SH3-fold-β-barrel domains of the chromo-like superfamily recognize epigenetic marks in eukaryotic proteins. Their provenance has been placed either in archaea, based on apparent structural similarity to chromatin-compacting Sul7d and Cren7 proteins, or in bacteria based on the presence of sequence homologs. Using sequence and structural evidence we establish that the archaeal Cren7/Sul7 proteins emerged from a zinc ribbon (ZnR) ancestor. Further, we show that the ancestral eukaryotic chromo-like domains evolved from bacterial versions, likely acquired from early endosymbioses, which already possessed an aromatic cage for recognition of modified amino-groups. These bacterial versions are part of a radiation of secreted SH3-fold domains, which spawned both chromo-like domains and classical SH3 domains in the context of peptide-recognition in the peptidoglycan or the extracellular matrix. This establishes that Cren7/Sul7 converged to a “SH3”-like state from a ZnR precursor via the loss of metal-chelation and acquisition of stronger hydrophobic interactions; it is unlikely to have participated in the evolution of the chromo-like domains. We show that archaea possess several Cren7/Sul7-related proteins with intact Zn-chelating ligands, which we predict to play previously unstudied roles in chromosome segregation during cell-division comparable to the PRC barrel and CdvA domain proteins. In the absence of statistically-significant sequence similarity, the detection of structural equivalences can be used to assess evolutionary relatedness5. However, the evidence can, in some instances, be equivocal regarding structural convergence versus divergence: the moot point in these cases is whether the structural similarity in folds in question is a signal of a divergent origin from a common ancestor or independent convergence to a common scaffold9. Automated sequence- and structure-similarity search tools, though widely used for gauging relatedness among proteins, are often of limited utility in these cases. Tracing the correct relationships demands careful case-by-case analysis10 and on multiple occasions, has helped untangle convergence from extreme divergence, which had otherwise eluded automated similarity search tools17. In this work, we present such a case regarding the SH3 fold and certain zinc ribbons (ZnRs), with bearing on the function and evolution of key domains involved in chromatin structure and chromosome segregation in archaea, recognition of epigenetic marks in eukaryotes, and bacterial cell-wall dynamics.Three-dimensional structures or folds of proteins are evolutionarily less prone to change than their sequencesSrc homology 3 (SH3) is a small β-barrel domain, comprised of five or six β-strands that are tightly packed into two orthogonal β-sheets18. The eponymous SH3 domains are involved in eukaryotic signaling pathways where they mediate protein-protein interactions by binding proline-rich peptide sequences via a conserved cluster of aromatic residues20. The discovery of bacterial homologs of the SH3 domain presented an interesting contrast as they were primarily found as extracellular domains in periplasmic or cell-wall associated proteins23. Members of the larger SH3-like β-barrel fold (hereinafter SH3 fold) include a vast collection of superfamilies found in diverse biological functional contexts. The best characterized of them are implicated in a variety of key protein-protein interactions via recognition of short peptide motifs24. SH3-fold β-barrels also mediate interactions with nucleic acids25. For example, PAZ (Piwi Argonaut and Zwille), a SH3-fold β-barrel domain, found in the Piwi and the Dicer proteins in the RNAi system interacts with RNA28. Likewise, certain representatives of other families with the SH3 fold such as the CarD30, chromo31, and TUDOR domains33 have been shown to bind DNA.The chromatin organization modifier) domains, BAM/BAH, BMB/PWWP and Tudor-like domains36. The Tudor-like domains further include within them the Tudor, MBT , Agenet, DUF3590, DUF1325, RAD53BP, Tudor-knot, AuxRF(PF06507) and the MORC C-terminal domains38 (PFAM clan CL0049). The conserved structural core of the chromo-like domains features a SH3-fold β-barrel with 5 strands that is often capped by a C-terminal helix35. They share a broadly conserved mode of interaction with peptides, specifically recognizing covalent modifications of positively charged side-chains via cation-π interactions with conserved aromatic residues39. While most members bind peptides with methylated lysines, representatives of the Tudor-like domains specialize in binding peptides with methylated arginines.In recent years, it has become clear that in eukaryotes a large superfamily of domains with the SH3 fold, the chromo-like superfamily, plays a key role in recognition of short peptide-motifs, especially those with covalently modified side-chains in chromatin (chiefly histones) and RNA-processing proteins. This superfamily includes the classical chromo 49, and the ubiquitin-binding ZnRs of deubiquitinating peptidases of the UBCH family (PDB: 2GFO).ZnRs are small domains that lack an extensive hydrophobic core beyond the presence of two β-hairpins, being primarily stabilized by chelation of a metal ionnRs Fig. , the C-t51. Despite these structural differences the two classes of ZnRs are likely to be related because they possess a common four-stranded core, conserved geometry of the Zn-chelating residues and can be structurally superimposed.In type-2 ZnRs the N-terminal β-hairpin is extended into a β-strand that forms hydrogen bonds with the C-terminal β-hairpin resulting in a sheet formed by a three-stranded β-meander Fig. . Type-2 . For example, a DALI52 search initiated with the ZnRs from the RNA polymerase subunit RBP9 (PDB: 1QYP_A), in addition to retrieving various ZnRs , recovered several distinct SH3-like β-barrel domains albeit with lower Z-scores such as the classic SH3 domains , the Tudor domain  and the chromo domain . Several SH3-like β-barrel folds were also retrieved in structural searches initiated with the ZnRs of peptide:N-glycanase (PDB: 1X3W_A), ORF131 of Pyrobaculum spherical virus (PDB: 2X5C_A), anaerobic ribonucleotide-triphosphate reductase (PDB: 1HK8_A) and lysyl-tRNA synthetase (PDB: 1IRX_A). Further, visual inspection confirmed this relationship, indicating a topological similarity in arrangement of the β-strands in type-2 ZnRs and the β-barrel of certain domains with the SH3 fold .Interestingly, while developing this classification of ZnRs, we noticed a consistent structural similarity between type-2 ZnRs and SH3 domains Fig. . For exaold Fig. .45. The interacting residues often emanate from strands β3 and β4 and frequently have aromatic or charged side chains   Fig. 41,57,58 41, DALI searches initiated with Cren7 protein (PDB: 3KXT_A), recovered multiple SH3-like fold domains such as chromo domains, Tudor domains and myosin S1 fragment SH3 domains41. These searches also recovered the Sul7 protein42 (PDB: 1WVL_A), which is believed to be structurally and functionally related to Cren7 as one of the hits . Concurrently, the search also retrieved hits to ZnRs, such as those in the sarcosine oxidase delta subunit , PNGase , ZPR1 , peptide:N-glycanase  and 50 S ribosomal protein L44E . Manual structural superimposition of Cren7 (PDB: 3KXT_A) and ZnRs  perfectly aligned all secondary structure elements of the Cren7 β-barrel onto the core of the ZnR fold (RMSD = 1.5 Å over 35 pairs of backbone Cα atoms), where the turns of β1/β2 and of β4/β5 of Cren7 recapitulate the position of knuckles in ZnRs. Similar results were obtained by automated pairwise structural alignment of Cren7 (PDB: 3KXT_A) and ZnRs  using TM-align59 and Fr-TM-align60 which gave a TM score of 0.52 , indicating a ‘fold level’ similarity between the two.The above observations hinted that Cren7 with structural features resembling both the SH3 fold and ZnR domains might help better understand the connections between the two folds. Consistent with earlier findingsS. solfataricus Cren7 (PDB: 3KXT_A) using the PSI-BLAST61 and the JACKHMMER62 programs. Interestingly, these searches recovered multiple orthologous sequences from crenarchaeota and bathyarchaeota that contained two to four cysteine residues at positions corresponding to turns between strands β1/β2 and β4/β5   typified by an extended C-terminal region, most of whose members possess intact Zn-chelating cysteines  found matches to many ZnR proteins such as  the lysine biosynthesis protein LysX/ArgZ , ZitP pilus assembly/motility regulator  and transcriptional regulator MqsA . Further, even among the Cren7Znr proteins some representatives show loss of one of the Zn-chelating cysteines exactly paralleling the situation observed among the classic Cren7 proteins  Fig. . The posnes Fig. . Profileins Fig. .Figure 2S. solfataricus Cren7. Although the Sul7 proteins were not retrieved in these sequence searches, they show several features that suggests their derivation from Cren7 proteins. (1) They are so far only found in a limited number of crenarchaeal lineages  unlike Cren7, which is conserved across crenarchaeota and the bathyarchaeota64. (2) They share a common DNA-compaction function in crenarchaeal chromatin58, which is mediated via a similar protein-DNA interface, with positionally- and chemically- equivalent residues involved in DNA contact. (3) Experiments to make the Cren7 protein more “Sul7d-like” by mutating the loop between strands β3/β4 results in an exact superposition of the DNA-binding interface58.Thus, multiple lines of evidence support Cren7-like proteins (PDB: 3KXT_A) being ZnRs, with secondary loss of Zn-chelating residues in some versions such as the 46. Consistent with this, we have found versions of Cren7, which still retain the ancestral Zn-chelating residues. The strengthening of the hydrophobic core due to interactions in the strand regions appear to have facilitated the loss of Zn-chelation in more than one version of the family. This appears to have concomitantly supported the emergence of a more β-barrel-like geometry that converged to a SH3-like state. This was followed by the emergence of Sul7 only in Sulfolobaceae as a specialized DNA-packaging protein from a Cren7-like protein that had already lost its Zn-chelating residues.Presence of Cren7/Sul7 proteins only in crenarchaeota and bathyarchaeota, together with their provenance from ZnRs, which we establish above supports the following evolutionary scenario: the ancestral Cren7 proteins likely arose from a DNA-binding ZnR of which several are found in the pan-archaeo-eukaryotic transcription apparatus65. In bathyarchaea, we found a fusion of the Cren7 domain to an N-terminal FtsZ domain (KYH38952.1), a key component of the cell-division apparatus related to the tubulin-like cytoskeletal proteins. These architectures suggest that representatives of the Cren7 family play a specific role in cell-division, probably in anchoring the DNA. All Cren7Znr proteins contain a conserved C-terminal motif with an absolutely conserved acidic residue beyond the core Cren7 domain, which is likely to adopt an extended conformation. It is possible that this region also helps in specific interactions with cell-division components. We also found an instance of bathyarchaeal Cren7 (KPV62179.1) fused to the URI domain (GIY-YIG endonuclease) and an uncharacterized enzymatic domain of the OrfY-like superfamily66, and a nanoarchaeal Cren7 (AMD29662.1) with a C-terminal REase fold nuclease domain  where it is combined with an N-terminal CdvA-like coiled coil domain and a central small 3-stranded domain. In crenarchaea, the CdvA-like coiled-coil domain proteins are components of the cell-division system and form filamentous double-helical complexes with DNAn KYH3895.1, a key38. To better understand the origins of the bacterial chromo-like domains, we ran iterative profile searches from the versions we had previously detected in bacteria38. Our current searches, greatly extended the phyletic spread of bacterial chromo-like domains, retrieving them from several different lineages such as proteobacteria (mainly α and δ), cyanobacteria, Thermus/Deinococcus, bacteroidetes, planctomycetes, and spirochaetes and in rare cases in euryarchaea. Firmicutes and actinobacteria showed a strong under-representation of this domain . This suggests that the bacterial versions are closer to the Tudor-like chromo domains and that the Tudor-like versions are likely to be closer to the ancestral mode of binding peptides. The plausible evolutionary relationship shared by the various chromo-like domains is depicted in Fig. The above rooting of the provenance of Cren7/Sul7-like proteins within the archaeal radiation of ZnRs and evidence for convergent acquisition of the SH3-like barrel morphology questions the evolutionary relationship of these archaeal chromosomal proteins and the chromo-like superfamily of SH3 fold, given the previous identification of bacterial versions of the latterins Fig. , we posiig. Fig. . This su23, again in contrast to their strictly intracellular location of the eukaryotic counterparts19. These bacterial SH3 domain proteins are distributed across a much wider phyletic range of taxa compared to the chromo-like domains. Recent studies have shown that secreted bacterial SH3 domains are likely involved in binding peptides in the peptidoglycan cell wall23. These parallels suggest that the bacterial chromo-like domains might function similarly to the bacterial SH3 in binding-specific peptides. However, they are likely to possess specificity for those containing methylated lysine-like moieties, in the murein or extracellular matrix peptides in bacteria with Gram-negative cell walls (given their near absence in firmicutes and actinobacteria).We observed that the bacterial chromo-like domains, in contrast to their eukaryotic counterparts, are consistently present in secreted proteins across the diverse lineages containing them. Some proteins with bacterial chromo-like domains also display cysteines likely to form disulphide bonds typical of extracellular proteins. Further, systematic analysis of their domain architectures revealed contexts unlike any seen in eukaryotes Fig. : in addiAnabaena . Here the chromo domain is fused C-terminal to the polyprotein containing pepsin, reverse transcriptase, RNase H, integrase and SH3 domains. Given the association of certain fungi with cyanobacteria  this probably represents a transfer facilitated by such an association. (2) The other case is found in a single species, where a eukaryotic chromodomain is inserted into a mobile element of a bacterium with a ParB and HNH domains   a Ty-3 family retrotransposon, which is commonly found in fungi is also found in multiple copies in 71. Such developments are seen in the form of the type-1B ZnRs, which developed an additional β-strand and finally the type-2 ZnRs, which appear to be related to the type-1B versions via a circular permutation. Here, the additional β-strand appears to have been further incorporated into the core of the fold to form a contiguous β-sheet . More structured ZnRs with β-hairpins developing around these Zn-knuckles at their turns Fig.  likely eeet Fig. . Indeed,51. We also observed that the type-2 ZnR at C-terminus of the nicotinate phosphoribosyltransferase converged to a SH3-like fold with the Zn-chelating sites lost alongside the evolution of compensatory hydrophobic interactions , with the SH3 fold emerging via loss of Zn-chelation from a ZnR via acquisition of stronger hydrophobic interactions.In this study, we capture the evolutionary stages in one such transformation: the Cren7 domain, previously considered a SH3 fold β-barrel, actually emerged from a metal-binding ZnR domain. We find evidence that such convergence between ZnRs and SH3 fold domains might have happened independently more than once. For example, the segment-swapped Ku-bridge domain and its homolog: the C-terminal all-β domain of the MarR-like transcription factors resemble segment-swapped SH3-like folds ; however, sequence analysis clearly indicated the provenance of these domains from type-2 ZnRs82. However, in crenarchaeaota and at least certain bathyarchaeota, the ancestral histones appear to have been displaced by a heterogeneity of chromatin-compacting proteins, namely Cren7, Sul7d and CC183. Based on structural considerations it was earlier suggested that these archaeal chromosomal proteins might have an evolutionary a relationship to the SH3-fold domains of the chromo-like superfamily, which are a hallmark of eukaryotic chromatin proteins40. Here, we present a clear evolutionary scenario for the convergent origin of a SH3-like morphology for Cren7/Sul7d-like proteins from ZnRs. We also present evidence for a functional diversification of these ZnRs in crenarchaeaota and bathyarchaeota with potential roles in cell-division comparable to the CdvA-like proteins with the PRC-barrel domain84.Our findings help clarify the origins and new functions of key domains in chromosomal proteins. Compaction of genomic DNA into the limited intracellular space is a universal challenge for which multiple solutions have been selected across cellular life. In asgardarchaea (believed by many to be the closest sister group of eukaryotes), eukaryotes and euryarchaea this function is carried out by the α-helical histone fold proteins and in bacteria by the HU/IHF superfamily of proteinsOn the other hand, we present evidence that eukaryotic chromo-like domains have no close relationship to Cren7-like archaeal chromosomal proteins. While there are several SH3 fold β-barrels, eukaryotic chromo-like domains specifically share a peptide-binding function with the classical SH3 domains. We show that both these superfamilies are present in bacterial extracellular proteins and based on the evidence from the bacterial SH3 domains, we posit that the bacterial chromo-like domains too bind peptides in the peptidoglycan or in the periplasm. Further, sequence analysis suggests the bacterial chromo-like domains likely acquired specificity for methylated side-chains of basic amino acids even in these bacterial versions. Further, they might have even interacted with nucleic acids founds in bacterial extracellular matrices. These observations suggest that the two SH3 fold superfamilies initially diverged and radiated primarily in the context of binding different peptides in the bacterial peptidoglycan. Notably, both these show parallel domain architectures Fig.  and are 38. Thus, the chromo-like superfamily was present in the stem eukaryote diversified prior to the common ancestor of all extant eukaryotes. This suggests that the eukaryotes possibly acquired their chromo-like and classical SH3 domains from the α-proteobacterial mitochondrial progenitor. Their presence in the extracellular matrix might have facilitated interactions with “cytoplasmic” proteins and nucleic acids of archaeal component of the ancestral eukaryote upon endosymbiosis. Hence, we posit that this was the likely scenario that favoured their recruitment as intracellular peptide-binding domains in the ancestral eukaryote. In eukaryotes, the chromo-like and classical SH3 domains radiated extensively due to the “opening” of entirely new niches in the form of peptide-substrates from histone tails, positively charged RNA-binding complexes and cytoskeletal proteins respectively. This appears to have gone hand-in-hand with the radiation of a diverse suite of enzymes covalently-modifying histones and other proteins38. We show that the bacterial chromo-like domains already possessed the capacity to bind peptides through the aromatic cage in the open mouth of the β-barrel; thus, they were pre-adapted to binding methylated peptides in the eukaryotic chromatin niche.These observations have important implications for the origin of eukaryotes. Bacterial chromo-like domains are found only in certain bacterial lineages, including alphaproteobacteria, unlike the bacterial SH3 domain which is more widely distributed. However, chromo-like domains are rare or absent in archaea (including asgardarchaeota). In contrast, we can trace at least 3 paralogous families of chromo-like domains in the LUCAWe believe the Cren7-like and chromo-like domains identified in this study might help further experimental characterization of the biochemical and biological diversity of these domains.85. Additional sequence similarity searches were performed using the HHpred program63 . Multiple sequence alignments were constructed using Kalign86 followed by manual corrections based on structural alignments. Sequence similarity-based clustering was performed using the BLASTCLUST program (http://ftp.ncbi.nih.gov/blast/documents/blastclust.html), by adjusting the length (L) and score (S) parameters based on need. Automated structure similarity searches were performed using the DALI server52. Structures were compared and superimposed in the molecular visualization program PyMOL by manually defining equivalent regions using the pair fitting wizard. Automated pairwise structure superimposition was performed using TM-align and Fr-TM-align tools60.The PSI-BLAST and JACKHMMER programs were used for iterative sequence profile searches against the National Center for Biotechnology Information (NCBI) non-redundant (NR) and locally generated databases (e.g. nr.50: NR sequences clustered at 50% sequence identity)87.Domain architectures and other contextual information about the protein sequences were generated using both Pfam and a custom set of profiles. This analysis was automated using scripts in PERL. Graphs in Fig. 63 and the Z-scores using the DALI program. These searches were respectively run using representative sequences or structures for each of the clades against a library of HMM profiles developed from alignments in the Pfam database or structures in PDB. The e-values and Z-scores were recorded for the hits to all other clades. The clades were then clustered using single linkage clustering based on these values and the consensus clustering is rendered as a tree in Fig. The tree of relationships between different clades of chromo-like domains was generated thus: given that these domains are small in size and show rapid divergence they are not amenable to analysis by conventional phylogenetic methods. Hence, we established the relationships between them using two methods, namely the e-values reported by the profile-profile comparisons with the HHpred programSupplementary Material.


Aggregatibacter actinomycetemcomitans (Aa) has been shown to sequester and uptake cytokines. We have identified two (OMPs) of Aa, bacterial interleukin receptor I (BilRI) and OM secretin channel (OMS), which interact with several cytokines, the interaction with IL-8 being the strongest. The NMR studies confirmed that BilRI is intrinsically disordered, i.e. it does not have stabile fold without ligand. The structure of the extramembranous domains of OMS (emOMS) solved with X-ray crystallography showed that the C-terminal domain of emOMS displayed a type I KH domain fold. Crosslinking experiments indicate that one lysine of this domain is located in the IL-8 interaction site. Neisseria meningitides PilQ (NmPilQ) has role in the uptake of IL-8. Although the part of the NmPilQ which interacts with IL-8 is unidentified, the N1-domain of NmPilQ shows sequence homology and adopts the same fold as the C-terminal domain of emOMS. Thus the type I KH domain fold might be involved in the cytokine interaction in both these bacterial proteins. It would be interesting to learn if type I KH domains can be found from other periodontal pathogens than Aa.Various gram-negative pathogens possess secreted or outer membrane proteins (OMPs) which interact with host cytokines. Of periodontal pathogens only


Our understanding of isoform-specific activities of phosphatidylinositol 3-kinase (PI3K) is still rudimentary, and yet, deep knowledge of these non-redundant functions in the PI3K family is essential for effective and safe control of PI3K in disease. The two major isoforms of the regulatory subunits of PI3K are p85α and p85β, encoded by the genes PIK3R1 and PIK3R2, respectively. These isoforms show distinct functional differences that affect and control cellular PI3K activity and signaling . In this We recently reported an oncogenic activity of wild type p85β in cultures of avian embryonic fibroblasts . This acHere we have investigated the biological and biochemical activities of p85 domains comparing the α and β isoforms. The results provide new insights into the functions of the p85 domains. These include an inhibitory action of the SH3 domain and essential roles of the RhoGAP and cSH2 domains in oncogenic signaling. These studies also reveal functional differences between the p85α and p85β cSH2 domains; the hinge domain located between iSH2 and cSH2 contributes to these differences.We cloned two N-terminal truncations of both the human PIK3R1 and PIK3R2 genes; in one, the SH3 domain was deleted, in the other, both SH3 and RhoGAP domains were deleted Figure . These cWe also analyzed C-terminal truncations of the p85 isoforms for their transforming and signaling activities. These constructs are depicted in Figure We then exchanged the cSH2 and the iSH2 domains of p85α and p85β Figure . The cSHThe iSH2 and cSH2 domains of p85α and p85β are linked by a hinge region Figure . In p85αThe levels of free p85β in the cell are controlled by the F-box protein FBXL2 and the tyrosine protein phosphatase PTPL1 . FBXL2 iWe cloned the p85α iSH2 (iSH2α) and p85β iSH2 (iSH2β) domains with an N-terminal HA-tag into the RCAS(A) vector and expressed these constructs in CEF. The expression of the iSH2 domains could not be detected by standard Western blotting using the HA-tag Figure , but wasIn vitro biochemical studies have supported these two functions extensively as have studies of mutations and truncations of PIK3R1 [The classic view of p85 functions is that it has two roles in the PI3K complex. Binding of p85 to the ABD domain stabilizes p110, and binding of the nSH2 domain inhibits the catalytic activity of p110. f PIK3R1 , 16–22.In this study, we analyzed truncations, deletions and domain hybrids of p85α and p85β for oncogenicity and signaling activity, documenting novel functional differences between the two isoforms. Our data show that all of the domains of p85α and p85β have functional consequences on PI3K activity and oncogenic potential in the context of whole cells.We find that the SH3 domain exerts an inhibitory effect on oncogenic potential and PI 3-kinase signaling. In the case of p85α, this effect could be explained by the role of the SH3 domain in p85α homodimerization which in turn is required for the interaction between p85α and PTEN , 11, 23.At the C-terminus of p85, the truncation of the cSH2 domain of p85α and p85β revealed an unexpected difference between the two isoforms. The cSH2 domain of p85α appears to have an inhibitory effect on oncogenic potency and signaling, whereas the cSH2 domain of p85β functions in the opposite direction: it is required for optimal transforming activity and functions as a stimulator of signaling. These opposing activities of the cSH2 domains are confirmed by domain exchange experiments: p85α with the cSH2 domain of p85β becomes oncogenic; in contrast, p85β with the cSH2 domain of p85α loses oncogenicity. These results are in contrast to the findings of Zhang et al.  who analLarger truncations of C-terminal domains greatly attenuate the effects of the single cSH2 domain deletions, resulting in constructs of low activity for p85α as well as p85β. Interacting with p110, the nSH2 domain binds to the helical domain and the iSH2 domain associates with the adapter-binding and C2 domains of p110 to stabilize and inhibit the catalytic subunit , 20, 21.The observation that oncogenic and signaling activities are not affected by protein stability is counterintuitive. It could reflect a low minimal concentration threshold for an oncogenic protein to score in the cell system used. That requirement could be satisfied by the high rate of protein synthesis directed by the RCAS vector. Thus, the differences in protein stability would not affect transformation or signaling.In summary, in this study we document important and distinct properties of the p85α and p85β isoforms. We confirm and extend the inhibitory function of the SH3 domain for PI3K-mediated transformation and signaling. We discover contrasting properties of the cSH2 domains and find no correlation between protein stability and oncogenicity as well as signaling activity in the investigated cell system.Chicken embryo fibroblasts (CEF) were prepared from single, pathogen-free White Leghorn embryos  as previously described . TransfeTransformation assays were carried out in six-well plates. CEF were transfected with DNA of the recombinant RCAS(A) constructs specified below using Lipofectamine 2000  and were kept in growth medium for 24 h before being overlaid with nutrient agar containing Ham's F-10 medium with 20% Earle's balanced salt solution, 0.6% SeaPlaque Agarose, 3% FBS, 1% heat-inactivated chicken serum, 9% tryptose phosphate broth, 1.8 mM glutamine, 89 U/mL penicillin, 89 μg/mL streptomycin, 1.1% DMSO. This mixture was applied every 2-3 days for 10 days, at which point the overlay was removed and the cell layer stained with crystal violet for quantitative evaluation of oncogenic activity by focus formation , 40. In cDNA of the human PIK3R1 and PIK3R2 genes was used to construct the expression vectors using the RCAS(A) backbone . The C-tThe experiments reported here were carried out with primary cells. For repeat experiments, cells routinely have to be derived from different avian embryos. Because starting conditions are then not identical, standard statistical analyses based on mean +/− standard deviation are not applicable. However, all these tests were repeated three times with consistent results, and the figures shown are from representative experiments.CEF were infected with the RCAS(A) vector expressing HA-iSH2α and HA-iSH2β at a MOI of 1; non-infected CEF cells were used as negative controls. After two passages in growth medium, total cellular RNA was isolated using TRIZOL  according to the manufacturer's protocol. First strand cDNA was synthesized using a reverse transcription kit  with a random primer. PCR amplification was performed using Platinum Pfx DNA Polymerase  with a HA-tag-specific primer and iSH2α and iSH2β C-terminal-specific sequence primers . The PCRCEF were infected with RCAS(A)-expressing p85β, p85α, or the specified p85 mutants at a MOI of 1. After two passages in growth medium, cells were switched for overnight incubation to Ham's F-10 medium containing 0.5% FCS  and 0.1% chicken serum, and this was followed by 2 h in basal F-10. At this point, protein samples were harvested. Western blotting was performed as described , with miCEF were infected with RCAS(A)-expressing p85β, p85α, p85β Y655A, Y655E, Y655F, p85β/αcSH2 or p85α/βcSH2 at a MOI of 1. After two passages, the cells were treated with the translation inhibitor cycloheximide at 50 μg/ml to stop protein syntheses. Total protein samples were harvested using ice-cold RIPA buffer at 0, 0.25, 0.5, 1, 4 and 24 h after cycloheximide treatment for analyzing expression levels relative to GAPDH by Western blotting. We used the untreated sample as a standard. Relative protein stabilities were determined from Western blots and converted into line graphs. The error bars indicate standard deviations obtained in three independent experiments.CEF were infected with RCAS(A) virus that expresses HA-tagged p85β, p85α, p85β/αcSH2 or p85α/βcSH2 at a MOI of 1. After two passages, cells were rinsed with phosphate-buffered saline (PBS) and lysed in immunoprecipitation lysis buffer  and 1× Complete protease inhibitor mixture; Roche, Indianapolis, IN). Cell lysates (0.5 to 1 mg of protein) were mixed with primary HA-tag antibody  and incubated overnight at 4°C with gentle agitation. The lysates were then incubated with protein A/G beads  for 1 h at 4°C with agitation. The beads were washed three times with washing buffer , bound proteins were eluted by boiling in 4XSDS sample  and analyzed for p85-p110α interaction by Western blotting. The antibody against p110 used in the co-immunoprecipitation experiment was #4255 from Cell Signaling, Danvers, MA.


We hypothesize that Mgm101 could, in Rad52-like manner, preferentially bind single-stranded DNAs , stabilize them and mediate single-strand annealing-like homologous recombination event to prevent them from converting into toxic structures.Mgm101 has well-characterized activity for the repair and replication of the mitochondrial genome. Recent work has demonstrated a further role for Mgm101 in nuclear DNA metabolism, contributing to an S-phase specific DNA interstrand cross-link repair pathway that acts redundantly with a pathway controlled by Pso2 exonuclease. Due to involvement of FANCM, FANCJ and FANCP homologues , this pathway has been described as a Fanconi anemia-like pathway. In this pathway, Mgm101 physically interacts with the DNA helicase Mph1 and the MutSα (Msh2/Msh6) heterodimer, but its precise role is yet to be elucidated. Data presented here suggests that Mgm101 functionally overlaps with Rad52, supporting previous suggestions that, based on protein structure and biochemical properties, Mgm101 and Rad52 belong to a family of proteins with similar function. In addition, our data shows that this overlap extends to the function of both proteins at telomeres, where Mgm101 is required for telomere elongation during chromosome replication in  MGM101  was originally identified in a screen for nuclear genes required for maintenance of functional (ρ+) mitochondrial DNA (mtDNA) in baker's yeast Saccharomyces cerevisiae. The MGM101 gene, identified by complementation of the temperature sensitive mgm101-1ts mutant strain,bona fide component of mtDNA-protein complexes called mitochondrial nucleoids (mt-nucleoids),S. cerevisiae Mgm101 (ScMgm101) is required for propagation of mtDNA in cells containing wild-type (ρ+) and neutral petite genomes, as disruption of MGM101 blocks initiation of mtDNA replication in these cells.- genomes enriched for GC-rich motifs (dubbed ori/rep) are stably maintained in mutants lacking a functional copy of MGM101, indicating that these mtDNA derivatives use a distinct mechanism for replication initiation.ScMgm101 can participate in ori/rep-independent mtDNA replication and/or repair by mediating formation of recombination intermediates containing 3′ single-stranded DNA (ssDNA) overhangs.Arabidopsis thaliana suggesting that it may represent a functional homolog of Mgm101.Mgm101 is a poorly conserved eukaryotic protein with homologs found in many fungal lineages, several amoebas, slime molds and marine animals  but not in insects, nematodes or vertebrates.S. cerevisiae. In contrast to most of other fungi, S. cerevisiae is a petite-positive organism tolerating the loss of functional mitochondrial genome, however there is evidence that the mitochondrial functions of Mgm101 do not dramatically differ between petite-positive and -negative yeast species. In line with this opinion, it has been shown that the orthologs of Mgm101 from Kluyveromyces lactis (KlMgm101) and Candida parapsilosis (CpMgm101) functionally complement defects in the metabolism of mtDNA associated with the mgm101-1ts mutant in S. cerevisiae.The majority of our current knowledge about Mgm101 functions is derived from studies of the yeast ScMgm101, the mitochondrial targeting sequence consists of 22 AA.via protein oligomerization and/or the stability of oligomers.ScMgm101 shares structural flexibility distributed eventually on ring surface of protein.mgm101-1ts allele.et al. showed that ScMgm101 preferentially binds to ssDNA compared to double-stranded DNA (dsDNA) and catalyzes the annealing of ssDNA pre-complexed with the mitochondrial ssDNA-binding protein, Rim1.ScMgm101 have been analyzed. The C-terminus consists of highly conserved aromatic and basic AA residues that are able to fold into 2 ß-sheets followed by a short unstructured C-terminus.MGM101 with mutant codons for K251, K252, K260 or Y266. Finally, W257A and R259A substitutions impaired conformation and ring structure. The authors concluded that short C-terminus of ScMgm101 is essential for ssDNA-binding, oligomeric state and protein stability in vivo.Comparative sequence analysis of Mgm101 orthologs from yeast revealed a highly conserved C-terminus consisting of 165 amino acids (AA). This region of Mgm101 corresponds to its functional core that is composed of 23% positively charged AA residues and that specifically and tightly binds to (negatively charged) DNA. In contrast, the N-terminus of the protein is not conserved. This region contains a cleavable signal for import into mitochondria. In ScMgm101 consists of 14 homo-oligomeric rings of approximately 200 Å diameter in solution and that binding to ssDNA promotes formation of large condensed helical nucleoprotein filament structure. Moreover, ScMgm101 with substitutions C216A C217A exhibits destabilized rings responsible for emergence of stable but defective filaments.in vitro but functional relevance of this higher order organization is still unknown.ScMgm101 (purified in fusion with MBP domain to increase the yield of soluble protein) was shown to form helical filaments with diameter of approximately 20 nm and to possess ssDNA-binding activity in vitro.et al. found that the ring structure disappears when ScMgm101 binds ssDNA.ScMgm101 were unstable in solution because they aggregate quickly and are toxic for bacterial cells.Biochemical analysis revealed that C. parapsilosis.CpMgm101 shares many features with ScMgm101, but these proteins also exhibit several differences. Both proteins form homo-oligomers in solution, bind to ssDNA with a similar affinity and prefer ssDNA over dsDNA substrates. However, CpMgm101 binds more strongly to dsDNA which can account for its permanent association with mitochondrial nucleoids and it has been demonstrated that upon binding to 5′ ssDNA overhang of a model mt-telomere it generates ring-shaped structures with the DNA being wrapped around the protein. Such differences indicate a specific role in C. parapsilosis. In this light, it is interesting that single-strand annealing (SSA) activity of CpMgm101 can mediate the invasion of the ssDNA overhang into dsDNA region of mt-telomeres resulting in a formation of t-loop structure and the protein binding to a range of replication and recombination intermediates suggests a role in both mtDNA replication and maintenance of mt-telomeres.in vivo.A recent study characterized the Mgm101 protein from the yeast ScMgm101 also participates in repair of nuclear DNA. Previous studies employing cell fractionation approaches suggest that ScMgm101 is present in both the nuclear and mitochondrial fractions.ScMgm101 within the nucleus  and Alexa 488 (ScMgm101-Myc). Single section images were taken from the middle of the nucleus to reduce the possibility of mitochondrial fluorescence above or below the nucleus appearing to be in the nucleus. Consistent with its localization in the mitochondria and cytoplasm, we observed strong Alexa 488 fluorescence spots and diffuse staining outside the nucleus (ScMgm101-Myc accumulated around the edge of the nucleus (an example can be seen in ScMgm101-Myc in the nucleus (ScMgm101-Myc (data not shown). In silico analysis by PSORT IIScMgm101 were deduced from a dual localization of a fraction of the protein and, by inference, its similarity to Rad52:In addition to its role in mitochondria, there is increasing evidence that  nucleus . In cell nucleus . However nucleus , G and H nucleus . NotablyScMgm101 has been shown to be involved in repair of oxidative DNA damageScMgm101 in ICL repair (see below).S. cerevisiae,ScMgm101 contributes to the protection of yeast cells against genotoxic effects of formaldehyde, a known protein-DNA crosslinking agent.MGM101 or RAD52 lead to an increase in sensitivity to nitrogen mustard (HN2) when cells were synchronized in S-phase  that is independent of Rad6-Rad18-controlled branch of PRR.via DNA-protein and protein-protein interactions.There is evidence to suggest that ICL repair factors contribute to the maintenance of nuclear telomeres , an ortholog of Pso2/SNM1A, on mammalian telomeres and its interaction with the major telomere-binding protein TRF2rad52 cells.rad52 cells resulted in the recovery of the wild-type telomere length. It is likely that in the absence of the ICL repair factors telomeres are not maintained by ALT mechanism and the shortening of telomeres in the corresponding cells may be caused by decreased levels of telomerase activity or by inefficient recruitment of telomerase to the chromosomal ends. Importantly, a combination of pso2 and/or msh2 with the deletion of YKU70 did not induce a shortening in TRF lengths  analysis in cells deficient for these factors . Our dat lengths  typical rad52 defective cells. In the light of recent biochemical and structural data,pso2 msh2 mgm101 rad52 quadruple mutant (but neither pso2 mgm101 rad52 nor msh2 mgm101 rad52 triple mutant) exhibit shorter telomeres demonstrating that the Pso2-dependent and FA-like pathway cooperate with Mgm101 in telomere maintenance in rad52 cells. The future experiments aimed at testing a physical association of Mgm101 with telomeric repeats in the wild-type cells as well as rad52 deletion mutants will address this possibility in a more detail.Double mutant lacking both Mgm101 and Rad52 exhibits normal length of TRFs, suggesting that Mgm101 and Rad52 likely have biochemically very similar  functions at telomeres during S-phase. In other words, it appears that Mgm101 is involved in a process of telomere elongation during chromosome replication in Data accumulated over last few years suggests that Mgm101 and Rad52 could have overlapping roles in a yeast cell, at least in the context of nuclear DNA metabolism. This overlap includes a function of both proteins (i) at the site of stalled replication forks where Mgm101 could contribute to repairing DSBs that arise as a consequence of replication fork collapse, and (ii) at telomeres where Mgm101 could be involved in a process of telomere elongation during chromosome replication. We hypothesize that in a Rad52-like manner  Mgm101 could preferably bind ssDNAs, stabilize them and mediate SSA-like HR event to ensure that they are not processed into structures that can be toxic.


Here, we report a grain-oriented (with 95% <001> texture) modified PbTiO3 ceramic that has a high Tc (364 °C) and an extremely large g33 (115 × 10−3 Vm N−1) in comparison with other known single-phase oxide materials. Our results reveal that self-polarization due to grain orientation along the spontaneous polarization direction plays an important role in achieving large piezoelectric response in a domain motion-confined material. The phase field simulations confirm that the large piezoelectric voltage coefficient g33 originates from maximized piezoelectric strain coefficient d33 and minimized dielectric permittivity ɛ33 in [001]-textured PbTiO3 ceramics where domain wall motions are absent.A rapid surge in the research on piezoelectric sensors is occurring with the arrival of the Internet of Things. Single-phase oxide piezoelectric materials with giant piezoelectric voltage coefficient ( High piezoelectric voltage coefficients drive the sensitivity of piezoelectric sensors. Here, the authors synthesized textured Sm- and Mn-doped PbTiO3 ceramics and demonstrate significant enhancement in voltage coefficient. Among them, PZT-based piezoelectric ceramics have been widely utilized due to their superior piezoelectric performance and they can be easily tailored to meet the requirements for different application through compositional modificationsg33 of PZT ceramics is usually in the range of 20 to 30 × 10−3 Vm N−1, as shown in 3 ferroelectric single crystals have ultrahigh piezoelectric strain coefficient d33 and electromechanical coupling factor k33 on the order of 2,000 pC N−1 and 0.9, respectively5g33 coefficient is still <40 × 10−3 Vm N−1. Considering the relation between d and g (g=d/ɛ), it is challenging to have higher value of g, because, any increase in piezoelectric response d is usually accompanied by even larger increase in the dielectric permittivity ɛ.Most of the state-of-the-art piezoelectric materials are based on perovskite-structured ferroelectrics, such as BaTiOg33, the most widely used method has been fabrication of piezoelectric composites containing high d piezoelectric material  with low ɛr polymers )g33 due to a very small ɛr To achieve high d33 and g33 of piezoelectric ceramics can be simultaneously improved by a cost-effective texturing process called templated grain growth (TGG)91011d33 of <001> textured PMN-PT and PMN-PZT ceramics was found to exceed 1,000 pC N−1, which is about two to five times higher than that of the non-textured ceramics9g33 magnitude also increased by a factor of two compared to that of the non-textured ceramics, as shown in d33 was attributed to engineered domain state in <001> textured ceramics in similar fashion as that of <001> oriented single crystal. In a rhombohedral single crystal, the domain configurations consisting of the equivalent <111> polarizations exhibit high piezoelectric response along the <001> direction. The enhanced g33 was related to the reduced dielectric constant of textured materials due to the presence of templates with low dielectric permittivity9g33 obtained in piezoelectric single crystal–polymer composite. Although <001> textured PMN-PT and textured PMN-PZT ceramics exhibit relatively large g33 than that of their non-textured counterparts, the temperature range of application is limited by phase transition between rhombohedral and tetragonal phases (TR-T)1314g33 of these textured ceramics was also much lower than that of piezoelectric ceramic/single crystal–polymer composite.Prior studies have shown that g33–high Tc material by considering anisotropy/composition/phase structure selection, tailored microstructure and domain engineering. First, we select tetragonal PT as starting composition and phase. PT has high Curie temperature (Tc=490 °C), small dielectric constant, and large piezoelectric anisotropyc/a=1.064), it is very difficult to sinter PT ceramics. It is also challenging to pole pure PT ceramics due to low resistivity and high coercivity. To overcome these challenges, several dopants have been attempted and it has been shown that the mechanical properties and electrical resistance of PT ceramic can be improved via Sm and Mn dopingTc (364 °C) and an extremely large g33 (115 × 10−3 Vm N−1) in comparison with other known single-phase oxide materials, as shown in In this work, we provide fundamental insight in the design of high 4Ti4O15 (PBiT) precursors were first synthesized and then converted into perovskite structured PT. Layered structured PBiT microcrystal can be easily grown into plate-like shape due to its strong structural anisotropy. 3 by the topochemical chemical conversion reaction with a little impurity phase denoted by the extra peak near 31° (2θ). It should be noted here that the synthesis of pure PT is much more difficult than BaTiO3, NaNbO3 and so on, using same topochemical conversion methodet al.2O3/PbO flux system. The results show that the presence of Na+ in the reaction flux will facilitate the perovskite phase formation because the incorporation of Na+ charge balances Bi3+ on the perovskite A-site (forming PbTiO3-Na0.5Bi0.5TiO3 solid solution).The textured SM-PT ceramic was fabricated by TGG method. In this process, PT plate-like template crystals were aligned in SM-PT ceramic matrix powder by the tape casting method. During sintering, the SM-PT matrix grains grew from aligned PT templates and resulted in textured/grain-oriented SM-PT ceramics. The perovskite PT templates were synthesized using topochemical conversion method. Direct synthesis of PT templates with high aspect ratio morphology is difficult due to its cubic symmetry at the synthesis temperature . To fabricate the perovskite PT template, layered perovskite structured PbBipc  peak was the highest for the randomly oriented (or non-textured) ceramics. With increasing degree of texture, the intensity of (110)pc Bragg reflections continuously decreased while the intensity of (001)pc and (002)pc reflections increased, manifesting strong preferred crystallographic orientation along <001>pc. To achieve high density and high texture, final samples were sintered at 1,230 °C for 10 h. The samples showed 99% relative density and 95% texture degree in terms of Lotgering factorTc of PbTiO3 from 490 °C to 343 °C for the non-textured sample. The textured ceramic had a little higher Tc (364 °C) due to slightly less concentration of Sm and Mn (template had no Sm and Mn). However, the Curie temperature of SM-PT ceramic was much higher than most of the PZT based ceramics P–E) hysteresis plots for the non-textured and textured samples. It can be seen that the polarization of the textured samples was higher than that of the non-textured samples. Theoretically, the intrinsic polarization value Ps along the polar axis of the mono-domain crystal for the tetragonal phase follows the relationship Ps,<001>= (3)1/2Ps,<111>  to 127 pC N−1 in textured ceramics. More importantly, a large magnitude of g33 (115 × 10−3 Vm N−1) was obtained in textured samples, which was significantly higher than that of PT, PZT and PMN-PT ceramics or single crystals as shown in d33 increases, ɛr decreases, resulting in a large magnitude of g33 in textured samples.θ away from the polar axis is given as  of the doped PT random ceramics may be not a property of the single crystal but of the ceramic26d33/d31 (or g33/g31) has been reported to be over 10; however, this ratio (d33/d31) is about 4.7 for a PT single crystal. g33/g31 in <001> textured PT ceramics as a function of texture degree, where it can be found that the ratio of g33/g31 is about 10 at low texture degree, but rapidly decreases to 4.8 when the ceramic is highly textured (more than 0.9). In this study, the new finding is the record-breaking value of large g33, and our focus is to maximize g33 based on crystallographic anisotropy (first type) through maximized ratio of d33 and ɛr, not g33/g31. Nevertheless, it is worth noting that a combination of both large g33 (about 70 × 10−3 Vm N−1) and large g33/g31 (about 11) can be achieved at partial texture around f=0.8, which is an attractive performance.It should be noted that for PT materials, two types of anisotropy need to be considered. The first type of anisotropy refers to the magnitude change of a particular parameter, such as 3 single crystal based on polarization rotation, we further investigated the effect of domain switching and domain wall motion on piezoelectric response in SM-PT ceramics. Domain switching and domain wall motion is generally used to explain the extrinsic contribution towards the piezoelectric response in polycrystalline ceramics. −1 are only 0.19% and 0.07%, for textured and non-textured ceramics, respectively, which suggests the absence of 90° domain switching. If 90° domain were switchable, SM-PT could have exhibited large electric field-induced strain around 6% in textured ceramics and around 2.5% in non-textured ceramics as shown by phase field model below. Li et al.In addition to the calculation of intrinsic piezoelectric anisotropy  of tetrapc  peak splits into two peaks, (002) and (200). The relative intensity ratio of (002) and (200) peaks indicates the percentage of c-domain and a-domain. On the basis of the X-ray diffraction pattern shown in c-domain and a-domain was not changed under electric field during poling process. Furthermore, for non-textured sample, the theoretical intensity ratio of (002) and (200) is 1:2. Interestingly, we noticed that the percentage of c-domain (Ic) in textured ceramic is much larger than a-domain (Ia). This phenomenon indicates that the <001>-textured SM-PT ceramic exhibits a strong polarization self-alignment or c-domain preferred orientation.To understand the non-180° domain switching, the X-ray diffraction patterns were recorded on poled and unpoled samples. Due to large tetragonality of PT ceramics, (002)3, as discussed earlier. High activation energy of 90° domain switching can be also indicated from the domain structures of textured SM-PT ceramic under transmission electron microscopy (TEM) as shown in To experimentally observe the domain motion under electric field, vertical and lateral piezoresponse force microscopy (PFM) was performed for both non-textured and textured samples, as shown in 3 ceramics, we adopted a phase field model for ferroelectrics O3 polycrystals with the composition of x=1 (PT) and x=0.6 (PZT) at room temperature for comparison. PZT with x=0.6 has an equilibrium tetragonal phase as PT does but is closer to the morphotropic phase boundary and has a reduced electrocrystalline anisotropy than PT, thus serving as a good case study for comparison. The same grain structure D and gij=(∂ɛj/∂Di)σ, respectively, which are equivalent thermodynamic definition via Maxwell's relationσ=0) in the phase field simulation, thus we simulated the converse piezoelectric effect to evaluate g33=∂ɛ3/∂D3=d33/ɛ33 through the piezoelectric strain coefficient d33 and dielectric permittivity ɛ33.To further quantitatively investigate the domain-level mechanisms for the enhanced piezoelectric voltage coefficient in [001]-textured PbTiOtructure  and the tructure  are usedThe simulated polarization distributions and domain structures in non-textured and textured ceramics of PZT and PT that are poled in vertical direction were compared . In the −1) was applied on textured PT to observe domain switching without considering dielectric breakdown or mechanical cracking, which is not possible in real samples. Domain switching is also difficult in non-textured PT, since the applied electric field is usually below its coercive field (above 400 kV cm−1). As discussed earlier, if switchable, PT could exhibit large electric field-induced strain around 6% in textured ceramics and around 2.5% in non-textured ceramics, which were not observed as shown in d33 and ɛ33 were obtained from the simulated strain-electric field and polarization-electric field responses, respectively, from which the piezoelectric voltage coefficient was evaluated using relation,g33=d33/ɛ33. The simulated value of 130 × 10−3 Vm N−1 agrees well with the experimental value of 115 × 10−3 Vm N−1, thus confirming the giant piezoelectric voltage coefficient in [001]-textured PT ceramics. To further reveal the underlying mechanisms of such a significant enhancement in g33, simulations were also performed with domain walls frozen by setting the gradient coefficient β=0 in d33 and ɛ33 in non-textured PT ceramics, thus have negligible effect on g33. On the other hand, domain wall motions do not play a role in textured PT ceramics, because polarizations are well aligned in [001] direction. The comparison study between textured and non-textured PT ceramics shows that PT has an intrinsically high value of d33 and low value of ɛ33 along [001] axis due to its high electrocrystalline anisotropy, which is in agreement with the experimental measurements shown in ɛ33 resulting in a significant enhancement in g33 of [001]-textured PT ceramics. The simulations show that similar mechanisms also improve g33 in textured PZT, but the achievable value is less than in textured PT. Thus, appropriate choice of PT composition combined with texture engineering and domain engineering effectively controls the grain structures and domain processes in [001]-textured PT ceramics to achieve giant piezoelectric voltage coefficient, which provides rational design and synthesis of piezoelectric materials for targeted applications.3 material with high Tc (364 °C) and an extremely large g33 (115 × 10−3 Vm N−1) as compared with other known single-phase oxide materials. Diffraction and scanning probe microscopy studies reveal that self-polarization due to grain orientation along the spontaneous polarization direction plays an important role in achieving large piezoelectric response in a domain motion-confined material. The simulations using phase field model confirm that the large piezoelectric voltage coefficient g33 originates from maximized piezoelectric strain coefficient d33 and minimized dielectric permittivity ɛ33 in [001]-textured PbTiO3 ceramics where domain wall motions are absent.We report a grain-oriented (with 95% <001> texture) modified PbTiO0.8725Sm0.085)(Ti0.98Mn0.02)O3. The SM-PT matrix powder was synthesized by conventional solid state reaction method. For this, PbO , Sm2O3 , TiO2 (Ishihara Sangyo Kaisha Ltd.), and MnO2  was mixed and ball-milled in ethanol for 24 h. The mixture was dried at 80 °C and then calcined at 850 °C for 4 h. The calcined powders were ball-milled again with 1.5 wt% excess PbO for 24 h. The templates for texturing SM-PT ceramic are plate-like <001> PbTiO3 microcrystals. The <001> PbTiO3 templates were synthesized by topochemical microcrystal conversion method. In the first step, PbBi4Ti4O15 precursor was synthesized in molten salt. In the next step, PbBi4Ti4O15 precursor was mixed with PbO and NaCl salt, and then heated to 1,050 °C for 3 h. Bi3+ in PbBi4Ti4O15 was substituted by the Pb2+ from PbO, yielding PbTiO3 template and Bi2O3 byproduct. The Bi2O3 byproduct was removed by diluted nitric acid. To fabricate textured ceramics, the ceramic slurry was prepared by ball milling the SM-PT matrix powders with organic binder (Ferro 73225), and toluene/ethanol solvents. Next 5 wt% of PT templates were mixed into the slurry by magnetic stirring. Afterwards, the slurry was casted at the rate of 40 cm min−1 by using doctor blade with height of 250 μm. The dried green tapes were cut, stacked, and laminated at 80 °C under 20 MPa pressure for 15 min. The green samples were heated to 400 °C for 2 h with a heating rate of 0.3 °C min−1 to remove organic solvent and binder, and then isostatically pressed at 200 MPa for 1 min. Samples were subsequently sintered at 1,000–1,300 °C for 2–10 h. The detailed process for the synthesis of grain oriented/textured ceramics is provided elsewhereThe composition of SM-PT was (Pbθ range of 20–60° by Lotgering factor method. The dielectric properties of poled samples were measured as a function of temperature by using a multi-frequency LCR meter (HP4274A). The piezoelectric properties of samples were obtained by resonance and anti-resonance technique using impedance/gain phase analyzer (HP 4194A) and d33-meter . Piezoresponse force microscopy  was used to image the ferroelectric domain structures. Conductive Platinum-Iridium silicon cantilevers  were used for the PFM characterization. Standard grinding and ion-milling method was used to prepare the electron transparent TEM specimens, and FEI Titan 300 microscope was used to capture TEM images.The phase and microstructure were characterized using X-ray diffraction  and SEM . The degree of pseudo-cubic <001> texture was determined from the X-ray diffraction pattern in 2The data that support the findings of this study are available from the corresponding authors on request.How to cite this article: Yan, Y. et al. Giant piezoelectric voltage coefficient in grain-oriented modified-PbTiO3 material. Nat. Commun.7, 13089 doi: 10.1038/ncomms13089 (2016).Supplementary Figures 1-4, Supplementary Notes 1-2 and Supplementary References.


L and VH. These exhibit a conserved ß-sheet structure and comprise highly variable loops (CDRs). Little is known about the contributions of the framework residues and CDRs to their association. We exchanged conserved interface residues as well as CDR loops and tested the effects on two Fvs interacting with moderate affinities (KDs of ~2.5 µM and ~6 µM). While for the rather instable domains, almost all mutations had a negative effect, the more stable domains tolerated a number of mutations of conserved interface residues. Of particular importance for Fv association are VLP44 and VHL45. In general, the exchange of conserved residues in the VL/VH interface did not have uniform effects on domain stability. Furthermore, the effects on association and antigen binding do not strictly correlate. In addition to the interface, the CDRs modulate the variable domain framework to a significant extent as shown by swap experiments. Our study reveals a complex interplay of domain stability, association and antigen binding including an unexpected strong mutual influence of the domain framework and the CDRs on stability/association on the one side and antigen binding on the other side.The antibody Fv module which binds antigen consists of the variable domains V L) and of the heavy chain (VH) which associate non-covalently to form the so-called Fv-fragment. Three hyper-variable regions (complementarity determining regions or CDRs) in VL and VH comprise the residues interacting with antigens. They account for approximately 25% of the variable domains1. CDR-H3  is the most diverse of these six regions concerning length and amino acid sequence2. Apart from the CDRs, both variable domains exhibit a conserved β-barrel framework stabilized by an internal disulfide bridge4. They are composed of two ß-sheets, one with four strands  and one with six strands , with the strands GFC’C involved in the VH/VL interface. Several studies on the VH/VL packing geometry have shown that residues within the framework as well as interface contributing residues of the CDRs can influence the interface6. 75% of the interface residues are constituted by framework ß-sheets and 25% by the CDRs 3. The interface contribution of the hypervariable loops comprises CDR1 and especially CDR3 residues3. In this context, the CDR-H3 can affect VH/VL orientation due to small position re-adjustments by the varying number of interface contributing residues3. An additional influence by hypervariable loop residues is exhibited by the so called proximate zone, which is situated at the base of the antigen binding site, comprising residues that do not actively participate in the interface3. Several studies on the VH-VL packing geometry showed that residues within the framework as well as interface contributing residues of the CDRs can influence the interface7. As the association of VL and VH is crucial for antigen binding3, understanding the underlying principles is of great importance.In the humoral immune response, antigen recognition is mediated by immunoglobulins, specifically by the N-terminal variable domains of the light chain . Generally, the majority of the most strongly conserved amino acids identified in this study were positioned at the VH/VL interface9. For VL, amino acids Y36, Q37, P44, A43, L46 and F98 were found to be highly conserved, with all residues except Q37 directly in contact with VH. On the VH side, amino acids V37, R38, G44, L45, E46, W47 and W103 could be identified in the VH/VL interface with all residues except E46 and R38 in direct contact with VL. VH W47 seems to be the central node based on the number and strength of its covariations with other interface residues, the same holds true for Y36 and P44 for the VL domain. Further computational analyses revealed two main modes of interaction for VH and VL which is either characterized by a proline or a medium/large hydrophobic residue at position 44 in VL7. Concerning VH, W47 seems to be essential as it was previously mutated for improved solubility and stability but none of the mutations  were favorable. Interface residues can also affect antigen binding13 by influencing the positioning of hypervariable loops. Additional experimental studies addressed the influence of the exchange of particular conserved amino acids on the association of VH and VL via the stabilities of covalently linked scFv and Fab fragments18. While these studies lay the groundwork, we are still far from a detailed and comprehensive understanding of the organization of the Fv interface. In this context, it is important to determine the affinity of the association of VL and VH directly. Strikingly, for the other domain interactions in IgG the KD values differ by several orders of magnitude. For the CH3 dimer a KD < 10−10 M was determined using SEC20. and the KD for the interaction between CH1 and CL was 6.2 µM obtained by the CL induced change in intrinsic fluorescence of CH121. This analysis is largely lacking for the VL/VH interaction.For the VL and VH domains of the human monoclonal antibody 1HEZ (κ/IgM subclass) and the murine monoclonal antibody MAK33 (κ/IgG1 subclass) as well-studied model systems23 to analyze the contribution of different factors on VL and VH structure and function. The application of variable domains from different species and subclasses should reveal to which extent the mutation-associated effects are conserved. Their sequences contain all the conserved residues identified by Wang and coworkers except for an alanine at position 43 in MAK33 VL, which is exchanged to serine and a glycine at position 44 in MAK VH, which is an arginine in MAK33. Since the relative importance of the conserved residues for structure, stability, association and antigen binding is not clear, we mutated every conserved residue against alanine and analyzed the properties of the variants in a comprehensive manner. Additionally, we generated two MAK33 VL double point mutations  to investigate the potential synergistic nature of the mutation. Importantly, we focused in the analysis on the isolated variable domains and the direct influence of point mutations on their interaction, and not on Fab or scFv fragments as in previous studies18 to draw conclusions concerning their stabilities and antigen binding properties. Furthermore, we performed CDR exchange experiments to address the contribution of these structural elements on domain architecture.Here, we chose to use the VL and VH domains.Our results on the effects of mutations on domain structure, stability, association and antigen binding together with CDR exchange experiments reveal complex relationships between structural and functional properties within the V9. Based on the results of Wang and coworkers we created six single  and two double point mutants for MAK33 VL (Y36A/P44A and Y36A/S43A) and five single mutants  for 1HEZ VL as well as seven single point mutations for MAK33 VH  and 1HEZ VH . As shown in Fig. L and VH. The single domains were expressed in E. coli, as insoluble proteins in inclusion bodies. During refolding and purification, it turned out that R38A and E46A in VH were unstable and aggregation-prone. Therefore, they were not considered further. For the determination of structural changes, we recorded CD spectra of the variants. FUV-CD spectra report on the secondary structure of proteins. All wt domains studied show a typical ß-sheet structure (minimum at 218 nm) with the exception of MAK33 VL, which exhibits a characteristic shape in agreement with previous studies in which we investigated the folding pathway of the protein24 and its amyloidogenic variants27  were generated in the MAK33 framework to investigate the potential synergistic nature of the observed effects. Indeed when incorporating the most stable MAK33 mutation, S43A, the stability of Y36A/S43A seemed slightly increased compared to Y36A. For Y36A/P44A, where P44A alone has wt-like stability, almost no difference in stability was visible compared to Y36A  unfolding transitions were performed Figs  and 2. A36A Fig. , Table 1H wt domains, we determined a midpoint for GdmCl-induced unfolding of 0.2 ± 0.1 M GdmCl for MAK33 and 0.9 ± 0.1 M GdmCl for 1HEZ. Thus, the investigated VH domains are less stable than their corresponding VL domains  form dimers, which usually enhances its intrinsic stability. In fact, dimerization adds an additional layer of complexity. However, homo-dimerization is a typical feature of many variable antibody domains and also a number of light chains. Therefore, this additional dimension of the VL-VH interplay was included to this study as it competes with hetero-dimerization and thus it is interesting to determine to which extent this equilibrium influences the effects of certain mutations on the interaction of the two variable dimers in the heterodimer.For the Vsis Fig. . The onlons Fig. . Thus, nL and VH domains was analyzed using scFvs or Fab fragments18. This is a relatively indirect approach, as the domains were either artificially linked (scFvs) or two additional covalently linked domains were present (CL and CH1 in the Fab). Here, we used assays that report directly on the formation of the VL and VH heterodimer. For SPR measurements, the wild type domain was immobilized on the chip and different concentrations of the corresponding mutant domains were added under a constant flow. With this setup, KDs of 2.0 ± 0.8 to 2.9 ± 1.1 µM and 4.2 ± 1.1 to 8.2 ± 1.4 µM were determined for the MAK33 and 1HEZ wild type VL and VH domains, respectively , which were generated to investigate whether the effects observed for single mutants are additive. Y36A/S43A, which is the combination of two point mutations with no change in KD, leads to the expected wt-like KD. But the point mutation P44A with the highest KD for the association with VH (58.0 ± 10.4 µM), exhibited improved binding when combined with Y36A, suggesting compensatory effects  showed a clear influence on the association, only the mutants V37A and L45A were affected in 1HEZ VH  and L45 (VH) as well as F98A (VL) and V37A (VH) where our analysis identified the most drastic effect on association for the point mutations P44A and L45A. Consequently, those VH point mutations in MAK33 and 1HEZ with an apparent effect on association but lacking a corresponding effect for the mutant affecting the partner residue in VL might be due to changes in secondary  show, besides 1HEZ W47A which exhibits a monomer-dimer equilibrium, a significantly decreased stability compared to 1HEZ wt . In this ELISA, a concentration-dependent increase in signal is only observed when VL and VH are combined . Antigen binding was found to be abolished for VH W47A. Apart from that, the VH mutation L45A led to the strongest reduction in the affinity for the antigen with a 12-fold increased KD compared to wt, while V37A and W103A exhibited a less pronounced decrease with a 2-fold higher KD  with MAK33 VL, a KD of 1.3 ± 0.1 µM was obtained. This corresponds to the value determined for the MAK33 wt domains , the chimera bound to MAK33 VL with a KD of 2.5 ± 0.3 µM. These observations lead to the conclusion that for MAK33 VL, the MAK33 and 1DHU VH domains are both suitable binding partners. But when grafting the MAK33 CDRs on the 1DHU framework, the association is slightly impaired, demonstrating that the CDRs can exhibit a marked influence. Concerning the VL grafting constructs, the observations are different: the 1DH5 VL domain binds to MAK VH with a roughly 30-fold lower affinity than the MAK33 VL domain. However, the 1DH5 grafting construct containing the MAK33 CDRs (MAK33_1DH5) shows an affinity similar to the MAK33 wt VL domain. In contrast, grafting of the 1DH5 CDRs onto the VL-MAK33 framework (1DH5_MAK33) resulted in strong binding impairment with a KD of 42.3 ± 9.8 µM, which is only slightly lower than the 1DH5 wt KD of 76.2 ± 12.8 µM. So for the VL domain, the CDRs and not the framework are the determining factor for the affinity towards VH wt.The KL while the spectrum of MAK_1DH5 was different from both wts  showed a FUV-CD spectrum similar to 1DH5 Vwts Fig. . So alrewts Fig.  but hereH domain, both grafting mutants showed similar FUV-CD spectra  has a slightly higher KD of 0.3 µM for creatine kinase whereas there is almost no antigen binding detectable for the VH grafting domain MAK _1DHU (>50 µM)  is the most flexible of these six regions concerning length and amino acid sequence2, additionally a mutant carrying a swapped CDR-H3 loop was analyzed. Concerning the VH/VL association the CDR-H3 mutation shows a wt-like KD of 1.8 ± 0.2 µM. Interestingly, this construct, MAK CDR-H3 1DHU VH showed a strongly impaired binding to the antigen with a 10-fold increased KD (Table As CDR-H3 (i.e. the third CDR of VH and VL mutations explicit solvent Molecular Dynamics (MD) simulations were performed for the heterodimer and for the individual domains. The set of simulations included point mutations that are associated with a significant reduction in VH/VL association  and, as a control, also substitutions that showed only modest effects on complex affinity . Simulations were started from the geometry of the wt structure . On the time scale of the simulations, none of the VH/VL complexes dissociated and the root-mean-square deviation (RMSD) of the complexes did not exceed 0.3 nm from the start structure . The structural distortion of the VL/VH complexes due to some of the mutations is also reflected in overall larger root mean square fluctuations of heavy atoms (RMSF) with respect to the mean structure. For the isolated mutated protein partners no significant difference to the wt was observed  showed increased fluctuations in regions at and near the binding interface but also in loops involved in antigen binding  was observed  Ving Figs  and S5. ner Fig.  which drner Fig. . Hence, H and VL domains much less than some of the interface point mutations (see above). During simulations on the time scale of 100 ns, these variants did not show significant differences of the calculated RMSF compared to the wt . In experiments these variants affected the binding affinity between V wt Fig. .H and VL is still unclear. This is an important aspect for understanding antibody architecture both as the basis of our immune system and also in the context of the engineering of antibodies for therapeutic purposes. In this context, it was found that in mutants an increase in affinity is often accompanied by a decrease in stability and vice versa - and these consequences are difficult to predict39. In our study, we analyzed the association of the variable domains for the first time directly. This allowed us to specifically define the contribution of framework and CDR mutations on the interaction of VH and VL. To determine whether the observed effects for the conserved residues can be transferred between different variable domains, the analysis was performed with two distinct pairs of variable domains from a murine IgG1 (MAK33) and a human IgM (1HEZ) antibody. For the VL wt domains, KDs of 2.0 ± 0.8 µM for the murine IgG1 and 8.2 ± 1.4 µM for the human IgM were determined. KDs for wild type VH domains were found to be 2.9 ± 1.1 µM and 4.2 ± 1.1 µM for IgG1 and IgM, respectively. Consequently, the two domains interact with a similar efficiency as CH1 and CL, but not as efficient as the CH3 homodimer21. This relatively weak interaction (in the absence of the CH1 and CL domains) necessitates the covalent linkage of Fv fragments via a peptide and thus creating a pseudo-monomeric fusion protein (scFv) used as a therapeutic agent41. Simulations of scFvs have shown that the stability of the interface between the two variable domains plays a critical role for the overall stability of an antibody (or fragment) as dissociation precedes unfolding42.The relationship between structure, stability and binding affinity of Vin silico analysis which showed that only very few residues (<10) are important for adopting an immunoglobulin fold43, the alanine mutants in the VL domain had only a relatively low influence on its secondary or tertiary structure compared to the VH domain. In contrast, VH structure is highly sensitive to the exchange of conserved residues in the interface. Two VH interface residues, E46 and R38, were identified to be essential for the folding of both, the MAK33 and 1HEZ VH domain. In the covariation analysis there was a very high ɸ-value for these two residues. In the crystal structure, both residues form a salt bridge9, R38 is buried and E46 does not interact with other VH interface residues, but might electrostatically affect VL binding9. Camelids and cartilaginous fish possess naturally occurring heavy chain antibodies lacking the light chain45. Interestingly a sequence alignment of MAK33 VH with the variable domain (VHH) of the camelid VHH (PDB entry 2XT1) and the variable domain of “monomeric” shark IgNAR (VNAR) (PDB entry 2I24) shows a match for the residues E46 and R38. Concerning the shark IgNAR these two residues are actually the only ones from the conserved network investigated in this study that are present at corresponding positions of this otherwise highly divergent sequence. For camelid VHH, a mutation of the hydrophobic VH/VL interface residues  in favor of hydrophilic ones was discovered45. The increase of hydrophilic residues in the framework also holds true for the VNAR which probably evolved from a cell surface receptor46. Both antibodies show high biophysical stability and their distinct structural patterns have by now been successfully applied to generate monomeric human VH domains49.Of special interest is the nature of the domain interface. It has to support the association of the two domains but also allow accommodating different CDRs and their repositioning in the context of antigen binding. Thus, an individual interface residue may be involved in one or more of these processes: 1) formation of the immunoglobulin fold, 2) domain stability, 3) interaction between the variable domains, or 4) antigen binding. Our alanine screen of conserved residues allowed us to address each of these possibilities and differentiate between them. Consistent with the results of L residues had predominantly less impact than observed for VH residues. As expected, MAK33 VL S43A and MAK33 VH R44A behaved similar to the wt. These are the only amino acid positions of the two antibodies studied which do not exhibit the conserved amino acid network identified by Wang and coworkers. In their covariation analysis an alanine is the conserved amino acid at VL position 43 and glycine is conserved at position 44 in VH, as can be seen for the 1HEZ variable domains.Mutations of conserved VL domains, the proline residue at position 44 showed the most prominent effects with an impaired VH/VL association as well as impaired antigen binding, followed by F98A. In contrast, P44 did not exhibit significant impact on VL stability. Generally, a change in stability did not necessarily coincide with a change in functionality. Surprisingly, MAK33 VL L46A exhibited a higher affinity for the VH domain than the wt but it is clearly less efficient in binding the antigen  adds another dimension to this study since it validates that the impact of highly conserved residues is preserved even in the presence of competing homo-dimerization. For the majority of the MAK33 VH point mutations, especially L45A, an increased KD for association with the VL domain could be detected, which correlated in most cases with an impaired binding of the antigen. But VH W47A, for example, showed no antigen binding while the VH/VL association was only slightly decreased. 1HEZ VH exhibited an impaired binding to VL only for two mutants L45A and V37A. According to the simulation data for MAK33 /P44(VL) and a contribution of V37(VH)/F98(VL). In terms of antigen binding, it has to be kept in mind that additionally to the stabilizing effect of the VL domain, antigen binding represents for the heterodimer an additional layer of stabilization. For the MAK33 VH domain, in isolation some of the conserved VH/VL interface residues are critical for structure formation and stability. This might cause the impaired association with the VL domain. In general, association and antigen binding do not necessarily correlate.The biophysical properties of isolated V33 Table , the ass27.Surprisingly, we could not find a general consistent relationship between secondary and/or tertiary structural changes, stability, association or antigen binding. Since it is known that antibody domains share the common ‘Ig fold’, the mutations most likely do not alter the highly conserved Ig fold, but rather lead to local structural changes in agreement with the MD simulations. Due to V(D)J joining and somatic hypermutations, immunoglobulin variable domains naturally experience an extreme sequence variation, except for some rare highly conserved residues. It thus makes sense that the Ig fold has an intrinsically high tolerance to structural changes. Interestingly, our results show that this is also the case for functionally important residues. However, in some cases natural mutations can disrupt the stable Ig topology, leading to the rare but severe disease AL amyloidosisH domains is a decisive, so far underappreciated factor. Especially concerning the observed MAK33 VH instability, the outcome of grafting experiments with stable human consensus sequences was interesting. The grafting constructs revealed that CDRs, in addition to antigen binding, affect variable domain structure strongly. This is especially true for CDR-H3  and slightly increases domain stability. The data for the grafting constructs show a very different picture for the VH and VL domain. For VL, the CDRs seem to be important for structure and VH/VL association but concerning antigen binding, also within the 1DH5 framework, the affinity is wt-like. The VH domain, though, did not exhibit such a strong CDR dependence for VH/VL association, only the framework had a slight effect. However, antigen binding was almost not detectable when grafting the CDRs to the 1DHU framework. Since exchanging only CDR-H3 of the VH MAK33 domain leads to a severe impairment of the antigen binding, this indicates a crucial role of the VH domain and especially the CDR-H3 in this process. An explanation for these observations could be the mentioned differences between MAK33 and 1DHU VH CDR-H3. Interestingly, grafting either the CDRs or framework from the human consensus sequences on MAK33 VL or VH, always led to an increase in stability. This might be an important aspect for CDR selection and the interplay with domain stability. Previous studies applying CDR graftings for antibody humanization approaches showed the importance and complexity of the influence of specific framework residues in the context of antigen binding and stability improvement51. Here we could show that vice versa, CDRs themselves can be considered as a crucial determinant of stability.It seems that during antibody biogenesis the effect of CDRs on the stability of VH3 Table . Compari50. Interestingly, we found both for the wild type but also for the mutants that regions involved in antigen binding change the flexibility pattern upon complex formation. Hence, the VL/VH association appears to lock some of the antigen binding loops into distinct conformations and associated flexibilities. This is in line with the observed subtle influence of some interface residues on antigen binding affinity despite the location distant from the antigen binding region. In some cases also increased solvation at the interface was observed. This was especially the case for the mutations with the most impaired VH/VL association . The presence of water molecules at the interface reduces intermolecular contacts between protein partners by giving the interface an increasing non-specific character. Thus, the experimentally observed changes in binding affinity and stability of the mutations are due to a combination of effects. A quantitative correlation with the experimentally observed change in antigen binding affinity due to the mutation is, however, not observed. It should be emphasized that this is also not expected since binding to the antigen is affected by each CDR loop differently and changes in loop mobility can in principle affect antigen binding affinity in an unpredictable way.Our MD simulations for the MAK33 variable domains indicate that the mutations altered conformational fluctuations of the isolated domains which cause structural and mobility changes at the binding interface. The non-optimal packing at the protein-protein interface leads to increased fluctuations at the interface which is also manifested in a reduction of interactions (reduced binding affinity) and also to fluctuations in the antigen binding loop regions which can reduce the binding affinity for antigens, in agreement with previous findings where it was shown that subtle changes in the interface can affect the affinity for the antigenH/VL association and the affinity for the antigen. The interplay between interface interactions and CDRs turned out to be complex with mutual influences on VH/VL association and antigen binding.Taken together our data indicate that multiple determinants regulate the VUnless otherwise stated, all experiments were carried out at 25 °C. Measurements were performed in 50 mM sodium phosphate buffer at pH 7.5H and VL were cloned into pET28 A  with NcoI and HindIII  and expressed in E. coli BL21 star . The transformed cells were grown in LB medium containing kanamycin at 37 °C until an OD600 of 0.6-0.8 was reached. The expression was induced by the addition of 1 mM isopropyl β-D-thiogalactopyranoside (IPTG). After 12 h, cells were harvested, and preparation of inclusion bodies was carried out as described previously23. The purification was performed according to the procedure described for the VL domain24. Protein purity was checked by SDS-PAGE  according to the manufacturer’s recommendations. Primers were ordered from MWG Operon . Intact protein was verified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.CD measurements were carried out using a Jasco J-720 spectropolarimeter  equipped with a Peltier element. Far-UV CD spectra were measured using 10 µM protein in 1.0-mm quartz cuvettes between 260 nm and 198 nm and near-UV CD spectra between 320 nm and 260 nm using 50 µM protein in 5-mm quartz cuvettes. The spectra were accumulated 16 times and buffer corrected.H and 2 nm and 5 nm for VL, respectively. All measurements were performed with 1 µM protein in a 1-cm quartz cuvette. The samples were incubated overnight at 20 °C at the different GdmCl concentrations prior to measurements.For denaturant-induced unfolding transitions, structural changes were monitored by fluorescence spectroscopy at 355 nm. Excitation wavelength was 280 nm and slit widths were 1 nm (excitation) and 3 nm (emission) for V52.Data evaluation was performed with Origin 8 G ; for GdmCl-transitions a two-state model was appliedH/VL association was measured by SPR experiments, performed with a Biacore ×100 . The wild type domain was immobilized on a CM5 chip by amine coupling chemistry. Subsequently, multi cycle runs with titrations of the mutant domains, ranging from 0–200 µM, were measured at 20 °C with an injection time of 60 seconds and a constant flow rate of 10 µl/min. After each cycle, bound protein was removed with 2 M NaCl regeneration solution.VL or VH with a C-terminal Flag-tag for detection. After the addition of 90 µl reaction mix I, the sample was incubated in a streptavidine-coated microwell plate to immobilize human biotinylated creatine kinase. Incubation was performed for 45 minutes with constant agitation at 20 °C or 10 °C for VL and VH mutants, respectively. Afterwards, the samples were washed with sterile pure water for three times. Then 100 µl/well of reaction mix II were added together with the detection antibody for the Flag-tag coupled to horseradish peroxidase in a 1:15.000 dilution. Afterwards, the samples were washed three times with water again and then 100 µl of reaction mix III was added. The product of the enzymatic reaction was monitored at 405 nm in a GENios plate reader  for 0.5–3 hours until a plateau was reached.Binding of the variable domains of MAK33 to the antigen creatine kinase was analyzed by ELISA. Assay components and microwell plates were from Roche . Samples were prepared in 10 µl volume. Different mutants were tested within a concentration range of 100 nM up to 50 µM against wild type VL/VH complexes as well as individual VL and VH domains were obtained by extracting the corresponding coordinates from the crystal structures 1FH550. Missing residues were added/corrected using the program PyMol50 with a final sequence corresponding exactly to the wild type sequence used in the experiments. Model start structures of all mutants were generated based on the wild type structure with residue substitutions generated in silico. All Molecular Dynamics (MD) simulations as well as the analysis of root-mean square deviation (RMSD) and fluctuations (RMSF) were performed using the Gromacs4.6 package54 in combination with the AmberSB99_ILDN force field55. Proteins were solvated in dodecahedral boxes including explicit ions (Na+ and Cl−) and explicit (TIP3P) water molecules56. The simulation systems were first energy-minimized  followed by heating up to 300 K at a constant volume with position restraints on the protein. Subsequently, a pressure equilibration at 1 bar with position restraints on the protein was carried out. All production simulations were performed at a temperature of 300 K and a pressure of 1 bar and extended to 100 ns. Root mean square deviation (RMSD) and root mean square fluctuations (RMSF) with respect to the mean structure were calculated with g_rms and g_rmsf modules of Gromacs. Snapshots were created using VMD57.Start structures of MAK33 VThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Information