AIDA1 links persistent chemical signaling events occurring at the neuronal synapse with global changes in gene expression. Consistent with its role as a scaffolding protein, AIDA1 is composed of several protein-protein interaction domains. Here we report the NMR structure of the carboxy terminally located phosphotyrosine binding domain (PTB) that is common to all AIDA1 splice variants. A comprehensive survey of peptides identified a consensus sequence around an NxxY motif that is shared by a number of related neuronal signaling proteins. Using peptide arrays and fluorescence based assays, we determined that the AIDA1 PTB domain binds amyloid protein precursor (APP) in a similar manner to the X11/Mint PTB domain, albeit at reduced affinity (∼10 µM) that may allow AIDA1 to effectively sample APP, as well as other protein partners in a variety of cellular contexts. Neurons receive chemical signals through a collection of over four hundred proteins that are organized into a network termed the postsynaptic density (PSD) AIDA1 derives its name from the ability to bind the carboxy terminal cytoplasmic region of amyloid precursor protein (APP), widely implicated in the development of Alzheimer’s disease. AIDA1 isoforms demonstrate differences in subcellular localization, affinity for APP and effect on the processing of APP to the Aβ40 nonpathologic fragment Previously, we determined the NMR structure of the AIDA1 SAM domain tandem and demonstrated that a nuclear localization signal was sequestered at the interface of the two domains Our initial attempt to perform NMR structural studies on the AIDA1 PTB domain were impeded by poor solubility regardless of solution conditions chosen. Expression of the PTB domain from Odin (81% identity), presented an even worse case, as this protein fragment could not be refolded from inclusion bodies. A strategy that we pursued to improve the solubility of the AIDA1 PTB domain by involved the progressive substitution of aromatic amino acids that were predicted to be solvent exposed.NdeI and EcoRI restriction sites and was subsequently inserted into pET28a (Novagen). The expressed protein contained an amino terminal 6xHis tag and intervening thrombin site. Other PTB domain fragments that lacked either the N-terminal 6xHis tag or the entire affinity tag along with 16 additional unstructured residues were also as insoluble as the fragment chosen for this study. To align the PTB domain described in this study with numerous AIDA1 isoforms, S1 in the PTB domain structure corresponds to S1045 in AIDA1b, the longest isoform. A one-liter fermentation in a minimal medium containing 1 g of 15NH4Cl and 4 g of 13C-glucose was sufficient to produce 5–10 mg of purified protein. Purification was achieved by Nickel-NTA affinity chromatography (Qiagen) and gel filtration chromatography on a S-100 HR 16/60 size exclusion column (GE Biosciences). Final buffer conditions were 20 mM Na-phosphate, pH 7.8, 0.15 M NaCl, 0.05% (w/v) NaN3. Five single aromatic-alanine substitutions  were produced from pET28-AIDA1-PTB using a service provided by Genscript . A 6xHis tagged PTB domain variant containing all five substitutions (PTB5M) was produced by DNA2.0  by direct gene synthesis in the expression vector, pJExpress401 (T5 promoter plus kanamycin resistance). A 6xHis-tagged, APP-peptide (GYENPTYKFFE) fused to the amino terminus of the AIDA1 PTB5M mutant with an intervening thrombin site was also synthesized by DNA2.0 in pJExpress401.A gene fragment encoding the PTB domain (aa. 1043–1195) of human AIDA1b was PCR amplified with 15N-HSQC spectra were used qualitatively. From experience, the wild type AIDA1 PTB domain was soluble for a least one day at room temperate at a concentration of 0.15 mM thereby permitting experiments to be performed but to the extent of a structure determination. Each aromatic-alanine substitution mutant was concentrated to 0.15 mM, assessed by NMR and then concentrated until increased resonance line broadening was observed or there was apparent turbidity.Since the objective of the aromatic-alanine substitutions was to improve solubility for a structure determination, m) was determined by heating samples from 20–90°C at 2°C/min and monitoring ellipticity at 222 nm.Far UV circular dichroism (CD) spectra were acquired with a Jasco J-810 instrument at a protein concentration of 50 µM using a rectangular cell with a 0.1 cm path length. Spectra were recorded from 260–200 nm with a scan rate of 50 nm/min and a 1.0 nm bandwidth. A midpoint denaturation temperature / was calculated by direct fitting the titration curves with a standard two-state relationship using proFit 6.2 (Quantsoft).Fluorescein isothiocyanate (FITC) labeled peptides spanning portions of APP were produced and purified by CanPeptide  for fluorescence anisotropy based binding studies at 25°C using an Agilent Eclipse spectrophotometer equipped with a manual polarizer accessory. Buffer conditions were similar to those used for NMR spectroscopy. Measurements were made under identical conditions and averaged. Anisotropy was calculated from the relationship A set of 12-mer peptides on a 150×100 mm cellulose membrane in a 10×30 array were synthesized using the SPOTS method 15N-edited HSQC spectra of the wild type PTB domain, mutants and protein-peptide complexes were acquired at 30°C on a Varian 600 MHz NMR spectrometer equipped with a salt tolerant cold probe. The use of a low protein concentration (0.10–0.15 mM) permitted assessment of all protein fragments regardless of intrinsic solubility. Chemical shift assignments on a uniformly 15N, 13C labeled sample of PTB5M at 0.8 mM were obtained using a conventional heteronuclear, triple-resonance strategy that incorporated non-uniform sampling for improved resolution and sensitivity. Backbone directed experiments: HNCACB, CBCA(CO)NH, HNCO, HNCACO, side chain directed experiments: H(C)(CO)NH, C(CO)NH, and 13C/15N-edited NOESY spectra were acquired on a Bruker Avance 900 MHz spectrometer equipped with a cold probe. Side chain HCCH-TOCSY, and aromatic HB(CBCG)CD, HB(CBCGCD)CE were acquired at 600 MHz. Protein solutions contained 10% D2O with the exception of the 13C-edited NOESY dataset in which the PTB5M sample was buffer exchanged into >95% D2O before data acquisition. Datasets were processed with NMRPipe From an initial set of 500 structures calculated with CYANA 3, the top 20 structures were selected with no NOE violations >0.3 Å and no torsion angle violations <5°. This ensemble was then subjected to additional refinement in explicit solvent with a Python script (wrefine.py) supplied with XPLOR-NIH 2.30. The top 15 structures according to lowest refinement energy was deposited as an ensemble in the Protein Data Bank with the accession code 2M38. The ensemble was aligned using MOLMOL 2K1 Cα RMSDs and alignments between the AIDA1 PTB domain and related proteins were performed with PDBeFold Starting from the AIDA1 PTB5M structure and APP peptide ligand placed in analogous position to that observed in the X11 PTB domain crystal structure Prior to the structure determination, a molecular model of the AIDA-1 PTB domain was made with HOMA m of the mutants was comparable to the wild type PTB domain suggesting that the alanine substitutions did not destabilize the fold. Like the wild type PTB domain, the Y6A and Y131A single mutants could only be concentrated to 0.1 mM before precipitation was observed. The remaining single mutants – F16A, F24A and Y70A – could be concentrated up to 0.5 mM; however, HSQC spectra at these concentrations suffered from line broadening and missing resonances. In contrast, the PTB5M mutant was very soluble at 0.8 mM, with line widths that were comparable to the single mutants acquired at low concentration. Thus, we observed a synergistic effect when multiple aromatic amino acids were substituted with alanine.As shown in 15N-edited HSQC spectrum of the bound PTB domain (∼120) versus the free PTB domain (∼131) suggesting that ligand and binding cleft were severely line broadened beyond detection .The impact of the APP ligand on solubility was also investigated. The APP ligand was added exogenously, as a 17-mer peptide and endogenously, by appending the sequence to the amino terminus of the wild type PTB domain. Tethering a peptide ligand to a protein is a useful approach to to shift binding kinetics from biomolecular to unimolecular and ensure stoichiometric binding. In either case, addition of the APP ligand enhanced thermostability by 8°C but did not affect solubility. A structural determination of the APP-bound AIDA1 PTB domain was not pursued because there were fewer HN resonances a 2O. Taken together, these observations suggested that A24 was the least surface exposed of the five mutants chosen for the study. Once the structure determination was completed  and an amino terminally extended 32 aa. (KD = 0.2 µM) cis and trans states of the adjacent proline (P669) that, in turn, affects the ability of Fe65 to engage its ligand. Titrations of long (APP32) and short (APP17) peptides showed no differences in binding affinity to the AIDA1 PTB domain suggesting that AIDA1, like many other PTB domains, binds an NPxY motif with a Kd of ∼10 µM  is 0.3 µM, or over 100× stronger than the AIDA1 PTB domain The K10 helix, strands β5/β6 and helix α2. Surveying down the cleft, the first tyrosine of the APP GYENPTYKFFEQ peptide is positioned such that it is predominantly making contacts with G138 and F141 in α2 Analysis of the ensemble of peptides bound to the cleft from the Rosetta based docking simulation identifies an almost equal population of rotamers that place the tyrosine in an analogous position to what is depicted in the X11-PTB/APP complex. The alternative rotamer would contact I134 and L135 in α2 of the AIDA1 PTB domain. The second tyrosine of the APP GYENPTYKFFEQ peptide is contacted by a disparate set of amino acids among AIDA1, X11 and Fe65. In AIDA1, these residues are N91 in 310 helix and K110 in β6. In X11 and Fe65, there is at least one supporting hydrophobic residue. The first of two consecutive phenylalanines in the APP GYENPTYKFFEQ peptide is supported by a tyrosine in all three PTB domain compared (Y145 in AIDA1).A comparison of the binding clefts of the AIDA1, X11 and Fe65 PTB domains is shown in D, and consequently a higher off-rate, would permit more ‘handshaking’ or sampling of potential protein partners to occur.A 12-mer SPOT peptide array  AIDA1 PTB5M protein and the (b) AIDA1 PTB5M protein with an APP binding sequence (GYENPTYKFFE) appended to the N-terminus along with a linker sequence (TLRPPNEATALQ) derived from the native AIDA1 protein. Both protein concentrations are 0.8 mM.(PDF)Click here for additional data file.Table S1A complete list of the 12-mer peptide sequences on the APP peptide array presented in .(DOCX)Click here for additional data file.


Recent genetic, biochemical and structural studies have established that eukaryotic-like Ser/Thr protein-kinases are critical mediators of developmental changes and host pathogen interactions in bacteria. Although with lower abundance compared to their homologues from eukaryotes, Ser/Thr protein-kinases are widespread in gram-positive bacteria. These data underline a key role of reversible Ser/Thr phosphorylation in bacterial physiology and virulence. Numerous studies have revealed how phosphorylation/dephosphorylation of Ser/Thr protein-kinases governs cell division and cell wall biosynthesis and that Ser/Thr protein kinases are responsible for distinct phenotypes, dependent on different environmental signals. In this review we discuss the current understandings of Ser/Thr protein-kinases functional processes based on structural data.  The involvement of eukaryotic-like serine/threonine kinase (STPKs) during cell division and cell wall biosynthesis is well documented ,2. STPK  xanthus ,13, but alactiae , S. pneueumoniae -17, S. ppyogenes ,19, S. m. mutans , and Bacrculosis .STPKs belong to the protein kinase family named as one-component signal transduction systems. Unlike the two-component systems, which are composed of two dedicated proteins (a sensor and a regulator), one-component systems combine both sensing and regulating properties . UsuallyActing both as sensors and regulators, STPKs typically share a modular structural organization, in which the sensing domain is extracellular and is connected to an intracellular kinase domain by a transmembrane linker . SequencM. tuberculosis (Mtb) has become one of the most studied STPKs [Mycobacteria and other Actinomycetes encode several eukaryotic-like kinases (namely PknA-L). Recently, PknB from ed STPKs -28. Alsoed STPKs . Due to ed STPKs -32. Thised STPKs ,34.2During the cell cycle and in preparation for division, bacteria replicate their DNA and segregate the newly formed chromosomes. A division septum then assembles at a predetermined site between the chromosomes, the cell constricts and ultimately, the mother cell splits into two identical daughters due to septum degradation -37. A maE. coli, cell division takes place at the mid-cell after the chromosomal replication and segregation into two daughter nucleoids. After the completion of chromosome segregation, the division process begins with the formation of the septal ring, called Z-ring, a polymer of the tubulin-like protein FtsZ [E. coli and B. subtilis, inhibitory mechanisms mediated by either the Min system [Bacterial cell growth and cell wall biosynthesis are mediated by a collection of proteins whose action is tightly coordinated at the level of septal ring . In E. cein FtsZ . FtsZ isein FtsZ ,41. The ein FtsZ . The rinein FtsZ . In the n system -46 and tn system ,47,48 haE. coli, only few hydrolases are known in mycobacteria, which possess a unique envelope structure with additional layers of arabinogalactan and mycolic acids [B. subtilis [E. coli [ripA depletion strains in M. smegmatis exhibit a decreasing growth and an abnormal phenotype, consisting in branching and chaining bacteria [1). Indeed, the crystal structure clearly reveals a zymogenic nature of RipA, a finding which is confirmed by cell wall degradation assays [Septal PGN is initially shared between daughter cells and must be degraded by PGN hydrolases to complete the division process. Whereas as many as 18 hydrolases are known to be involved in septum cleavage of ic acids ,49. Cellic acids , similarsubtilis  and Spr [E. coli . RipA habacteria . Crystaln assays . Interesn assays , a key cn assays -58. Furtn assays . This syn assays . It is t3pknA and pknB are organized in an operon that encodes other essential proteins involved in cell shape (Wag31) and envelope biosynthesis  [pknB or pknA genes alters cell phenotypes in different mycobacterial strains. In particular, mycobacterial cells in which pknB o pknA gene transcription was partially inhibited are highly elongated. These morphological changes have provided the evidence that these two kinases are key regulator of active cell replication and cell shape in mycobacteria [Cell division and cell wall synthesis are closely linked complex phenomena and play a crucial role in the maintenance and regulation of bacterial growth and virulence . ExcelleA, PbpA)  and whosA, PbpA) . Furtherbacteria . The role of PknA in regulating cell division in mycobacteria was also confirmed in another study showing modulation of FstZ activity by PknA . FurtherS. pneumoniae and B. subtilis possess only one and two STPKs, respectively. Despite these differences, bacterial STPKs sequences are more similar to each other than to their human homologues, with which they share low sequence identity (<30%) [S. pneumoniae and B. subtilis cell division. The kinase StkP from S. pneumoniae is essential for bacteria survival and virulence [B. subtilis, the kinase PrkC is responsible for resuscitation from dormancy induced by the presence of PGN fragments in the bacterial milieu [The availability of complete genome sequences has confirmed the presence of genes encoding PknB-like proteins in a broad range of gram-positive bacteria, whose genes number differs greatly from that observed in mycobacteria and other Actinomycetes. For example, y (<30%) . Recent irulence  and, simirulence , it locairulence . In B. sl milieu . The wid42). The frequent occurrence of trans-membrane kinases (TM-kinases) in most bacterial species is in line with their twofold role as receptor like kinases involved in direct interaction with the extra-cellular ligands [2B), whose strong conservation in STPKs suggests a key role in conserved signaling pathways. Crystallographic studies of STPKs in the last decade have helped understanding key mechanisms of regulation and substrate recognition (Table 1). Twenty-six structures of portions of STPKs have been currently deposited with the Protein Data Bank, most of them of the intra-cellular kinase domains (Table 1).Bacterial STPKs reveal high diversity in their domain organizations. A significant number of them contain trans-membrane spanning segments, different than eukaryotic Ser/Thr kinases . On analogy with eukaryotic kinases, these structures were defined as exhibiting a “closed” conformation, due to the relative orientation between both the N- and C-terminal lobes. A critical role in the control of the activity of protein kinases as well as in the recognition of their substrates is played by the activation loop (residues 164-177 in PknB). Furthermore, the hexapeptide 18-GFGGMS-23 forms a loop  in which the main chain amides of the glycines coordinate the phosphates.Two pioneering crystallographic studies of bacterial STPK kinase domain (KD) described complexes of PknB KD with two different ATP analogues ,28. Thes3). These similarities support the conservation of mechanisms of nucleotide binding and catalysis in both bacterial and eukaryotic STPKs [These structures have for the first time shown the conformation similarity of bacterial STPKs with eukaryotic Ser/Thr kinases. Indeed, conserved structural features comprise the enclosure of the N- and C- terminal lobes around the nucleotide, the P loop and the catalytic loop in which residues involved in phosphate transfer are located  by PknB [Given to the broad range of crucial cellular processes in which STPKs are involved, these proteins represent strong candidates for the development of novel drugs. Indeed, partial depletion of ed cells . The low by PknB ,82. ThisGarA Rv18 by PknB GarA Rv18 by PknB 6Despite the proven key role of STPKs in regulating environmental responses in bacteria and therefore the importance of the extracellular portions of these proteins (sensor domains) in sensing environmental changes, a molecular explanation for this process is still under debate. Also, only limited structural information on sensor domains is available. 4A). The authors suggested that regulatory ligands could bind the central part of the PknD β-propeller, denoted as the “cup”, and that binding could change the protein quaternary structure [The extracellular domain of the STPK PknD from Mtb was shown to form a highly symmetric six-bladed β-propeller . An overall linear shape of PknB extracellular region was determined by combining the NMR structural description of three overlapping portions PknB  with small-angle X-ray scattering (SAXS) experiments of the whole extracellular PknB . Parallel studies have led to the structure determination of two STPK PASTA-domain containing extracellular regions, PknB and PrkC from the human pathogens Mtb and of ectively ,71. PknBS. aureus is predicted to have a Ser/Thr kinase domain (residues 1-270), a region of unknown structure and function (residues 271-377) that includes a transmembrane helix (residues 349-373), and an extra-cellular region . The crystal structure of the extracellular portion of PrkC revealed that it consists of four consecutive domains, arranged sequentially such that only neighbouring domains interact with each other . Consistent with PknB solution structural studies [4C) [2B)[4C) [E. coli pilus subunit PapG, PrkC Ig-like domain may be involved in peptidoglycan binding [PrkC from ies [4C) . Interes[4C) [2B). Althoug[4C) [2B) revealed[2B)[4C) . A compa[2B)[4C) -88. On a binding ,85. Cons binding ,85.7B. subtilis, is essential for resuscitation from dormancy induced by muropeptides [B. subtilis spores germinate in response to DAP-type (diaminopimelic acid) muropeptides, which constitute B. subtilis cell wall, but not in response to L-Lys type muropeptides. This finding suggested that extra-cellular domains of PrkC exhibit specificity of muropeptide binding. However, the ability of muropeptides to physically bind the extracellular region of the protein was only very recently assessed [With the discovery that PrkC, a PASTA domain-containing STPK kinase from peptides , it has assessed ,89. B. subtilis [B. subtilis cell walls [et al. further showed that PknB is preferentially localized to the septum than to the cell poles, the sites of active PGN synthesis in mycobacteria, and that the PASTA domains of PknB are required for its localization [B. subtilis, whereas L-Lys-type muropeptides do not [B. subtilis to discriminate between DAP- and Lys-type muropeptides in bacterial revival [B. subtilis bacteria, which possesses a DAP-type PGN, can cross-talk and trigger resuscitation by its own cell wall turnover [In the last year, two studies have investigated the quantitative binding of muropeptide fragments to the extracytoplasmic regions of PknB from Mtb  and PrkCsubtilis . These sll walls ,89. Mir lization . Consists do not . A furths do not . In this revival . Using t8Streptococcus pneumoniae, which contains two C-terminal PASTA domains, each of them consisting of an alpha helix and three beta strands[PASTA domains exist in penicillin-binding proteins . The fira strands. A furtha strands. In thisa strands. The stra strands,89. Howe9B. subtilis [S. aureus [3) [The kinase domain of STPKs is highly homologous among several bacterial species. Common to PknB , PrkC frsubtilis  and PrkC. aureus , the kinreus [3) ,96. Ther4) containing either three (PrkC) of four (PknB) PASTA domains [S. pneumoniae, which suggested an antiparallel arrangement of the PASTA domains. On the other hand, the observed linear organization of PASTA domains is fully compatible with a muropeptide-dependent dimerization mechanism underlying STPK activation  [B. subtilis [in vitro [et al. [Four structural descriptions of STPK sensor domains are hitherto available. The sensor-domain structures of PknD and PknH of Mtb adopt globular structures ,32 where domains ,30. Thes domains , based o ,89. Howesubtilis  were proin vitro ,30. This [et al.  stating  [et al. .Commonly used drugs target structural features and metabolic characteristics of prokaryotes that are significantly different from those in eukaryotic cells. Drugs used to treat bacterial diseases can be grouped into categories based on their modes of action. In general, these drugs inhibit cell wall synthesis ,98, protThere is increasing evidence that STPKs are key mediators of developmental changes and host-pathogen interactions in bacteria ,104. Whifil rouge between cell wall hydrolysis, a process connected with both bacterial growth and resuscitation from dormancy [via post-translational modifications.The recent structural information on STPKs has provided insights into the involvement of STPK kinases in cell division processes. Indeed, the modular and linear organization of PASTA-domain containing STPK sensors and their ability to bind muropeptides ,30,71,89dormancy ,72,105,


Recent  Based on the finding that a single CaV can gate the fusion of an SV, our previous work predicted that the calcium sensor must be within the high-Ca2+ domain of the SV and, hence, physically attached or “tethered” to the channel (At fast synapses neurotransmitters are released by the fusion and discharge of synaptic vesicles (SV) at transmitter release sites within the active zone (AZ). Action potentials that invade the terminal open voltage gated calcium channels (CaV) and admit Ca channel . While i channel . leaner mice, that express a CaV2.1 truncated C-terminal (**P domain) in the distal third of the C-terminal  2138 to 2357 (in chick), a region we term C3. Our quantitative immunocytochemical analysis . The GST-tagged fusion protein C3Prox (aa 2138–2299) PCR fragment was subcloned into a pGEX-KG  vector and GST-FLAG-tagged proteins  into a pGEX-KG expression vector with a sub-cloned FLAG tag. The DNA sequence in frame was confirmed by sequencing after transformation into DH5α competent cells (Invitrogen). C3Strep was used on bead whereas GST fusion proteins were eluted using 20 mM reduced Glutathione (Bioshop), in 50 mM Tris-HCl pH 8.0. GST fusion proteins were concentrated using a 10K Microcon and stored in PBS .For C3rep see: , a PCR fStandard Western blots and pull-down assays were as described .2+ clamped to 10 nM  throughout. The beads were then washed and proteins solubilized for Western blot analysis. To test for SV-PD we selected integral proteins that could be used as markers for SV capture: SV2A, synaptotagmin (STG) and vesicle-associated membrane protein-2 (herein VAMP). Positive SV-PD capture was concluded if bands for two of these integral SV proteins were obviously darker than the vector control. Densitometry was also used to quantify individual protein bands for a particular data set, as described followed by statistical analysis (see below) for whether the mean was significantly above zero.The novel SV-PD method is described in detail in a recent report . Briefly**P region (PQTP in chick) and the C terminus PDZ ligand-domain region (DDWC) that included: RQLPQTPL ; HEADEDDWC ; DDWA (aa 2354–2357), and HEADE . Peptides were made as a 10 mM stock solution in HB or RIPA buffer, and 1–1.2 mM peptide was added to each sample for pull-down experiments.We synthesized mimetic or control peptides for the PThis method is described in detail in a recent paper . Imaging2+ is buffered to 0.1 μM . The ghosts were retrieved by a 30 min 20,000 × g spin and were resuspended in a 0.2 M sucrose HB with 1 mM EGTA. The suspension was then loaded onto a second (0.4 M/0.6 M/0.8 M/1.0 M sucrose) gradient and the ghosts were collected from the 0.8/1.0 M interface. Ghosts were pelleted at 16,000 × g for 1 h. The pellet was fixed, dehydrated, embedded, and sectioned for EM as described (Synaptosomes used for SSM ghost preparation were prepared in HB with 1 mM EGTA in place of 2 mM EDTA. SSM ghosts were prepared by hypotonic lysis of SSMs as for SV isolation except using a lysis buffer in which free Caescribed . ImagingV), as appropriate, controls for all test lanes and rejecting protein bands associated with contaminated control lanes (see t = 0). For SV-PD experiments, however, the primary question is discrete: whether or not the SV protein was detected, not its amplitude. To test this question we used a binomial approach. Individual immunoblots were scored as positive for the capture of a particular SV protein marker if the test band was unequivocally darker than its control, GST- or StrepV-only lane. A large data set with the control, native-sequence fusion protein, C3Strep, was used to predict the expected SV capture probability. This was then used to test the capture frequency observed with modified conditions  by binomial analysis (see text and Table 2). We scored individual SV-PD experiments as positive for SV capture based on the detection of at least two of our three integral SV protein markers  and tested if these were significantly different from the capture rate with C3Strep by binomial analysis.The study of cell membrane components in detergent-free conditions is prone to non-specific binding and high background protein staining. This challenge was dealt with primarily by running paired GST or strep vector (Strepanes see  and we tStrep) comprising the distal 220 aa of the channel C-terminal which are known to bind to the PDZ ligand domain , and SV capture was assessed by Western blot for signature integral proteins . In contrast to the full-length C3 fusion protein, C3Strep . Since the primary question in this study was whether SVs were captured and not how much protein was recovered, we scored each experiment as positive or negative for each protein and then used a discrete statistical analysis method. We used our large dataset with C3Strep to establish a predicted capture frequency for the normal distal C-terminal: n/N, where n is the number of experiments with positive recovery and N the total number of experiments. Binomial analysis showed that the SV capture with C3Prox was significantly less than with C3Strep, based either on individual SV marker proteins (Table 2A) or SV-PD . Thus, we concluded that a key SV binding site must be located within the missing 58 aa distal region of the C terminus.We recently used the SV-PD assay to demonstrate that SVs can be captured by a fusion protein (C3n MINT-1  and RIM d domain . We nextMobyle predictor). Secondary structure informatics (Phyre2 bioinformatics server) identified few alpha helices, beta sheets, or other ordered regions. Thus, allowing 0.36 nm/aa (the length of the polypeptide backbone) for disordered regions and 0.54 nm/3.6 aa’s for α-helices, 1/3rd of the disordered length for β-sheets (the length of these cannot be estimated with confidence but they were in any case minor) we can predict a maximum (limiting) length of up to 200 nm. Obviously other folding is possible and the terminal could be shorter but, nonetheless, this “back-of-the-envelope” calculation confirms that it is at least theoretically possible that an SV tethered to the end of the C-terminal could yet be located as far as ~4 SV diameters from its docking site within the AZ.The experiments above identify an SV binding site within the last 58 aa of the channel C-terminal. This terminal extends 641 aa from the cytoplasmic face of the transmembrane helix . However, we reasoned that if the untethered SVs and other contents of the SSM could be passively discharged, SVs that were physically attached to the surface membrane should remain. Such a model was at hand: SSM rupture and content-discharge, achieved by osmotic shock, is a routine step in our SV isolation protocol. We isolated the resulting nerve terminal membranes, “SSM ghosts” , ranging from intimately attached, and presumably docked  to relatively remote within the surrounding cytoplasm . By far the majority (>70%) of the cytoplasmic SVs in the AZ region could be seen to be linked to the AZ via fibrous processes, defined as morphological tethers . We measured the length and number of SV tethers, tracing the course of the fiber to the AZ or, if the SV was close to the surface membrane so that connections could not be seen, we simply measured the inter-membrane distance . A cumulative frequency histograms plot  indicates that 95% of these morphological tethers are up to 100 nm long and 99% are up to 175 nm, which we take as our estimate of the upper limit of the morphological tether lengths. Interestingly, a plot of the number of tethers against length indicated that the more distant SVs were linked to the AZ by a single tether  whereas SVs that were closer, less than ~45 nm , often exhibited multiple links .Active zones were identified primarily by two standard criteria: darkening of the surface membrane and by the residual “scab” of postsynaptic apparatus that frequently remains attached . Fuzzy fStrep from SSM lysate  which contains both reported RIM attachment sites  domain associated with RBP binding. However, C3Prox which includes the P**P domain, but lacks the terminus PDZ ligand domain , failed to capture RIM . These results argue that RIM does not bind to the C3 region via the P**P site in our assay .Thus far our results were not inconsistent with published models of C-terminal/SV tethering. We next set out to test SV binding to the distal C-terminal and to explore the role of RIM. As noted above, RIM was pulled down by C3nt sites : the PDZWildF and C3MutantF . C3WildF is identical to the native C3Strep but has an added distal C terminus FLAG tag, a mutation that can be predicted to eliminate PDZ ligand activity  or C3MutantF . We also noted weak, but detectable RIM pull down with C3Strep from SV lysate  consistent with binding of this protein to the PDZ ligand domain  0.001 U, N = 3, p = 0.367; C3MutantF 0.03 ± 0.02 U, N = 4, p > 0.1; Figure 4B), providing compelling support for the idea that the C-terminal can bind to RIM via a PDZ interaction . Further, SV capture by the two mutants was greater than with C3Prox using a similar binomial analysis, even with the relatively low N values of the latter.Thus far we confirmed that the normal distal C-terminal C3 region can capture SVs and that RIM binds to the PDZ ligand domain. We predicted that if RIM is critical for SV tethering the mutant C3 fusion proteins should fail to capture SVs. To our surprise, as assessed by SV-PD, both C3Strep . In contrast, control experiments using H-WC pre-PDZ-ligand domain region HEADE , an inactivated PDZ ligand domain peptide DDWA (with a mutated terminus cysteine), or the P**P-domain mimetic peptide RQLPQTPL, had little effect (Table 2B). Thus, H-WC was an effective and selective competitive blocker for the channel C-terminal PDZ ligand domain. Further, the lack of effect of the P**P domain peptide provided additional evidence that the SVs are not captured via the RBP site.The mutant fusion protein approach provides compelling evidence that SVs can bind to the distal C-terminal and that this can occur via a PDZ-RIM-independent mechanism. However, there was a remote possibility that the modified fusion proteins might snare SVs by an anomalous mechanism. We therefore devised a complimentary but independent analysis based on mimetic blocking peptides. Initial tests with a short four aa C-terminus peptide of the PDZ ligand domain alone, DDWC (aa 2354–2357), were abandoned due to non-specific blocking effects on unrelated proteins. However, as tested using SSM lysates, a longer nine aa C terminus mimetic peptide, HEADEDDWC , selectively inhibited MINT-1 pull down with C3Strep . However, H-WC did not inhibit SV-PD with C3Strep as assessed by SV marker protein capture . Thus, the blocking peptide results supported the conclusion that SVs can bind to the C-terminal independently of RIM or the PDZ domain.Consistent with the MINT-1 result and its interaction by PDZ binding, H-WC inhibited RIM pull down by C3in vitro binding experiments accurately reflect the molecular basis of CaV2.2 C-terminal-based tethering, we can predict that in the intact nerve terminal depolarization-gated SV turnover should persist in the presence of PDZ ligand domain or P**P mimetic blocker peptides. This hypothesis was tested in the chick SSMs using our novel “cryoloading” method  verifies the method  to mark cryoloaded SSMs. Uptake of H-WC into the SSMs was confirmed by two methods. First, the peptide was detected in the cryoloaded, but not the unloaded SSMs on the same plate by immunostaining using our anti-long-splice distal C-terminal antibody, L4569 . SSMs cryoloaded with H-WC alone or with inactive botulinum toxoid  exhibited depolarization/Ca2+ styryl dye uptake (FM4-64) uptake as observed in controls. In contrast, a large fraction of SSMs that were cryoloaded with H-WC spiked with Bot A-LC  failed in vitro . This argues against RIM linking to the C-terminal via RBP. Lastly, we did not detect any inhibition of SV recycling in situ when terminals were loaded with a mimetic P**P-site blocking peptide , a peptide that also had no detectable effect on SV-PD by the normal C3 fusion protein C3Strep (Table 2B). These results suggest that marked changes in transmitter release physiology observed to occur after deletion of RBP  had no detectable effect on SV recycling, as monitored by FM uptake, supporting the above conclusion and arguing against a critical role for the PDZ-ligand domain in SV turnover. As for other channels . The finding that there are a reduced number of short-range tethers in RIM knockouts (These findings implicate a novel SV tether site within the last 49 aa of the CaV2.2, proximal to the H-WC terminus. The idea that SVs are tethered to the calcium channel was predicted from the finding that SV fusion can be triggered by the opening of a single calcium channel . For thenel pore . While Cange one , to “locnockouts  raises tFigure 3H; Our model of C-terminal-based, G-tethering begs two immediate questions. First, how is the SV retracted to dock after binding to the distal C-terminal? Previous studies have identified motor proteins at the release site  that couThe 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.


Here we use a local approach, able to distinguish among the different backbone conformations within loops, Using a structural alphabet, we translated the 3 D structures of large sets of protein-protein complexes into sequences of structural letters. The shape of the secondary structures can be assessed by the structural letters that modeled them in the structural sequences. The distribution analysis of the structural letters in the three protein compartments  reveals that secondary structures tend to adopt preferential conformations that differ among the compartments. The local description of secondary structures highlights that curved conformations are preferred on the surface while straight ones are preferred in the core. Interfaces display a mixture of local conformations either preferred in core or surface. The analysis of the structural letters transition occurring between protein-bound and unbound conformations shows that the deformation of secondary structure is tightly linked to the compartment preference of the local conformations.The conformation of secondary structures can be further analysed and detailed thanks to a structural alphabet which allows a better description of protein surface, core and interface in terms of secondary structures' shape and deformation. Induced-fit modification tendencies described here should be valuable information to identify and characterize regions under strong structural constraints for functional reasons. Although classical secondary structure assignment methods attribute residues to either regular or non-regular secondary structures, secondary structures borders are transitional conformations between the two and can be characterized by the structural alphabet. They are classified as loops initially but are analysed separately in the following.HMM-SA is a library of 27 structural prototypes  of four   . HMM-SA α- and β-letters). The great number of loop- and α-letters at interface compared to β-letters in homodimers and obligates complexes, as well as the greater proportion at interface of β-letters compared to α-letters in heterodimers and transient complexes, is consistent with . In the second group, [D] is under-represented at interface (highly negative inter face/surfaceZ) whereas [F] shows preference for interface. The KLd values associated with border-letters are all significant:  are the most preferred on surface and the least in core while  display the opposite behavior.The first axis of the MCA plot separates loop-letters into two groups of letters Figure :  present low proportion of proline (from <7%) while [D] appears to be particularity enriched in glycine (55%) in agreement with  are the least exposed. In particular,  correspond to straight β-strand conformations (with the large d2). Distinction between α-letters in terms of structural descriptors is not clear  and β-letters (15%). It highlights that, although secondary structures are very stable upon complexation  displays a clear preference to be deformed towards straight core-letter [A] upon interaction (the least changed α-letter: 60%) while the inversed substitution is more likely to be due to protein flexibility being as observed at interface as in surface. Similarly for β-strands, non-interface-letter [N] (the most changed β-letter: 48%) is preferentially deformed towards the straightest core-letters  (the least changed β-letters: 29% and 34% respectively). Curved surface-letter [L] (deformed in 42% of cases) appears to be deformed towards [N]. For loop-letters, non-interface-letter [D] is the least changed letter (11%) and core-letter [R] the most one (45%). The fact that the least changed loop-letter [D] corresponds to a conformation avoided at interface suggests a non-flexible conformation interfering with efficient recognition or interaction with the other protein. 27% of the interface-letter [F] are deformed. No clear preferential deformation appears between specific loop-letters but they appear to be deformed towards letters with the same compartment preference: 70% of surface-letters  are changed towards surface-letters and 75% for core-letters  are changed towards core-letters. Although the deformation tendencies at interface of local conformations associated with regular secondary structures (from curved to straight conformations) agree with their compartment preference (non-interface-letter and surface-letter are deformed towards core-letters when interface residues become buried upon complexation), the expected deformation of loops from surface-letters to core-letters is not observed. Instead deformation appears to be barely affected by solvent accessibility variation induced by the complexation with transitions between local conformations of the same compartment preference/structural characteristics. The relation between loop deformation and exposure to protein exterior is further analysed.The deformation of secondary structures is analysed by comparing the structural letter transitions from the proteins unbound to bound state. The local conformations are mainly unchanged in the three compartments, the majority of deformation occurred at interface  compared to surface 34%) and core (30%) in agreement with  between the unbound and bound states of each subunit of each complex. However, the low number of observations per complex does not allow any firm conclusions to be drawn.Example cases of protein-protein interaction are selected from the bound/unbound dataset to illustate the information that can be derived from the deformation tendencies described above. The two first examples illustrate induced-fit modifications that follow the deformation tendencies, the last four illustrate their violation.1KLU], chain A:58-72) displays a Cα RMSD of 0.26Å between its bound and unbound conformations  towards a straight one (run of [A])  in complex with the human Major Histocompatibility Complex molecule HLA-A2 ([PDB:1AKJ] chain D:228-232) corresponds to a curved β-strand (run of two [N]) in the unbound state that is deformed into a straight one ("TM") in the bound state . It ill) Figure . The five Figure . This deAll the following example cases illustrate the violation of the deformation tendencies. In these examples, it appears that the observed deformations are associated with structural constraints directly related to the function of the proteins.αGS2 helix of the TGFβ receptor type I  upon interaction with FKBP12, an inhibitor of the TGFβ pathway . The phosphorylation site of the TβR-I is located in the GS loop surrounded by the two helices αGS1 and αGS2. When FKBP12 interacts with the αGS2, the helix nestles into the TβR-I structure and the GS loop formed an inhibitory wedge that inserts into a space in the protein core [αGS2 presents a Cα RMSD of 0.56Å between the unbound and bound states that the local approach reveals to correspond to the deformation of a straight conformation encoded by a run of [A] towards a curved one encoded by a run of [a]  belonging to the α1 domain loop of the hemocromatosis protein (HFE) is deformed upon interaction with the transferin receptor (TfR) from a curved conformation (modeled by core-letter [O]) to a straight conformation (modeled by surface-letter [P]) . It corresponds to the transition from a straight (modeled by surface-letter [H]) to a curved conformation (modeled by core-letter [O]). It appears that this deformation is due to residue S100 that is pushed towards the protein interior while interacting with the partner, inducing a rotation of P102  Figure . This ex2 Figure .1FAK] chains HL,T). Although the overall Cα RMSD between the bound and unbound states indicates a strong deformation upon interaction (3.71Å), the two EFG-like modules (EGF1 and EGF2) are structurally similar with respectively 0.58Å and 1.03Å Cα RMSD and 79% and 55% structural sequence identity. The EGF1 domain rotates ≈ 180° about the linker hexapeptide (positions 85-90) compared to its position in the unbound state thanks to a single change in the main-chain torsion angles of D88 [1FAK] chain L:82-90) corresponds to the linker region and is associated with a 2.05Å Cα RMSD. It characterizes the conformational modification required for the rotation of the EGF1 module from an helical conformation modified to a loop one "AV"→"BE"  indicates a deformation from a loop to a β-strand conformation "GEE"→"MNL"  is located in the C-ter of the protein.The last example regards the light chain of the coagulation factor VIIA (fVIIa) inhibited with a BTPI-mutant , HeteroTwo more additional datasets extracted from the version 2.4 of the benchmark proposed in ,64 are ucompartments: interface, surface and core. Residues are assigned to one of the three compartments according to their percentage of relative solvent accessibilities in the disjoint bound conformation (noted chainA), in the two-chain complex forming the interface of interest (noted interfA) and in the higher complex considering all chains described in the PDB entry (noted complexA). Core residues correspond to residues r that are buried in the core of the protein . Therefcaffolds  where thr is associated with four different fragments L1, ..., L4 where L1 corresponds to the four successive residues r - 3 → r and L4 to the four successive residues r → r + 3. Each four-residue fragment is associated with a structural letter describing its conformation, a protein structure of N residues is encoded in a sequence of N - 3 structural letters. The physico-chemical characteristics and the compartment assignment of the structural letter encoding the fragment r - 2 → r + 1 are determined according to the properties of the residue r as in  prefered in non-interface region and letter [F] preferred in interface regionClick here for fileMCA performed on β-letters for homodimers, heterodimers, obligate, transient complexes and protein chains in bound and unbound states. The first axis differenciates the surface-letters from the core-letters. Letters are similarly distributed around this axis for all the different datasets. Particularly, letters [L] and [N] are clearly associated with the surface and the non-interface region in the all seven datasets while [M] is associated with the core. The MCA plot obtained for transient complexes shows a difference for letter [T] which appears to be preferred in the non-interface region in opposite to its tendency to prefer interface in homodimers, heterodimers and obligate complexes. This contradictive behavior is less pronounced in the bound and unbound dataset.Click here for fileMCA performed on α-letters for homodimers, heterodimers, obligate, transient complexes and protein chains in bound and unbound states. Preferences of α-letters among the seven datasets are less stable than for the other structural letters. This agrees with other analysis of this study where α-letters display the weaker distribution signal and the most similar structural properties among them. Globally the first axis tends to differentiate between surface and core excepted for obligate complexes where it differentiates between interface and non-interface regions. However, the behavior of the two letters [a] and [A] are stable among the different datasets being preferentially distributed in non-interface region and in core respectively.Click here for fileDetailed evaluation of the percentage of secondary structures affected by the preferential distribution in the complete dataset. Counting of structural letters at interface, surface and core in the complete dataset. The observed (Obs) and expected (Exp) numbers of structural letters at interface, surface and core are given and the difference between the two is calculated (Diff). For each structural type, the sum of the difference is calculated to evaluate the proportion of the secondary structure affected by the preferential distribution.Click here for fileAmino acid composition of the structural letters associated with regular secondary structures. Amino acid composition at interface (white), on surface surface (grey) and in core (black) for α-letters , β-letters  and border-letters . No common amino acid specificities are observed between letters associated with identical compartment.Click here for fileAmino acid composition of the structural letters associated with loops. Amino acid composition at interface (white), on surface (grey) and in core (black) for loop-letters. Surface letters  present high proportion of proline and a small proportion of glycine and therefore present a similar amino acid composition profile to core-letter [R] than to surface-letter [U]. Interface-letter [F] present a high proportion of both residues glycine and proline while non-interface-letter [D] appears to be particularly enriched in glycine.Click here for fileDeformation matrices for surface and core compartments. Proportion matrix P  where ω is the letter in the unbound state (y-axis) and ψ the corresponding letter in the bound state (x-axis). Structural letters are separated according to their structural type with black lines, and differentiated according to their compartment preferences . Grey dotted lines separated surface loop-letters from core ones.Click here for file


The actions of prostacyclin are mainly mediated through its activation of the prostacyclin receptor or, in short, the IP. In recent studies, the cytoplasmic carboxy-terminal domain of the IP was shown to bind several PDZ domains of the multi-PDZ adaptor PDZK1. The interaction between the two proteins was found to enhance cell surface expression of the IP and to be functionally important in promoting prostacyclin-induced endothelial cell migration and angiogenesis. To investigate the interaction of the IP with the first PDZ domain (PDZ1) of PDZK1, we generated a nine residue peptide (KK411IAACSLC417) containing the seven carboxy-terminal amino acids of the IP and measured its binding affinity to a recombinant protein corresponding to PDZ1 by isothermal titration calorimetry. We determined that the IP interacts with PDZ1 with a binding affinity of 8.2 µM. Using the same technique, we also determined that the farnesylated form of carboxy-terminus of the IP does not bind to PDZ1. To understand the molecular basis of these findings, we solved the high resolution crystal structure of PDZ1 bound to a 7-residue peptide derived from the carboxy-terminus of the non-farnesylated form of IP (411IAACSLC417). Analysis of the structure demonstrates a critical role for the three carboxy-terminal amino acids in establishing a strong interaction with PDZ1 and explains the inability of the farnesylated form of IP to interact with the PDZ1 domain of PDZK1 at least in vitro.The prostanoid prostacyclin, or prostaglandin I Postsynaptic density protein 95 (PSD-95), DrosophilaDisks large tumour suppressor 1 (Dlg1), and Zonula occludens protein 1 (ZO-1). The PDZ domain typically spans approximately 80–90 residues and, structurally, is composed of six anti-parallel β-strands (βA–βF or β1–β6) sandwiched between two α-helices (αA/αB or α1/α2), with a highly conserved ‘GLGF sequence motif’ forming a hydrophobic binding pocket Scaffold or adaptor proteins play key roles in facilitating and integrating signal transduction in multicellular organisms. These proteins recruit and/or anchor their binding partners to a specific subcellular location, serving as a platform to mediate or regulate interactions between proteins involved in diverse signalling pathways +/H+ exchanger regulatory family (NHERF) and is predominantly expressed in proximal epithelial cells of the kidney and in hepatocytes, and at a lower level in other epithelial cells and endothelial cells PDZK1 is a member of the Na2, plays a central role in haemostasis, acting as a potent inhibitor of platelet aggregation and as an endothelium-derived vasodilator 386) of the human (h) IP 0 position ) or the P-2 position (Ser384) of the PDZ ligand within the human (h)IP disrupted its interaction with PDZK1. Furthermore, mutation of the ‘GLGF motifs’ within the hydrophobic binding pockets of the four PDZ domains of PDZK1 suggested that the IP can bind to PDZ domain 1 (PDZ1), PDZ3 and PDZ4, but not PDZ2, of PDZK1 in vitroThe prostanoid prostacyclin, or prostaglandin (PG) ICaaX motif (-CSLC) at its extreme carboxyl terminal cytoplasmic domain CaaX-containing proteins involves the initial isoprenylation of the target Cys through carbon (C)-15 farnesylation or C-20 geranylgeranylation followed by proteolytic cleavage, or –aaXing, of the three terminal residues and subsequent carboxy-methylation of the nascent terminal isoprenylated-Cys residue to form the fully processed farnesyl/geranylgeranyl-Cys-carboxymethyl ester CaaX motif (-CSLC) sequence may also serve as a ‘PDZ ligand’ and previous investigations have indicated that the interaction of PDZK1 with the IP is largely independent of its isoprenylation status The IP is unusual among GPCRs in that it undergoes isoprenylation or, more specifically, farnesylation within an evolutionarily-conserved in vitro with high affinity, the isoprenylated form of the receptor does not under the conditions tested.Herein, we have further characterized the interaction between the IP and PDZK1. Isothermal titration calorimetry (ITC) was used to measure the binding affinity of IP-derived synthetic peptides to a recombinant protein corresponding to the first PDZ domain (PDZ1) of PDZK1. In addition, we determined the high resolution structure of PDZ1 bound to the ‘PDZ ligand’ of the IP by X-ray crystallography. We show that while the non-prenylated form of the IP is capable of interacting with PDZ1 411IAACSLC417, ‘target peptide’); Peptide 2 consisted of an octapeptide corresponding to the carboxy-terminus of IP (K407SEAIAAC414) devoid of the 3 terminal amino acids (-aaX/-415SLC417) which would be removed by proteolytic cleavage following farnesylation 407SEAIAAC414) except that it was modified by the addition of a C-15 farnesyl group on the carboxy-terminal cysteine (C414) and a carboxy-methyl group on the terminal –COOH, thereby representing the farnesyl-Cys-carboxymethyl ester form of the IP C-terminus. Peptides 1 and 2 were synthesized and HPLC purified by the Tufts University Core Facility . Peptide 3 was synthesized by Thinkpeptides  and solubilized in DMSO. Briefly, 1.0 mM of the target peptides 1–3 was titrated against recombinant protein corresponding to PDZ1 or full length PDZK1 at concentrations of 0.03 mM in a buffer containing 150 mM NaCl, 25 mM Tris-pH 8.0 at 20°C under reducing conditions (0.5 mM tris (2-carboxyethyl) phosphine (TCEP)). Titration curves were analyzed and Kd values determined using ORIGIN 7.0 software (Origin Lab) with baseline correction. ITC experiments were performed in triplicate when binding of the peptide to the recombinant proteins was observed (peptide 1) and in duplicate when no binding was seen (peptides 2 and 3).Expression and purification of recombinant proteins corresponding to PDZ1 domain 1(PDZ1) of PDZK1 and to full length PDZK1 has been described previously 411IAACSLC417), was cloned into the pGEX-4T-3 vector and expressed in E. coli JM109 cells to produce a glutathione-S-transferase fusion protein. The recombinant protein was purified on glutathione-Sepharose 4B, released from GST using thrombin digestion and further purified by FPLC using a Superdex S75 column . The isolated, recombinant protein  contains an additional two residues  at the N-terminus that are not normally present in PDZK1. After initial screening for optimal crystallization conditions using the PEG suite (Qiagen), the PDZ1–IP chimera at 1 mM concentration was crystallized by the sitting drop vapor diffusion method at 18°C in a well containing 0.1 M MES pH 6.5 and 30% (w/v) PEG 300. Crystals were flash frozen directly into liquid nitrogen. Diffraction data were collected on beamline X29 at the National Synchrotron Light Source . The crystals belong to space group P21 with unit cell dimensions of a = 39.270, b = 68.477, c = 40.124 Å with β = 91.8°. The data were reduced and merged using the HKL2000 suite A DNA fragment encoding the chimeric PDZ1–IP recombinant protein (residues 7–106 of PDZK1 - including PDZ1 (residues 7–86), 20 residues from the region of the protein between the PDZ1 and PDZ2 domains (interdomain residues 87–106) and, fused to the C-terminus of this interdomain segment, the seven carboxy-terminal amino acids of mouse IP  with several PDZ domains of PDZK1, including PDZ1, PDZ3 and PDZ4 411IAACSLC417). The second peptide (K407SEAIAAC414) corresponded to the carboxy-terminal sequence of the IP, but did not include the most carboxy-terminal three amino acids (-415SLC417) which are classically proteolytically cleaved (-aaXed) in the mature IP following the initial isoprenylation step (407SEAIAAC414), except that the 414Cys was modified by the addition of both a farnesyl and a carboxymethyl group, thereby representing the mature, fully processed farnesyl-Cys-carboxymethyl ester form of the IP  carboxy-terminal peptide on PDZ1 (Kd of 8.2±1.3 µM) (407SEAIAAC414) (407SEAIAAC414-(Farnesyl)-O-Me) was observed (d of 60.2±14.2 µM) than that for the PDZ1 isolated domain (Binding of peptides 1–3 to a recombinant protein corresponding to PDZ domain 1 (PDZ1) and to full length PDZK1 was then evaluated by ITC. These experiments revealed the presence of a high affinity binding site for the IP (KK±1.3 µM) , but no IAAC414)  or its fobserved . Moreoveobserved , but witd domain . This co2 adrenergic and platelet-derived growth factor receptors 411IAACSLC417. In −6IAACSLC 0  which is commonly used for PDZ domain targeted peptide sequences is employed and will distinguish the target peptide residues of the PDZ ligand from those of PDZK1. We grew crystals, collected X-ray diffraction data at the NSLS  and solved the crystal structure at 1.70 Å resolution ). As has been described for target peptide binding to other PDZ domains −6IAACSLC 0) inserts into a groove formed between the β2 strand and the α2 helix, and the CBL formed by 14Lys… 20Tyr-21Gly-22Phe-23Phe-24Leu that connects the β1 and β2 strands. The backbone amides and carbonyls of the target peptide form classic anti-parallel β-sheet hydrogen bonding interactions beginning with carbonyl of 22Phe of the CBL and extending through β2 from 23Phe to 26Ile . While the crystal structure of numerous individual PDZ domains, either alone or in complex with target ‘PDZ ligand(s)’, have been reported solution  by molectructure . To facito 26Ile .0Cys makes hydrogen bonds with the amide nitrogens of 20Tyr, 21Gly and 22Phe in the CBL and, through water-mediated interactions, the carbonyls of 75Val and 76Arg of α2 and the carbonyl of 79Gly. In addition, the hydroxyl group of 20Tyr (CBL) forms hydrogen bonds with the carbonyl of 79Gly in α2 and the amide of 81Ser in β6, interconnecting multiple secondary structure elements of PDZ1. The amide nitrogen of 0Cys makes a hydrogen bond to the carbonyl oxygen of 22Phe (CBL) and the side chain fits into a deep hydrophobic pocket composed of the side chains of 22Phe and 24Leu of the CBL and 72Val, 75Val and 76Arg of α2 (−1Leu and the side chain of 76Arg (α2), the side chain of −2Ser and the side chain of 68His (α2), the amide nitrogen of −2Ser and the carbonyl oxygen of 24Leu, the carbonyl of −2Ser and the amide of 24Leu, the amide nitrogen of 26Ile and the carbonyl oxygen of −4Ala and the amide of −4Ala and the carbonyl oxygen of 26Ile (19Asn and 22Phe (CBL) with 0Cys, 23Phe (CBL) with −1Leu, 23Phe (CBL) and 72Val (α2) with −2Ser, 25Arg (β2) with −3Cys, 68His (α2) with −4Ala and 28Lys with −6Ile . There of 26Ile . There ath −6Ile . The surth −6Ile , showingThe structure provides important clues for the understanding of the ITC data and highlights the role of IP’s three carboxy-terminal amino acids (-SLC) in particular for the stability of the interaction.in vitro molecular analysis of the interaction between IP and the first PDZ domain of PDZK1 (PDZ1) revealed that only the non-isoprenylated form of the carboxy-terminus of IP is capable of interacting with PDZ1 suggesting a alternative role for the isoprenylated form, presumably anchoring IP in the plasma membrane in vitro at least. The basis of the apparent inconsistency between the two studies is currently unknown but may possibly be due to differences between the experimental approaches used and is most likely due to unexplained differences between that which occurs in vitro and in vivo in the cell. The binding affinity  of peptide 1 corresponding to the PDZ ligand of the IP to interact with PDZ1 in vitro appears to be in the same order as that of SR-BI binding to PDZ1  of PDZK1 d, 60.2±14.2 µM) than that for PDZ1 alone. These findings are consistent with the ability of the IP to bind to multiple PDZ domains within the full length PDZK1 itself PDZK1 is a multi-PDZ domain-containing adaptor protein known to play a role in the expression, localization and function of several cell surface receptors and ion channels In addition, these findings further contribute to the evidence that the binding for PDZ ligands within full-length multiple PDZ domain-containing proteins can differ substantially from that of the individual PDZ domains in isolation 20Tyr, which played a major role in the SR-BI/PDZ1 and SR-BI/PDZ3 interactions, using a complex hydrogen bond-water molecule network between the hydroxyl group of 20Tyr and the carboxy-terminal last amino acid (0Leu) of SR-BI, is not participating in the interaction with the carboxy-terminal last amino acid (0Cys) of IP. 68His (α2), which did not participate in the SR-BI/PDZ1 interaction appears to play an important role in the IP/PDZ1 interaction, forming an hydrogen bond with the side chain of −2Ser. There is a hydrophobic interaction between 19Asn and 0Cys in the IP/PDZ1 interaction which did not exist in the SR-BI/PDZ1 interaction. However, 76Arg does not appear to form a hydrophobic interaction with the IP carboxy-terminal peptide, while such interaction was taking place with the carboxy-terminal leucine (0Leu) of SR-BI. The structure also highlights the importance of the last three carboxy-terminal amino acids of IP, responsible for the majority of hydrogen bonds formation and hydrophobic interactions between IP and PDZ1, a function that could not be assumed by a farnesyl group (We recently solved the structures of the interactions between the HDL receptor SR-BI with the first and with the third PDZ domains of PDZK1 by X-ray crystallography yl group  and 5.Collectively, these findings highlight the importance of the information gathered from the crystal structure and may serve as the basis for the identification of new compounds, such as peptidomimetics, that may either selectively stabilize or disrupt the IP/PDZ1 interaction, with the ultimate goal of designing new therapeutic modalities for the treatment of some of the cardiovascular disease etiologies in which prostacyclin and its receptor, the IP, are most widely implicated.


Hence, the structural differences between individual E6 and their malleability appear, together with HPV type-specific surface exposed side-chains, to provide the structural basis for the different interaction networks reported for individual E6 proteins. Furthermore, the interaction of 51Z2 with a PDZ domain of hDlg was analyzed. Human Dlg constitutes a prototypic representative of the large family of PDZ proteins regulating cell polarity, which are common targets of high-risk HPV E6. Nine C-terminal residues of 51Z2 interact with the second PDZ domain of hDlg2. Surface plasmon resonance in conjunction with the NMR spectroscopy derived complex structure (PDB 2M3M) indicate that E6 residues N-terminal to the canonical PDZ-BM of E6 significantly contribute to this interaction and increase affinity. The structure of the complex reveals how residues outside of the classical PDZ-BM enhance the affinity of E6 towards PDZ domains. Such mechanism facilitates successful competition of E6 with cellular PDZ-binding proteins and may apply to PDZ-binding proteins of other viruses as well.The high-risk human papilloma virus (HPV) oncoproteins E6 and E7 interact with key cellular regulators and are etiological agents for tumorigenesis and tumor maintenance in cervical cancer and other malignant conditions. E6 induces degradation of the tumor suppressor p53, activates telomerase and deregulates cell polarity. Analysis of E6 derived from a number of high risk HPV finally yielded the first structure of a wild-type HPV E6 domain (PDB 2M3L) representing the second zinc-binding domain of HPV 51 E6 (termed 51Z2) determined by NMR spectroscopy. The 51Z2 structure provides clues about HPV-type specific structural differences between E6 proteins. The observed temperature sensitivity of the well-folded wild-type E6 domain implies a significant malleability of the oncoprotein  Infections are responsible for 17.8% of all cancers The HPV oncoproteins E6 and E7 of tumor-associated so-called “high-risk” HPVs prevent the differentiation of HPV-infected keratinocytes and immortalize primary, cultured human keratinocytes The HPV oncoprotein E6 has no known enzymatic activity. It interacts with numerous cellular proteins and these interactions contribute to reprogramming keratinocytes so as to prevent normal differentiation and to maintain cellular replication competence in order to amplify the HPV genome The inactivation of p53 is a general hallmark of tumorigenesis and tumors frequently show mutations and/or reduced p53 levels via induction of telomerase reverse transcriptase at the transcriptional level e.g. HPV 16, 18 and 51 A second common feature of tumors constitutes an increase in telomerase activity via its C-terminal PDZ-binding motif (PDZ-BM) in vivo mouse model resulted in smaller cervical tumors Furthermore, E6 of high-risk types interacts with a number of PDZ-domain containing proteins (PDZ proteins) in vivo is the multi-domain protein hDlg (human Dlg/hDlg1/SAP-97) in vitro degradation assay for a number of high-risk HPVs, among them HPV 16, 18 and 51 One PDZ domain containing protein targeted by E6 for degradation 29-CXXC Full-length E6 consists of approximately 150 residues and includes two zinc-binding domains (ZBDs) each coordinating one zinc ion via cysteines arranged in a motif of the form CXXC-XUp to now, no structural data on a wild-type HPV E6 are available. So we reasoned that addressing wild-type E6 proteins of high-risk types other than HPV 16 could on the one hand lead to a structure of a wild–type E6 and on the other hand shed light on structural similarities and differences among the E6 proteins including their interaction with target proteins. Here we determined the solution structure of the wild-type C-terminal zinc-binding domain of E6 derived from the high-risk HPV 51 and unraveled the structural basis of its interaction with the PDZ domain 2 of hDlg.6-tagged, recombinant E6 constructs derived from the high-risk types 16, 18, 26, 31, 45, 51, 59 and 97 as well as for the non-tumorigenic cutaneous type 1a was performed after bacterial expression in presence of 10 µM of Zn2+ as solubility of E6 constructs is dependent on low µM concentrations of zinc E. coli had been employed .Assessment of solubility of amino-terminally Hisemployed . The sol1H,15N]-HSQC spectra were recorded (see SI for details). If such a spectrum contained an acceptable signal distribution, homogeneous peak intensities and a signal number consistent with the expected number of observable amide groups, the respective protein was considered amenable to further NMR spectroscopic analysis. In the end, only 26Z2 and 51Z2 fulfilled these criteria . In the calculated structure, Ile88 Hδ2 is located directly above the aromatic ring of Trp132, while Ile88 Hδ1 is proximal to the aromatic ring of Phe125. Thus, ring current effects The calculated structural ensemble  exhibits10 helix. One anti-parallel β-sheet on 51Z2 is composed of the strands β1, β4 and β5, stabilized by charge-charge interactions and side chain hydrogen bonds, e.g. from R81 Hη12 to E127 Oε1. A second short anti-parallel β-sheet consisting of β2 and β3 is arranged almost perpendicular to the first sheet. This allows for the formation of hydrogen bonds between R102 HN and W132 O, H104 HN to G134 O and G134 HN to R102 O which stabilize the arrangement of both β-strands  of HPV 51 E6  and represents the first structural characterization of a non-mutated HPV E6 domain. The International Agency for Research on Cancer classifies HPV 51 as high-risk in vitro suggest protein motion at a timescale that causes line broadening at higher temperatures, whereas the zinc coordinating residues remain rigid. These findings derived from a wild-type E6 domain allow us to suggest that in cervical epithelial cells, where the E6 protein is expressed at 37°C, this particular domain and potentially the entire E6 protein may be more malleable in vivo than their published structures suggest at first glance. As the fold is re-adopted upon cooling down  E6 in complex with the LD1 motif of paxillin reveals an identical general topology for E6 . Thus, iE6 types  but rareE6 types . The coni.e. the target spectrum of closely related E6 is more similar than that of evolutionary more distantly related E6. Thus, although the E6 proteins may share a generally identical structural topology, subtle structural variations could explain the altered target spectrum of different E6 proteins Interestingly, a direct correlation between E6 phylogeny and their protein-protein interaction networks exists vide supra). In 51Z2 I128 is proximal to the conserved residues H126 and S82 on β4 and β1, respectively. HPV 51 is, as HPV 16, frequently detected in premalignant neoplasias, but proportionally significantly less present in cervical cancers e.g. E6AP or paxillin. Since the E3 ubiquitin ligase E6AP is often involved in E6-mediated target degradation Our analysis allows the identification of significant local structural differences between individual E6 proteins. Particularly, β4 and β5 of 51Z2 position differently as compared to the unbound ZBD2 of HPV 16 E6, but similar to the corresponding region of the only available full-length E6 structure in a complex, the BPV E6 bound to LD1 of paxillin . The convia contacts to bona fide PDZ domain residues [this study] or to residues outside the classical PDZ domain 51Z2 in the unbound state adopts a globular fold with a solvent exposed and flexible C-terminus  and S3 cversus E6CT6 and located far away from the peptide interface might have been perturbed due to the presence of such a long range network. This raises the question if the HPV 51 E6 extended PDZ-BM may even induce changes in places remote from the PDZ 2 of hDlg as the PDZ domains 1 and 2 of the hDlg-related PSD-95 are organized as a supramodule and retain their relative orientation in the context of the full-length protein A crystal structure of the C-terminal 11 residues of APC (APC_CT11) in complex with hDlgPDZ2 reveals only one peptide per asymmetric unit containing in turn five PDZ domains. Here, only the C-terminal six APC peptide residues gave rise to interpretable electron density in vivo with regard to interactions, such as e.g. with E6AP, may be addressed based on our findings in order to solve the puzzle of how the E6 proteins exert their malignant potential via interaction with a multitude of cellular targets.In summary, the first structure of a wild-type E6 domain and of the complex between its complete PDZ-BM and the PDZ domain 2 of hDlg revealed a number of E6 properties common for high-risk HPV E6 but also important differences between individual E6. The fully characterized interaction of E6’s extended PDZ-BM to one PDZ domain suggests future routes towards the elucidation of PDZ-supramodule E6 interaction. The implications of the E6 structural variations and the impact of the probable malleability of E6 E. coli BL21 (DE3). Cells were disrupted and protein solubility in cell-lysates was assessed. Soluble proteins were FPLC-purified and initially characterized by CD spectroscopy, dynamic light scattering, gel filtration chromatography and NMR spectroscopy. The affinity of E6-derived peptides to hDlgPDZ2 was elucidated utilizing Surface Plasmon Resonance. hDlgPDZ2 was generated by bacterial expression and FPLC purification. The generation of the labeled E6CT11 peptide was performed as previously described A detailed description of methods is provided in Figure S1Spectral changes of 26Z2 over time. Freshly prepared 15N-labeled 26Z2 (250 µM) was subjected to -HSQC NMR spectroscopy (blue con-tours). After 10 days at 4°C, the spectra (red contours) showed significant differences. Low peak-dispersion suggests an increased proportion of unfolded protein. Both spectra were recorded with 16 scans at a Bruker Avance III 750 MHz NMR spectrometer. Sample conditions were 135 mM NaCl, 45 mM L-Arg, 45 mM L-Glu, 9 mM DTT, pH 7.4, 4°C.(TIF)Click here for additional data file.Figure S2Biophysical characterization of 51Z2. For experimental details, see supplementary text 1. A Analytical gel-filtration. The chromatogram of 51Z2 run on a TSK gel G3000SWxl column is presented in blue, the column calibration is shown in orange with molecular weights of reference proteins indicated. 51Z2  eluted as a 9 to 10 kDa sized protein indicating a monomeric state. B Dynamic light scattering. 51Z2 exhibits a hydrodynamic radius of 1.58 nm, which corresponds to an approx. 10 kDa sized protein assuming a globular shape. C Circular dichroism spectrum of purified 51Z2. D The secondary structure content of 51Z2 was estimated from the CD spectrum C using CDNN (TIF)Click here for additional data file.Figure S3Sequence alignment of oncogenic E6 proteins. ClustalW2 alpha-papillomaviridae and to the species indicated on the left table-side.(TIF)Click here for additional data file.Figure S4Alignment of the high-risk HPV 51 E6 to representative low-risk and cutaneous E6 proteins. The residues corresponding to conserved positions among high-risk types Click here for additional data file.Table S1E6 domain architecture and solubility of recombinant E6 constructs.(PDF)Click here for additional data file.Table S2Final screening results of E6 constructs for NMR spectroscopy.(PDF)Click here for additional data file.Table S3Details of NMR experiments and samples for the 51Z2 structure determination.(PDF)Click here for additional data file.Table S4Details of NMR experiments and samples for the hDlgPDZ2-E6CT11 complex structure determination.(PDF)Click here for additional data file.Text S1Methods.(PDF)Click here for additional data file.


Epigenetic reading domains are involved in the regulation of gene expression and chromatin state by interacting with histones in a post-translational modification specific manner. A detailed knowledge of the target modifications of reading domains, including enhancing and inhibiting secondary modifications, will lead to a better understanding of the biological signaling processes mediated by reading domains.We describe the application of Celluspots peptide arrays which contain 384 histone peptides carrying 59 post translational modifications in different combinations as an inexpensive, reliable and fast method for initial screening for specific interactions of reading domains with modified histone peptides. To validate the method, we tested the binding specificities of seven known epigenetic reading domains on Celluspots peptide arrays, viz. the HP1ß and MPP8 Chromo domains, JMJD2A and 53BP1 Tudor domains, Dnmt3a PWWP domain, Rag2 PHD domain and BRD2 Bromo domain. In general, the binding results agreed with literature data with respect to the primary specificity of the reading domains, but in almost all cases we obtained additional new information concerning the influence of secondary modifications surrounding the target modification.We conclude that Celluspots peptide arrays are powerful screening tools for studying the specificity of putative reading domains binding to modified histone peptides. Epigenetic signals include the methylation of DNA, the post-translational modification (PTM) of the N-terminal histone tails and non-coding RNAs. In eukaryotes, these epigenetic marks are involved in the regulation of gene expression and chromatin state. The most studied histone tail modifications are acetylation of Lys, methylation of Lys or Arg leading to mono-, di- (symmetric or asymmetric in the case of Arg) or trimethylation in the case of Lys and phosphorylation at Ser or Thr -4. TheseAcetylated histones are recognized by Bromo domains, an about 110 amino acid residues long domain folded into a left-handed four α-helical bundle. The family of Bromo domains has more than 70 identified members which are found in many chromatin-associated factors, including histone acetyltransferases or chromatin-remodeling factors. The Bromo domain containing protein 2 (BRD2) belongs to the Bromo Domain And Extra-Terminal Domain (BET) family, members of which contain two Bromo domains and an additional conserved terminal domain. It was reported that the tandem Bromo domains of BRD2 bind to H4K12ac [Protein domains belonging to the Royal family include among others Tudor, Chromo and MBT domains. They are known to interact with methylated lysine residues. The Chromatin Organization Modifier (Chromo) domain is about 50 amino acids in size which are folded into a small ß-finger flanked by one α-helix. The Chromo domain family consists of more than 120 identified members. The Chromo domain of the Heterochromatin protein 1 beta (HP1ß) binds specifically to H3K9me3 and with weaker affinity to H3K9me2 and it is involved in the establishment of heterochromatin ,9. AnothThe Tudor domain folds into a ß-sandwich flanked by one α-helix. Members of this domain family are for example the p53 binding protein 1 (53BP1), which has been shown to interact with H4K20me2 ,15 and HHowever, a specific interaction with modified amino acids is possible in other families as well. For example, the PWWP domain (Proline Tryptophan Tryptophan Proline Motif) present in DNA methyltransferase 3a was shown to read H3K36me3  and the The investigation of the PTM specific binding of reading domains to peptides requires testing of binding to as many peptides with different PTMs as possible which is impeded by the high costs of synthetic modified peptides. Recently, we described the application of Celluspots peptide arrays for the quality assessment of commercial antibodies . PeptideIn the present study, the binding specificities of seven known reading domains were analyzed using Celluspots peptide arrays comprising 384 peptides from 8 different regions of the N-terminal histone tails, viz. H3 1-19, 7-26, 16-35 and 26-45, H4 1-19 and 11-30, H2A 1-19 and H2B 1-19. The arrays are commercially available from Active Motif and feature 59 post-translational modifications  in many different combinations  [In the structure of HP1ß bound to the H3K9me3 peptide (pdb entry 1KNE)   [3QO2) [An inhibiting effect of some secondary modifications was seen for most of the studied reading domains. For example, HP1ß binding to H3K9me3 was prevented by H3R8Citr, H3S10ph and H3T11ph. All of these modified amino acids are either close or adjacent to the target trimethyl lysine, but an additional modification at an adjacent residue does not necessarily influence binding as seen in the case of the MPP8 Chromo domain. Even though binding was inhibited by H3S10ph and H3T11ph similarly as for HP1ß, H3R8Citr did not have any effect on MPP8 Chromo domain binding to H3K9me3. Trying to understand that difference, we superimposed the structures of HP1ß (pdb entry 1KNE)  and MPP8) [3QO2)  in complWe observed with several domains that the presence of one or more additional modifications improved binding to peptides which carried the primary mark. This effect could be due to technical problems like unequal peptide synthesis or surface binding. It could also mean that these combinations of PTMs are biologically important, like in the case of HP1ß only binding to H3K9me3 if S10 is not phosphorylated  or the A2QQS) [Another example of the inhibition of binding by a secondary modification was seen with the JMJD2A double Tudor domain binding to H4K20me3. While H4K12ac and H4K16ac had no effect on the signal intensity, we observed that asymmetric or symmetric methylation of the arginine 19 reduced binding of the JMJD2A double Tudor domain to H4K20me3 severely. This observation can be explained in the light of the crystal structure of the JMJD2A double Tudor domain in complex with the H4K20me3 peptide (pdb entry 2QQS)  , the Chromo domain of human MPP8 (residues 58-112), the double Tudor domain of human JMJD2A (residues 856-1047), the tandem Tudor domain of human 53BP1 (residues 1490-1608), the PHD finger of human Rag2 (residues 311-520) and the second Bromo domain of human BRD2 (residues 348-455) were amplified from cDNA derived from HEK293 cells and cloned as GST fusion proteins into the pGEX-6P-2 vector . The sequence encoding the PWWP domain of murine Dnmt3a (residues 279-420) was subcloned as GST-fusion protein into the pGEX-6P-2 vector . The GST-tagged proteins were expressed and purified as described  . They are now commercially available from Active Motif Cat. No. 13001). The peptide sequences and PTMs are specified in the Additional File 3001. Thehttp://www.activemotif.com/catalog/667.html or from the authors upon request.The array was blocked by incubation in TTBS buffer  containing 5% non-fat dried milk at 4°C overnight, then washed two times with TTBS buffer, one time with interaction buffer , and incubated with purified GST-tagged reading domains: HP1ß full length protein (10 nM), MPP8 Chromo domain (0.5 μM), JMJD2A double Tudor domains (10 nM), 53BP1 tandem Tudor domain (1 μM), Dnmt3a PWWP domain (50 nM), Rag2 PHD finger (2 nM) and BRD2 Bromo domain 2 (10 nM) at room temperature for 2 hours in interaction buffer. After washing with TTBS buffer three times, the array was incubated with goat anti-GST antibody  1:5000 dilution in TTBS buffer containing 1% non-fat dried milk for 1 h at room temperature. Then, the membrane was washed three times with TTBS and incubated with horseradish peroxidase conjugated anti-goat antibody (Invitrogen #81-1620) 1:12000 in TTBS containing 1% non-fat dried milk for 1 h at room temperature. Finally, the membrane was washed four times with TTBS and submerged in ECL developing solution (Thermo Fisher Scientific) and image was captured on an X-ray film. Typical exposure times were 0.5-5 min. Analysis was done using the Array Analyze program, which was also used to prepare the graphs shown in Figures Peptide binding of the MPP8 Chromo domain was analyzed by fluorescence depolarization using a Varian Carry Eclipse fluorescence spectrophotometer as described . PurifieIB, AD, SK, GK and RT conducted and analyzed the experiments. IB, AD and AJ designed the study. IB and AJ were involved in data analysis and interpretation and drafted the manuscript. All authors read and approved the final manuscript.Supplemental information S1.xls. Compilation of the peptides presented on the Celluspots arrays. Excel file showing the sequences, modifications and positions of all peptides on the Celluspots arrays.Click here for file


Human platelet antigens (HPAs) are polymorphisms in platelet membrane glycoproteins (GPs) that can stimulate production of alloantibodies once exposed to foreign platelets (PLTs) with different HPAs. These antibodies can cause neonatal alloimmune thrombocytopenia, posttransfusion purpura, and PLT transfusion refractoriness. Most HPAs are localized on the main PLT receptors: 1) integrin αIIbβ3, known as the fibrinogen receptor; 2) the GPIb-IX-V complex that functions as the receptor for von Willebrand factor; and 3) integrin α2β1, which functions as the collagen receptor.We analyzed the structural location and the evolutionary conservation of the residues associated with the HPAs to characterize the features that induce immunologic responses but do not cause inherited diseases.We found that all HPAs reside in positions located on the protein surface, apart from the ligand-binding site, and are evolutionary variable.Disease-causing mutations often reside in highly conserved and buried positions. In contrast, the HPAs affect residues on the protein surface that were not conserved throughout evolution; this explains their naive effect on the protein function. Nonetheless, the HPAs involve substitutions of solvent-exposed positions that lead to altered interfaces on the surface of the protein and might present epitopes foreign to the immune system. Polymorphisms in platelet (PLT) membrane glycoproteins (GPs) are responsible for alloantibody production upon exposure to PLTs with different human PLT antigens (HPA). These antibodies can cause neonatal alloimmune thrombocytopenia (NAIT), posttransfusion purpura, or PLT transfusion refractoriness after exposure to unmatched PLTs. The molecular basis of the 24 serologically defined antigens had been resolvedMost HPAs are localized to the main PLT receptors, namely, integrin αIIbβ3 that is also known as the fibrinogen receptor, the complex GPIb-IX-V that functions as the receptor for von Willebrand factor (VWF) and integrin α2β1—the collagen receptor. Exceptional is HPA-15, which is carried by the glycosylphosphatidylinositol-linked protein CD10942-terminal ectodomain of the α and β subunits assembles into an ovoid “head” and two “legs” composed of several domains. The remaining segments form two tails that span the plasma membrane.7Integrins are adhesion receptors that mediate vital bidirectional signals within the cell.Integrin β3 subunit is known as PLT membrane GPIIIa that forms heterodimers with αIIb and αv integrins subunits; αIIbβ3 is the most abundant PLT receptor with approximately 80,000 copies per PLT. The structure of the inactive extracellular region of αvβ3 was the first one reported (PDB ID 1JV2http://www.ebi.ac.uk/ipd/hpa/freqs_1.html). The antigen most commonly implicated in alloantibodies production is HPA-1a, a Leu33Pro substitution in integrin β3. Approximately 80% of NAIT cases are caused by anti-HPA-1a. The second frequent antigen causing NAIT is HPA-5b (10%-15% of NAIT cases), while all the rest cause NAIT very rarely.Eleven HPAs are located on the most abundant receptor on PLTs surface: integrin αIIbβ3. Nine of the HPAs are mapped to the β3 subunit that was found to be the most polymorphic GP on the PLT membrane.Interestingly, although most HPAs are nonsynonymous SNPs and HPA-14w is even a single-amino-acid deletion, they probably inflict a minor effect on PLT receptors function since they do not cause inherited diseases, and even their influence on thrombus formation is controversial.In this article we analyzed the structural features and evolutionary conservation of the HPAs to define the characteristics that lead to immunologic problems but not to inherited disease. We demonstrate that all HPAs studied here involve residues located on the surface of the protein far from the ligand-binding site and are not evolutionary conserved. These criteria imply that the substitutions in these positions present a minor effect on the structure and function of the protein and therefore, do not represent disease-causing mutations. Moreover, their location on the surface suggests that they could play a role in presenting epitopes.http://consurf.tau.ac.il)http://conseq.tau.ac.il/).The evolutionary conservation analyses were calculated using the Bayesian methodhttp://pfam.sanger.ac.uk/). This alignment corresponds to Residues 481 to 921 in αIIb and was used to calculate the conservation scores for the HPAs in the calf domain of αIIb. The last two strands of the calf2 domain constitute a very variable region among α-integrins and therefore were not included in the Pfam alignment. Since one of the HPAs in α2 is located on the C-terminus of the calf2 domain, we generated a smaller MSA via ConSeq, using α2 human as the query sequence to collect 50 homologs from the Swiss-Prot databaseA multiple sequence alignment (MSA) of the β-propeller domain in various α-integrins was constructed as described previously.Human β3 (Swiss-Prot entry ITB3_HUMAN) was used as query to collect homologous sequences from the Swiss-Prot databaseHuman GPIbα 19Since the structure of human CD109 is not available (Swiss-Prot entry: CD109_HUMAN), the ConSeq Web server19The analyses were based on the crystal structures of αVβ3 (PDB ID 1JV2),17The solvent-accessible surface area (SASA) was calculated using the SURFV program with a probe sphere of radius 1.4 Å and default parameters.Data are expressed as mean ± standard deviation. Kruskal-Wallis test was used to analyze the difference between groups using computer software . A p value of less than 0.05 was considered as a significant difference. In case that the SASA was calculated in various structures, the mean value was used for the statistical analysis.Integrin β3 ectodomain is composed of eight domains—the plexins, semaphorins, and integrins (PSI); βA; hybrid; four epidermal growth factor (EGF) domains; and a β-tail domain (βTD). There are nine HPAs located in integrin β3; seven of them  are visible in the inactive structures of both αvβ3 (PDB ID 1JV2) and αIIbβ3 (PDB ID 3FCS). Structural analyses of the crystal structures revealed that all HPAs are exposed to the solvent  and, morIntegrin activation is a multistep process that involves transitions between multiple conformations.Some HPAs are located close to the interface with adjacent domains on the β subunit. For example, Arg633, the polymorphic residue of HPA-11, and Arg636, the polymorphic residue of HPA-8, both located in the βTD, are close to the hybrid domain. In the inactive complex αVβ3, Arg633 forms a salt bridge with Asp393 from the hybrid domain and is surrounded by Leu389, Gly388, and Cys374. In the structure of αIIbβ3, also in the inactive conformation, there are some conformational variations with respect to the inactive β3 in the αVβ3 complex. In αIIbβ3, Arg633 is facing away from the hybrid domain, yet might be still bound with Glu378 on the hybrid domain. Arg636 is in close proximity to Met387 and Gly388 from the hybrid domain in the αIIbβ3 complex, while in the αVβ3 structure, Arg636 is close to Asn376. Arg489 of HPA-6, located on the EGF-1 domain, is close to the Calf1 domain of the αIIb subunit (in the inactive conformation) but is not in direct contact.Five polymorphic residues involved in the formation of HPA-1, -4, -7, -10, and -16 are visible in the crystal structures of both the active  is unique among other HPAs because it shows an intermediate evolutionary conservation. Nonetheless, this position can be occupied by a variety of amino acids and is not restricted to proline in β-integrins from other species . Pro407 HPA-14, the single-deletion polymorphism, Lys611del, is located within the βTD, at the C-terminus of the ectodomain. The region encompassing Lys611 forms a helix in the αVβ3 structure; thus such a deletion might disrupt the structure. However, this helix is partially unwound in the αIIbβ3 inactive conformation (PDB ID 3FCS) raising uncertainties about its stability and contribution to the overall fold.Recently, two additional low-frequency SNPs, not yet approved as HPAs, were reported within integrin β3: Lys137Gln and Glu628Lys.Integrin αIIb (PLT membrane GPIIb) comprises the β-propeller, thigh, and two calf domains. Two HPAs (HPA-3 and -9) were found in the αIIb integrin. Both related residues  are located in the calf2 domain , within Recently, a low-frequency HPA was found in αIIb corresponding to Thr619Met substitutionIntegrin α2 (PLT membrane GPIa) forms hetrodimers with integrin β1 and, similarly to the integrins discussed above, is important for PLT function. Three polymorphic residues in the α2 integrin form antigens, HPA-5, -13, and -17, localized to the β-propeller, calf1, and calf2 domains, respectively. Since the only crystal structure of α2 is for the I-domain in the β-propeller , affecting Lys505, is located on one of the loops of the β-propeller domain, on the surface of the protein, far from the interface with the β subunit . Lys505 The GPIb-V-IX complex, another PLT receptor, is composed of four Type I membrane spanning proteins that belong to the leucine-rich repeat (LRR) family of proteins: GPIbα, GPIbβ, GPIX, and GPV. HPA-2 and HPA-12 are localized to the two first GPs, respectively. Crystal structures are available only for the LRR domain of GPIbα . HPA-2, affecting Thr145, is located in this region, on the surface of the protein, facing the solvent. It is located far from the interface with its ligand, the VWF .The GPIb-V-IX complex is known to regulate thrombin function by distinct interactions.35CD109 antigen, also known as PLT-specific Gov antigen, is a TGF-β1 binding protein for which no structure is available. The closest homolog with known structure has 26% sequence identity, which is not enough for accurate structural modeling. Tyr703 (Residue 682 in the mature protein), associated with HPA15, is evolutionary variable.http://sinaicentral.mssm.edu/intranet/research/glanzmann/menu). Single-nucleotide sequence alterations responsible for both HPAs and GT result in nonsynonymous amino acid substitutions. The interesting question is why some amino acid substitutions cause disease while others are naive polymorphisms.Most HPAs are localized to integrin αIIbβ3, specifically to the β3 subunit.Our structural analysis revealed that the SNPs responsible for the HPAs affected nonconserved positions located on the surface of the protein, far away from the ligand binding site and not in direct contact with other subunits. In contrast, disease-causing mutations within integrins often disturb highly conserved and buried positions in the proteins' coresTaken together, we can conclude that although disease-causing mutations and SNPs responsible for HPAs can be localized to the same domain, they differ in their evolutionary conservation and solvent exposure. Interestingly, although most HPAs result in substituting chemically dissimilar residues, they have no dramatic effects on the apparent function of the proteins. The observations that these positions are evolutionary variable and surface exposed explain their tolerance to substitutions for residues of various properties. Actually, in most cases, the amino acid of the polymorphic residue was found in equivalent positions within homologous proteins . It is iLeu33 involved in HPA-1 is located in a loop of the PSI domain of integrin β3, totally exposed to the solvent in both the active and the inactive conformations of αIIbβ3 . HPA-1 w45Integrins such as αIIbβ3 were shown to be in a bent inactive conformation on the resting PLT. After physiologic activation, the integrins shift to the extended active conformation leading to exposure of multiple new epitopes named LIBS (ligand-induced binding site).45The rearrangements of interdomain orientation during activation impinge on the presentation of the HPA epitopes. Then again, some HPA polymorphisms can have an effect on the dynamic of the conformational changes involved in the activation process. For example, Arg633 in β3, the residue substituted in HPA-11, was shown to be important for constraining αIIbβ3 in a low-affinity state. Correspondingly, the polymorphism Arg633His can enhance the separation of domains. Interfering with interdomain interactions can increase protein flexibility and initiate the extension of the integrin.In conclusion, our analyses provide a unique perspective to the molecular basis of amino acid substitutions leading to the formation of epitopes for auto-/alloantibodies. The HPAs involve SNPs at evolutionary variable positions located on the protein surface. These polymorphisms do not cause diseases but lead to immunologic disorders. Interestingly, some HPAs might also affect the activation process of the protein. The integrins form hetrodimers, whereas each subunit comprises multiple domains. This allows a regulation mechanism that involves multiple conformations of the protein on the cell surface. Our analyses show that some HPAs involve substitutions at positions located at the interface between domains and thereby might affect interdomain rearrangement and the activation dynamics.