. We show that the Rps14a-T119A mutant exhibits impaired growth at 16 and 20 °C, and is defective in cytoplasmic 20S pre-rRNA processing, uncovering a critical role for phosphorylation of this region during eukaryotic ribosome assembly.We have previously studied domain phosphorylation hotspots across 10 example domain types11 models across all species (Methods) and phosphosites were matched to these regions. Of all phosphosites, 83,359 phosphosites occur within Pfam domain regions  was used globally to identify domain regulatory hotspots. A cut-off of an average of 2 phosphosites per position was also used to avoid significant positions with a low effect size difference. Contiguous positions were merged to identify domain regions of interest that were defined as phosphorylation hotspot regions (Methods). Using this procedure a total of 1999 positions corresponding to 241 domain regions were identified as hotspots within 162 Pfam domain families  sites from tyrosines (Y). We were able to analyze a set of 983 S/T and 317 Y phosphosites with known regulatory functions in human, as defined in PhosphoSitePlus, out of a total of 8270 S/T and 1395 Y human phosphorylated positions within the same domains.Under the assumption that strong conservation of protein phosphorylation within a region of a domain is predictive of functional relevance we would expect to find an enrichment of phosphosites with known functions at predicted phosphorylation hotspot regions. We therefore tested if the hotspot predictions could discriminate between human phosphosites with known function from other human phosphosites with unknown function. For each human phosphosite within the protein domains analyzed we assigned the hotspot p-value was a strong predictor of know regulatory phosphosites (AUC = 0.78 for S/T and 0.62 for Y). In line with this, the defined hotspot regions show significant enrichment over random for human phosphosites of known function  curve Fig.  and summion Fig. . Overall13. For each domain family we discarded structures with short sequences, performed structural clustering, and selected a representative from the most populated structural cluster (Methods). The protein sequences from the selected structural models were added to the alignments and a total of 116 hotspots regions were mapped to a 3D model for 85 domains. We provide the structural representation of these hotspot regions and enrichment plots in Supplementary Data The phosphorylation hotspots are of functional and structural interest as these are very likely to have regulatory potential that should often be a general property of the domain family. To study these regions in the context of protein structures we have collected structures available for Pfam domains in PDB14. Another well characterized example is the regulation of the pyruvate dehydrogenase complex (PDC) which is primarily composed of multiple copies of pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipamide dehydrogenase (E3). PDC activity is regulated by phosphorylation of E1 in positions that overlap with our identified phosphorylation hotspot  region, which overlaps directly with the hotspot region. Phosphorylation of this loop region is known to induce a conformational change in the loop that causes enzyme inhibition17. We expect that the identified hotspots from other domain families will be of regulatory importance in analogy to the activation loop of kinases and the phosphorylation loops of pyruvate dehydrogenase.The protein kinase activation loop is the prototypical example of a regulatory hotspot Fig. , top. Ovsferase E, and dih18 implicating the phosphorylation of this region in the regulation of Ras-effector interactions. Supporting this hypothesis, the phosphorylation of human KRas in this region at Y64 regulates the interaction between KRas and AGO219. Similarly, phosphorylation of the same region in Rab7 (S72) and in RAC1 (Y64) is associated with changes in interaction partners21. We suggest that this is a commonly used regulatory feature of Ras domains.A clear phosphorylation hotspot was found for the Ras domain family Fig. . This sm22. The orientation of VDAC domains in the membrane is contentious with conflicting reports suggesting that the C-terminal may point towards the cytoplasm23, the mitochondrial inter-membrane space (IMS) or that is may occur in both orientations24. The phosphorylation hotspot between β6 and β7 is on the opposite side of C-terminal region, suggesting that this loop most likely or most often will face the cytoplasm placing the C-terminal towards the IMS.A different mode of phospho-regulation has been observed in the voltage-dependent anion channel (VDAC). This 19 beta sheet beta barrel domain is a class of porin ion channel spanning the outer mitochondrial membrane. The major phosphorylation hotspot in the VDAC domain is predicted for a loop region between β6 and β7 Fig. . The regThese examples further illustrate how our analysis recovers well known regulatory regions as well as shows some ways in which domain function is regulated by protein phosphorylation . We next tested if some of the regulatory mechanisms found in these examples can be generalizable to other domain families.25 and for catalytic residues based on UniProt annotations26 (Methods). In addition, we also analyzed surface accessibility, defined as >20 relative surface accessibility (RSA), and predicted disorder (from DISOPRED27 (Methods)). As expected from prior studies of protein phosphorylation28 the hotspot positions are more likely to be surface exposed  and within disordered elements  when compared to other residues. We next measured distances between hotspot positions and catalytic residues. Compared to other residues within enzymes, hotspot positions are 3 times more likely to be catalytic residues ; 3.4 times more likely to be within 5 amino-acid distance  and 5 times more likely to be within 5 Å distance  to catalytic residues. For enzyme domains 3.3% of hotspot residues are within 5 Å distance of catalytic residues compared with 0.97% for other residues.Regulation of interactions and catalytic activities may be general mechanisms of domain regulation. To study this across all domains we used annotations for interface residues from the 3DID databasep-value < 2.4 × 10−9). 39% of accessible hotspot residues are interface positions compared with 26% for other accessible residues. Some hotspot regions overlap with interaction regions that can make contacts with a large number of different types of domains as determined by crystal structures. For example, the hotspot region in Ras described above contacts 42 other domain types, some of which are illustrated in Fig. For each domain position we identified interface contacts found in 3D structures with any other protein domain based on 3DID, excluding intra-domain contacts. For the interface residue enrichment test, we considered only surface accessible residues (>20% RSA) to avoid an enrichment simply due to accessibility of both interface and hotspot positions. Controlling for surface accessibility hotspots are 1.8 more likely to be interface positions  catalyze the exchange of terminal phosphate between different nucleoside diphosphates (NDP) and nucleoside triphosphates (NTP). A NTP serves as a donor and the reaction proceeds via a phospho-histidine intermediate in the NDK active site. The main hotspot in this domain occurs just next to this active site histidine Fig. . The pho32 but the position or functional role of IMPDH phosphorylation has not been established. In structures of this domain a serine residue can be found in this hotspot . The IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+. In human cells Akt has been shown to interact with IMPDH and phosphorylate the protein in vitropot Fig.  pointing34 but the position or mechanism of this regulation has not been determined. The hotspot identified for TAL is very likely to alter the accessibility of the substrate to the active site. In structures of this domain a serine residue within this hotspot can be found just at the entrance to the substrate pocket  is in a position that could influence the access to the catalytic centre. TAL is an enzyme of the nonoxidative part of the pentose phosphate pathway (PPP). The active site, located in the center of the barrel is formed of a lysine that holds the sugar in place and a glutamate and aspartate that act as proton donors and acceptors. There is evidence that TAL activity can be regulated by phosphorylationket Fig.  and phos35). A phosphorylation hotspot of unknown function was identified in the C-terminal tail of this protein family  we observed re-current detection of phosphorylation of the catalytic residue or neighboring amino-acids. These cases may often represent phosphorylation that is not catalyzed by protein kinases but instead an intermediate enzymatic step or autophosphorylation. This suggests also that mass spectrometry based approaches can be used to track such enzyme reactions via their intermediate phosphorylation states.40. The C-terminal region of uS11 triggers activation of Fap7-ATPase, whose essential function is to pre-fabricate an uS11:eS26 complex, and co-deposit the ribosomal proteins onto the assembling pre-ribosome in the nucleolus38. The ribosomal protein eS26 clamps the 3′ end of rRNA at the site where the endonuclease Nob1 cleaves the 20S pre-rRNA into a mature 18S rRNA41. eS26-depletion impairs 20S pre-rRNA processing in the cytoplasm41 suggesting that the clamping of the 3′ end of rRNA is critical for the endonucleolytic cleavage. Thus, failure to deposit eS26:uS11 on the pre-ribosome does not affect nuclear export of these particles to the cytoplasm. Moreover, these aberrant 40S pre-ribosomes engage with mature 60S subunits to form 80S-like particles, but are unable to process 20S pre-rRNA and therefore accumulate in the cytoplasm40. Thus, it is tempting to speculate that the underlying 20S pre-RNA processing impairment could arise due to the inability of the T119A mutant to efficiently activate Fap7-ATPase, a critical event to deposit uS11:eS26 onto the assembling pre-ribosome. Notably, even though Rps14a-S123A phosphomutant shows slow growth phenotype at 39 °C and is sensitive to CHX, we did not observe any impairment in nuclear export of 40S pre-ribosomes or cytoplasmic 20S pre-RNA processing. However, given the CHX sensitivity, it could be that this region within uS11 plays a critical role during translation.With our analysis we were able to identify in total 241 domain phosphorylation hotspots. In this study, we analyzed phenotypes associated with two point mutants within the C-terminal tail of uS11 (yeast Rps14). We found that the T119A mutant exhibits reduced growth rate at 20 and 16 °C and accumulates 20S pre-rRNA in the cytoplasm. This conserved residue is located at the base of uS11 C-terminal tail. Mutants in the C-terminal tail have been previously shown to also accumulate 20S in the cytoplasm43 should make these studies more feasible. Such studies can in-turn spur the rational design of novel phosphorylation switches.The identification of phosphorylation hotspots across a diverse set of domains suggests a widespread ancient role for control of protein domains by phosphorylation in eukaryotic species. It remains to be studied how these regulatory regions arise during evolution. These hotspot regions and annotations are provided in Supplementary Data Homo sapiens, Mus musculus, and Rattus norvegicus were obtained from the PhosphoSitePlus database5. Phosphorylation data for 6 Drosophila species  was obtained from the iProteinDB in FlyDB database44. Two additional metazoan phosphoproteomes were obtained from published studies for Caenorhabditis elegans45 and Trichoplax adhaerens46. Phosphosites for 18 fungal species  pastoris, Meyerozyma guilliermondii, Candida albicans, Scheffersomyces stipitis, Schizosaccharomyces pombe) were obtained from Studer and colleagues9. An additional fungal phosphoproteome was added for Magnaporthe oryzae47. Plant phosphosites for 4 species  was retrieved from the P3DB database48 and additional plant species were retrieve from selected publications for Brachypodium distachyon49, Chlamydomonas reinhardtii50, and Selaginella moellendorffii51. Information for 3 Apicomplexa species  were compiled from phosphoproteomic studies for these species53. For all species we removed potential redundancies to avoid assigning the same phosphosites to multiple sequences which could cause false enrichments in proteins rich in isoforms. For H. sapiens, M. musculus, and R. norvegicus data was retrieved from PhosphoSitePlus, we removed isoform redundancy by using only the canonical sets of proteins as defined by UniProt. For other species we filtered out redundant peptides by removing identical 11 amino-acid peptides centered on reported phosphosite positions. The total list of phosphosites compiled for this study is shown in Fig. Phosphorylated residues 54. In order to identify alignment regions containing more phosphosites than expected by chance we used a permutation strategy to generate a null background. We first count the observed phosphosites for a given region of a Pfam domain using a rolling window with a fixed size of 5 positions. We chose to use a window instead of individual positions due to the uncertainty in phosphosite localization within the phosphopeptides; evolutionary drift whereby the phosphorylation of nearby residues could have the same outcome; potential uncertainty in the alignment. To generate a null background model we randomly select phospho-acceptor residues  within the alignment respecting the total number of acceptor residues for the 3 amino-acid types. Permutations were repeated 100 times and for each position in the alignment and an expected median and standard deviation of phosphorylation were calculated. The observed values were converted to z-scores using the permutation information and then to p-values using the survival function of the normal distribution from scipy (scipy.stats.norm.sf)55. Only enrichment over random was considered, not depletion and the Bonferroni correction was used to account for multiple testing globally. In addition, to avoid the identification of positions with a low effect size a cut-off of an average of 2 phosphosites per position was used. Finally, contiguous positions were merged to identify domain regions of interest with added ±2 positions on either side that were defined as phosphorylation hotspot regions.For all of the analyzed protein sequences we used PfamScan to predict Pfam domains. The PfamScan option for predicting catalytic residues was used to retrieve annotations on these types of residues. For each Pfam domain, all corresponding sequences having at least 1 phosphorylation site mapped to them were selected and aligned using MAFFT http://www.sbg.bio.ic.ac.uk/~maxcluster) and for each domain one structure was selected from the cluster representing the most common conformation. In order to map the hotspot information to structures, the sequence of the representative structure of each domain was aligned to the corresponding Pfam domain sequences using MAFFT. In some instances the structural model did not cover a predicted hotspot region.For each Pfam domain having a significant hotspot region we obtained 3D structures available in the PDB. We then identified and selected the corresponding Pfam domain regions within each structure excluding the remainder. The structures were filtered to exclude those with gaps larger than 1 amino-acid and those shorter than 70% of the longest structural model for a given domain family. Each set of structures representing a domain were structurally clustered with MaxCluster with single linkage . For interface contact information we used data from 3DID56. For a given Pfam domain in 3DID we calculated, for each residue, the number of times this residue is found in contact of another protein chain (intra-chain contacts were ignored). We considered a position within a Pfam domain family to be at an interface if it was found forming contacts with other proteins in 10 or more structures. This cut-off was used to obtain a high confidence list of Pfam domain interface positions but similar results were observed considering more lenient definitions of interface positions. A set of human phosphorylation sites known to have regulatory roles were obtained from PhosphoSitePlus57.For the selected structural models we obtained water accessibility using naccess, disordered prediction with DISOPREDMATα, his3Δ1; leu2Δ0; ura3Δ0; MET15+; LYS2+; can1Δ::STE2pr- SpHIS5; lyp1Δ::STE3pr-LEU2, from Krogan Lab - UCSF). SceI endonuclease  was integrated at the mutated leu2Δ0 locus . The point mutations T119 or S123 were introduced into the RPS14A endogenous locus and the URA3 marker after the stop codon. The URA3 marker was flanked by SceI recognition sites to enable its removal by the Galactose inducible SceI endonuclease58. Point mutations were verified by sequencing.Phospho-deficient mutants Rps14a T119A and S123A were constructed using the Y8205 background strain  96 format manual pinning tool. The agar plates were incubated for 48 and 72 h and imaged.Yeast strains were grown on agar plates and individual colonies of each strain were picked and arrayed in 96-well plates containing the synthetic SC medium and incubated overnight. The strains were then serially diluted four times at one in 20 dilutions in 96-well plates filled with 160 µl sterile ddH59. DNA was stained with 0.5 μg/ml DAPI. Early biogenesis defect of small ribosomal subunit was determined by localization of uS5-GFP expressed from centromeric plasmid pRS316-RPS2-GFP60. Cells were visualized using a DMI6000 microscope  equipped with a HCX PL Fluotar 63×/1.25 NA oil immersion objective . Images were acquired with a fitted digital camera  and Openlab software .Cells were grown to mid-logarithmic phase and fixed with 4% formaldehyde in 0.1 M potassium phosphate buffer. Cells were then converted to spheroplasts using 0.1 M potassium phosphate buffer containing 1.2 M sorbitol and 500 μg of zymolase. Spheroplasts were washed in 2× SSC buffer and incubated overnight at 37 °C with Cy3-labeled oligonucleotide probe (5′-Cy3-ATG CTC TTG CCA AAA CAA AAA AAT CCA TTT TCA AAA TTA TTA AAT TTC TT-3′) that is complementary to the 5′ portion of ITS1RNA was extracted with Trizol reagent following a standard protocol. Three micrograms of total RNA were separated on a 1.2% agarose/formaldehyde gel for 1.5 h at 200 V. For northern blot analysis, rRNA were blotted onto a Hybond-XL  membrane by capillary transfer and probed for 20S (5′-GGTTTTAATTGTCCTATAACAAAAGC) using radioactively labeled probes (synthesized by Microsynth). rRNA was detected using phosphoimaging screens .Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting SummarySource Data