Fibrinogen and elastin bind to the same region within the A domain of fibronectin binding protein A, an MSCRAMM of Staphylococcus aureus

The fibronectin binding protein, FnBPA, is a multifunctional microbial surface component recognizing adhesive matrix molecule (MSCRAMM) that promotes bacterial adherence to immobilized fibrinogen and elastin via the N-terminal A domain. The binding site for fibrinogen and elastin was localized to subdomains N2N3. A three-dimensional structural model of FnBPA was created based on the known crystal structure of the domains N2N3 of clumping factor A (ClfA). The role of individual residues in the putative ligand binding trench was examined by testing the affinity of mutants for fibrinogen and elastin. Two residues (N304 and F306) were crucial for binding both ligands and are in the equivalent positions to residues known to be important for fibrinogen binding by ClfA. A peptide comprising the C-terminus of the gamma-chain of fibrinogen and a monoclonal anti-rAFnBPA antibody were potent inhibitors of the FnBPA-elastin interaction. This suggests that FnBPA binds to fibrinogen and elastin in a similar manner. Amino acid sequence divergence of 26.5% occurred between the A domains of FnBPA from strains 8325-4 and P1. Most variant residues were predicted to be located on the surface of domains N2N3 while few occurred in the putative ligand binding trench and the latching peptide explaining limited immunocross reactivity while ligand binding activity is conserved.     

A 'Collagen Hug' model for Staphylococcus aureus CNA binding to collagen

The structural basis for the association of eukaryotic and prokaryotic protein receptors and their triple-helical collagen ligand remains poorly understood. Here, we present the crystal structures of a high affinity subsegment of the Staphylococcus aureus collagen-binding CNA as an apo-protein and in complex with a synthetic collagen-like triple helical peptide. The apo-protein structure is composed of two subdomains (N1 and N2), each adopting a variant IgG-fold, and a long linker that connects N1 and N2. The structure is stabilized by hydrophobic inter-domain interactions and by the N2 C-terminal extension that complements a beta-sheet on N1. In the ligand complex, the collagen-like peptide penetrates through a spherical hole formed by the two subdomains and the N1-N2 linker. Based on these two structures we propose a dynamic, multistep binding model, called the 'Collagen Hug' that is uniquely designed to allow multidomain collagen binding proteins to bind their extended rope-like ligand.     

Sequence analysis of alpha 1(VI) and alpha 2(VI) chains of human type VI collagen reveals internal triplication of globular domains similar to the A domains of von Willebrand factor and two alpha 2(VI) chain variants that differ in the carboxy terminus.

Amino acid sequences of human collagen alpha 1(VI) and alpha 2(VI) chains were completed by cDNA sequencing and Edman degradation demonstrating that the mature polypeptides contain 1009 and 998 amino acid residues respectively. In addition, they contain small signal peptide sequences. Both chains show 31% identity in the N-terminal (approximately 235 residues) and C-terminal (approximately 430 residues) globular domains which are connected by a triple helical segment (335-336 residues). Internal alignment of the globular sequences indicates a repetitive 200-residue structure (15-23% identity) occurring three times (N1, C1, C2) in each chain. These repeating subdomains are connected to each other and to the triple helix by short (15-30 residues) cysteine-rich segments. The globular domains possess several N-glycosylation sites but no cell-binding RGD sequences, which are exclusively found in the triple helical segment. Sequencing of alpha 2(VI) cDNA clones revealed two variant chains with a distinct C2 subdomain and 3' non-coding region. The repetitive segments C1, C2 and, to a lesser extent, N1 show significant identity (15-18%) to the collagen-binding A domains of von Willebrand factor (vWF) and they are also similar to some integrin receptors, complement components and a cartilage matrix protein. Since the globular domains of collagen VI come into close contact with triple helical segments during the formation of tissue microfibrils it suggests that the globular domains bind to collagenous structures in a manner similar to the binding of vWF to collagen I.     

The A domain of fibronectin-binding protein B of Staphylococcus aureus contains a novel fibronectin binding site

The fibronectin-binding proteins FnBPA and FnBPB are multifunctional adhesins than can also bind to fibrinogen and elastin. In this study, the N2N3 subdomains of region A of FnBPB were shown to bind fibrinogen with a similar affinity to those of FnBPA (2 μM). The binding site for FnBPB in fibrinogen was localized to the C-terminus of the γ-chain. Like clumping factor A, region A of FnBPB bound to the γ-chain of fibrinogen in a Ca(2+)-inhibitable manner. The deletion of 17 residues from the C-terminus of domain N3 and the substitution of two residues in equivalent positions for crucial residues for fibrinogen binding in clumping factor A and FnBPA eliminated fibrinogen binding by FnBPB. This indicates that FnBPB binds fibrinogen by the dock-lock-latch mechanism. In contrast, the A domain of FnBPB bound fibronectin with K(D) = 2.5 μM despite lacking any of the known fibronectin-binding tandem repeats. A truncate lacking the C-terminal 17 residues (latching peptide) bound fibronectin with the same affinity, suggesting that the FnBPB A domain binds fibronectin by a novel mechanism. The substitution of the two residues required for fibrinogen binding also resulted in a loss of fibronectin binding. This, combined with the observation that purified subdomain N3 bound fibronectin with a measurable, but reduced, K(D) of 20 μM, indicates that the type I modules of fibronectin bind to both the N2 and N3 subdomains. The fibronectin-binding ability of the FnBPB A domain was also functional when the protein was expressed on and anchored to the surface of staphylococcal cells, showing that it is not an artifact of recombinant protein expression.     

Molecular Characterization of the Interaction of Staphylococcal Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMM) ClfA and Fbl with Fibrinogen

The ligand-binding domain of Fbl (the fibrinogen binding protein from Staphylococcus lugdunensis) shares 60% sequence identity with ClfA (clumping factor A) of Staphylococcus aureus. Recombinant Fbl corresponding to the minimum fibrinogen-binding region (subdomains N2N3) was compared with ClfA for binding to fibrinogen. Fbl and ClfA had very similar affinities for fibrinogen by surface plasmon resonance. The binding site for Fbl in fibrinogen was localized to the extreme C terminus of the fibrinogen γ-chain at the same site recognized by ClfA. Isothermal titration calorimetry showed that Fbl and ClfA had very similar affinities for a peptide mimicking the C-terminal segment of the fibrinogen γ-chain. The peptide also inhibited binding of Fbl and ClfA to fibrinogen. A series of substituted γ-chain variant peptides behaved very similarly when used to inhibit ClfA and Fbl binding to immobilized fibrinogen. Both ClfA and Fbl bound to bovine fibrinogen with a lower affinity compared with human fibrinogen and did not bind detectably to ovine fibrinogen. The structure of the N2N3 subdomains of Fbl in complex with the fibrinogen γ-chain peptide was modeled based on the crystal structure of the N2N3 subdomains of the ClfA-γ-chain peptide complex. Residues in the putative binding trench likely to be involved in fibrinogen binding were identified. Fbl variant proteins with alanine substitutions in key residues had reduced affinities for fibrinogen. Thus Fbl and ClfA bind the same site in fibrinogen by similar mechanisms.     

Crystal structure of the E. coli Hsp100 ClpB N-terminal domain

E. coli Hsp100 ClpB can disaggregate denatured polypeptides by employing ATP hydrolysis. The ClpB N-terminal domain (ClpBN) has been proposed to play important roles in ClpB molecular chaperone activities. We have determined the crystal structure of ClpBN to 1.95 A resolution by MAD methods. The ClpBN monomer contains two subdomains that have similar folds. The crystal structure revealed a hydrophobic groove on the molecular surface. We have constructed ClpB mutants in which the hydrophobic residues within the putative peptide binding groove were replaced by glutamine. These ClpB mutants exhibited severe defects in molecular chaperone activity but retained the wild-type ATPase activity.     

Structural organization of the fibrinogen-binding region of the clumping factor B MSCRAMM of Staphylococcus aureus

The clumping factor B (ClfB) of Staphylococcus aureus is a surface protein that binds to fibrinogen (Ni Eidhin, D., Perkins, S., Francois, P., Vaudaux, P., Hook, M., and Foster, T. J., 1998 Mol. Microbiol. 30, 245-257). The ligand-binding activity is located in the approximately 500-residue A-region (residues 44-542), which represents the N-terminal half of the MSCRAMM protein. We now hypothesize that the ClfB A-region is composed of three subdomains, which we have named N1, N2, and N3, respectively. To examine this hypothesis, we expressed recombinant forms of the individual putative subdomains, the tandem motifs N12 and N23, and the full-length A-region N123. Far UV circular dichroism spectra showed that each subdomain is composed mainly of beta-sheets with little or no discernible alpha-helices. Heat-induced unfolding of individual subdomains occurred with a single state transition and was reversible, indicating that the subdomains can fold as discreet units. Gel permeation chromatography indicated that N2, N3, and N23 are globular. In contrast, domain N1 appeared to be elongated and conferred a somewhat elongated structure on segments containing this subdomain (i.e. N12 or N123). N123, N12, and N23 all bound to fibrinogen, but N23 had a higher affinity for fibrinogen than that observed for the full-length A-region; N123 or for N12. However, an extended N terminus of N23 was required for ligand binding. A form of N23 that was generated by proteolytic processing and lacked the N-terminal extension was unable to bind fibrinogen. Recombinant forms of individual subdomains did not bind fibrinogen. The addition of recombinant N23 effectively inhibited ClfB-mediated bacterial adherence to fibrinogen, and N123 caused some reduction in bacterial attachment, whereas N12 was essentially inactive. Antibodies raised against the central N2 domain of the A-region were the most effective at inhibiting bacterial adhesion to immobilized fibrinogen, although anti-N3 or anti-N1 antibodies also caused some reduction in ClfB-mediated adherence to fibrinogen.     

Mosaic structure of globular domains in the human type VI collagen alpha 3 chain: similarity to von Willebrand factor, fibronectin, actin, salivary proteins and aprotinin type protease inhibitors.

Human collagen alpha 3(VI) chain mRNA (approximately 10 kb) was cloned and shown by sequence analysis to encode a 25 residue signal peptide, a large N-terminal globule (1804 residues), a central triple helical segment (336 residues) and a C-terminal globule (803 residues). Some of the sequence was confirmed by Edman degradation of peptides. The N-terminal globular segment consists of nine consecutive 200 residue repeats (N1 to N9) showing internal homology and also significant identity (17-25%) to the A domains of von Willebrand Factor and similar domains present in some other proteins. Deletions were found in the N3 and N9 domains of several cDNA clones suggesting variation of these structures by alternative splicing. The C-terminal globule starts immediately after the triple helical segment with two domains C1 (184 residues) and C2 (248 residues) being similar to the N domains. They are followed by a proline rich, repetitive segment C3 of 122 residues, with similarity to some salivary proteins, and domain C4 (89 residues), which is similar to the type III repeats present in fibronectin and tenascin. The most C-terminal domain C5 (70 residues) shows 40-50% identity to a variety of serine protease inhibitors of the Kunitz type. The whole sequence contains 29 cysteines which are mainly clustered in short segments connecting domains N1, C1, C2 and the triple helix, and in the inhibitor domain. Five putative Arg-Gly-Asp cell-binding sequences are exclusively localized in the triple helical segment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular basis of the high insecticidal potency of scorpion alpha-toxins

Scorpion alpha-toxins are similar in their mode of action and three-dimensional structure but differ considerably in affinity for various voltage-gated sodium channels (NaChs). To clarify the molecular basis of the high potency of the alpha-toxin LqhalphaIT (from Leiurus quinquestriatus hebraeus) for insect NaChs, we identified by mutagenesis the key residues important for activity. We have found that the functional surface is composed of two distinct domains: a conserved "Core-domain" formed by residues of the loops connecting the secondary structure elements of the molecule core and a variable "NC-domain" formed by a five-residue turn (residues 8-12) and a C-terminal segment (residues 56-64). We further analyzed the role of these domains in toxin activity on insects by their stepwise construction onto the scaffold of the anti-mammalian alpha-toxin, Aah2 (from Androctonus australis hector). The chimera harboring both domains, Aah2(LqhalphaIT(face)), was as active to insects as LqhalphaIT. Structure determination of Aah2(LqhalphaIT(face)) by x-ray crystallography revealed that the NC-domain deviates from that of Aah2 and forms an extended protrusion off the molecule core as appears in LqhalphaIT. Notably, such a protrusion is observed in all alpha-toxins active on insects. Altogether, the division of the functional surface into two domains and the unique configuration of the NC-domain illuminate the molecular basis of alpha-toxin specificity for insects and suggest a putative binding mechanism to insect NaChs.     

Crystal structure of the E. coli dipeptidyl carboxypeptidase Dcp: further indication of a ligand-dependent hinge movement mechanism

Dcp from Escherichia coli is a 680 residue cytoplasmic peptidase, which shows a strict dipeptidyl carboxypeptidase activity. Although Dcp had been assigned to the angiotensin I-converting enzymes (ACE) due to blockage by typical ACE inhibitors, it is currently grouped into the M3 family of mono zinc peptidases, which also contains the endopeptidases neurolysin and thimet oligopeptidase (TOP). We have cloned, expressed, purified, and crystallized Dcp in the presence of an octapeptide "inhibitor", and have determined its 2.0A crystal structure using MAD methods. The analysis revealed that Dcp consists of two half shell-like subdomains, which enclose an almost closed two-chamber cavity. In this cavity, two dipeptide products presumably generated by Dcp cleavage of the octapeptide bind to the thermolysin-like active site fixed to side-chains, which are provided by both subdomains. In particular, an Arg side-chain backed by a Glu residue, together with two Tyr phenolic groups provide a charged anchor for fixing the C-terminal carboxylate group of the P2' residue of a bound substrate, explaining the strict dipeptidyl carboxypeptidase specificity of Dcp. Tetrapeptidic substrates are fixed only via their main-chain functions from P2 to P2', suggesting a broad residue specificity for Dcp. Both subdomains exhibit very similar chain folds as the equivalent but abducted subdomains of neurolysin and TOP. Therefore, this "product-bound" Dcp structure seems to represent the inhibitor/substrate-bound "closed" form of the M3 peptidases, generated from the free "open" substrate-accessible form by a hinge-bending mechanism. A similar mechanism has recently been demonstrated experimentally for ACE2.     

The role of FhuD2 in iron(III)-hydroxamate transport in Staphylococcus aureus. Demonstration that FhuD2 binds iron(III)-hydroxamates but with minimal conformational change and implication of mutations on transport

The fhuD2 gene encodes a lipoprotein that has previously been shown to be important for the utilization of iron(III)-hydroxamates by Staphylococcus aureus. We have studied the function of the FhuD2 protein in greater detail, and demonstrate here that the protein binds several iron(III)-hydroxamates. Mutagenesis of FhuD2 identified several residues that were important for the ability of the protein to function in iron(III)-hydroxamate transport. Several residues, notably Tyr-191, Trp-197, and Glu-202, were found to be critical for ligand binding. Moreover, mutation of two highly conserved glutamate residues, Glu-97 and Glu-231, had no affect on ligand binding, but did impair iron(III)-hydroxamate transport. Interestingly, the transport defect was not equivalent for all iron(III)-hydroxamates. We modeled FhuD2 against the high resolution structures of Escherichia coli FhuD and BtuF, two structurally related proteins, and showed that the three proteins share a similar overall structure. FhuD2 Glu-97 and Glu-231 were positioned on the surface of the N and C domains, respectively. Characterization of E97A, E231A, or E97A/E231A mutants suggests that these residues, along with the ligand itself, play a cumulative role in recognition by the ABC transporter FhuBGC2. In addition, small angle x-ray scattering was used to demonstrate that, in solution, FhuD2 does not undergo a detectable change in conformation upon binding iron(III)-hydroxamates. Therefore, the mechanism of binding and transport of ligands for binding proteins within this family is significantly different from that of other well studied binding protein families, such as that represented by maltose-binding protein.     

p75 Co-receptors regulate ligand-dependent and ligand-independent Trk receptor activation, in part by altering Trk docking subdomains.

Neurotrophins signal via Trk tyrosine kinase receptors and a common receptor called p75. Nerve growth factor is the cognate ligand for TrkA, brain-derived neurotrophic factor for TrkB, and neurotrophin-3 (NT-3) for TrkC. NT-3 also binds TrkA and TrkB as a heterologous ligand. All neurotrophins bind p75, which regulates ligand affinity and Trk signals. Trk extracellular domain has five subdomains: a leucine-rich motif, two cysteine-rich clusters, and immunoglobulin-like subdomains IgG-C1 and IgG-C2. The IgG-C1 subdomain is surface exposed in the tertiary structure and regulates ligand-independent activation. The IgG-C2 subdomain is less exposed but regulates cognate ligand binding and Trk activation. NT-3 as a heterologous ligand of TrkA and TrkB optimally requires the IgG-C2 but also binds other subdomains of these receptors. When p75 is co-expressed, major changes are observed; NGF-TrkA activation can occur also via the cysteine 1 subdomain, and brain-derived neurotrophic factor-TrkB activation requires the TrkB leucine-rich motif and cysteine 2 subdomains. We propose a two-site model of Trk binding and activation, regulated conformationally by the IgG-C1 subdomain. Moreover, p75 affects Trk subdomain utilization in ligand-dependent activation, possibly by conformational or allosteric control.     

The second type II module from human matrix metalloproteinase 2: structure, function and dynamics.

BACKGROUND: Matrix metalloproteinase 2 (MMP-2, gelatinase A, 72 kDa type IV collagenase) has an important role in extracellular matrix degradation during cell migration and tissue remodeling. It is involved in development, inflammation, wound healing, tumor invasion, metastasis and other physiological and pathological processes. The enzyme cleaves several types of collagen, elastin, fibronectin and laminin. Binding to collagen is mediated by three repeats homologous to fibronectin type II modules, which are inserted in the catalytic domain in proximity to the active site. RESULTS: We have determined the NMR solution structure of the second type II module from human MMP-2 (col-2). The module exhibits a typical type II fold with two short double-stranded antiparallel beta sheets and three large loops packed around a cluster of conserved aromatic residues. Backbone amide dynamics, derived from (15)N relaxation experiments, correlate well with solvent accessibility and intramolecular hydrogen bonding. A synthetic peptide with the collagen consensus sequence, (Pro-Pro-Gly)(6), is shown to interact with the module. CONCLUSIONS: Spectral perturbations induced by (Pro-Pro-Gly)(6) binding reveal the region involved in the interaction of col-2 with collagen. The binding surface comprises exposed aromatic residues Phe21, Tyr38, Trp40, Tyr47, Tyr53 and Phe55, and the neighboring Gly33-Gly37 segment.     

Structure of FBP11 WW1-PL ligand complex reveals the mechanism of proline-rich ligand recognition by group II/III WW domains

FBP11/HYPA is a mammalian homologue of yeast splicing factor Prp40. The first WW domain of FBP11/HYPA (FBP11 WW1) is essential for preventing severe neurological diseases such as Huntington disease and Rett syndrome and strongly resembles the WW domain of FCA, the essential regulator for flowering time control. We have solved the structure of FBP11 WW1 and a Pro-Pro-Leu-Pro ligand complex, and demonstrated the binding mechanism with mutational analysis using surface plasmon resonance. The overall structure of FBP11 WW1 in the complex form is quite similar to the structures of WW domains from Group I and IV in complexes. In addition, conformation of FBP11 WW1 does not change much upon ligand binding. The binding orientation of the ligand against FBP11 WW1 is the same as that of the Group IV WW domain-ligand complex, but opposite to that of the Group I complex. The ligand interacts with two grooves formed by surface aromatic residues. The Pro and Leu residues in the ligand interact with the grooves and the Loop I region of FBP11 WW1, respectively, which are necessary interactions for binding the ligand. Interestingly, the two aromatic grooves recognize the Pro residues in entirely different manners, which allows FBP11 WW1 to recognize shorter sequences than the SH3 domain. Combined with homology models of other WW domains, the present report shows the detailed mechanism of ligand binding by Group II/III WW domains, and provides information useful in designing drugs to treat neurodegenerative diseases.     

NMR structure of the sterol carrier protein-2: implications for the biological role

The determination of the NMR structure of the sterol carrier protein-2 (SCP2), analysis of backbone (15)N spin relaxation parameters and NMR studies of nitroxide spin-labeled substrate binding are presented as a new basis for investigations of the mode of action of SCP2. The SCP2 fold is formed by a five-stranded beta-sheet and four alpha-helices. Fatty acid binding to a hydrophobic surface area formed by amino acid residues of the first and third helices, and the beta-sheet, which are all located in the polypeptide segment 8-102, was identified with the use of the spin-labeled substrate 16-doxylstearic acid. In the free protein, the lipid-binding site is covered by the C-terminal segment 105-123, suggesting that this polypeptide segment, which carries the peroxisomal targeting signal (PTS1), might be involved in the regulation of ligand binding.     

Solution structure of the helicase-interaction domain of the primase DnaG: a model for helicase activation

The helicase-primase interaction is a critical event in DNA replication and is mediated by a putative helicase-interaction domain within the primase. The solution structure of the helicase-interaction domain of DnaG reveals that it is made up of two independent subdomains: an N-terminal six-helix module and a C-terminal two-helix module that contains the residues of the primase previously identified as important in the interaction with the helicase. We show that the two-helix module alone is sufficient for strong binding between the primase and the helicase but fails to activate the helicase; both subdomains are required for helicase activation. The six-helix module of the primase has only one close structural homolog, the N-terminal domain of the corresponding helicase. This surprising structural relationship, coupled with the differences in surface properties of the two molecules, suggests how the helicase-interaction domain may perturb the structure of the helicase and lead to activation.     

The two homologous domains of human angiotensin I-converting enzyme are both catalytically active.

Molecular cloning of human endothelial angiotensin I-converting enzyme (kininase II; EC 3.4.15.1) (ACE) has recently shown that the enzyme contains two large homologous domains (called here the N and C domains), each bearing a putative active site, identified by sequence comparisons with the active sites of other zinc metallopeptidases. However, the previous experiments with zinc or competitive ACE inhibitors suggested a single active site in ACE. To establish whether both domains of ACE are enzymatically active, a series of ACE mutants, each containing only one intact domain, were constructed by deletion or point mutations of putative critical residues of the other domain, and expressed in heterologous Chinese hamster ovary cells. Both domains are enzymatically active and cleave the C-terminal dipeptide of hippuryl-His-Leu or angiotensin I. Moreover, both domains have an absolute zinc requirement for activity, are activated by chloride and are sensitive to competitive ACE inhibitors, and appear to function independently. However, the two domains display different catalytic constants and different patterns of chloride activation. At high chloride concentrations, the C domain hydrolyzes the two substrates tested faster than does the N domain. His-361,365 and His-959,963 are established as essential residues in the N and C domains, respectively, most likely involved in zinc binding, and Glu-362 in the N domain and Glu-960 in the C domain are essential catalytic residues. These observations provide strong evidence that ACE possesses two independent catalytic domains and suggest that they may have different functions.     

Inhibitory antibodies to human angiotensin-converting enzyme: fine epitope mapping and mechanism of action

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the 3D-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E.mAb and E.S.mAb complexes, respectively; however, only complex E.S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the "closed" conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.     

Backbone dynamics of the C-terminal SH2 domain of the p85alpha subunit of phosphoinositide 3-kinase: effect of phosphotyrosine-peptide binding and characterization of slow conformational exchange processes

The backbone dynamics of the C-terminal SH2 domain from the regulatory subunit p85alpha (p85alpha C-SH2) of phosphoinositide 3-kinase has been investigated in the absence of, and in complex with, a high-affinity phosphotyrosine-containing peptide ligand derived from the platelet-derived growth-factor receptor. (15)N R(1) and R(2) relaxation rates and steady-state [(1)H]-(15)N NOE values were measured by means of (1)H-(15)N correlated two-dimensional methods and were analyzed within the framework of the model-free formalism. Several residues in the BC loop and in the neighbouring secondary structural elements display fast local dynamics in the absence of phosphotyrosine peptide ligand as evidenced by below-average [(1)H]-(15)N NOE values. Furthermore, residue Gln41 (BC3) displays conformational exchange phenomena as indicated by an above-average R(2) relaxation rate. Upon binding of the phosphotyrosine peptide, the NOE values increase to values observed for regular secondary structure and the exchange contribution to the R(2) relaxation rate for Gln41 (BC3) vanishes. These observations indicate a loss of backbone flexibility upon ligand binding. Substantial exchange contributions for His56 (betaD4) and Cys57 (betaD5), which are known to make important interactions with the ligand, are attenuated upon ligand binding. Several residues in the betaD'-FB region and the BG loop, which contribute to the ligand binding surface of the protein, exhibit exchange terms which are reduced or vanish when the ligand is bound. Together, these observations suggest that ligand binding is accompanied by a loss of conformational flexibility on the ligand binding face of the protein. However, comparison with other SH2 domains reveals an apparent lack of consensus in the changes in dynamics induced by ligand binding. Exchange rates for individual residues were quantified in peptide-complexed p85alpha C-SH2 from the dependence of the exchange contributions on the CPMG delay in an R(2) series and show that peptide-complexed p85alpha C-SH2 is affected by multiple conformational exchange processes with exchange rate constants from 10(2) s(-1) to 7.10(3) s(-1). Mapping of the exchange-rate constants on the protein surface show a clustering of residues with similar exchange-rate constants and suggests that clustered residues are affected by a common predominant exchange process.     

Crystal structure of the carbohydrate recognition domain of p58/ERGIC-53, a protein involved in glycoprotein export from the endoplasmic reticulum

p58/ERGIC-53 is an animal calcium-dependent lectin that cycles between the endoplasmic reticulum (ER) and the Golgi complex and appears to act as a cargo receptor for a subset of soluble glycoproteins exported from the ER. We have determined the crystal structure of the carbohydrate recognition domain (CRD) of p58, the rat homologue of human ERGIC-53, to 1.46 A resolution. The fold and ligand binding site are most similar to those of leguminous lectins. The structure also resembles that of the CRD of the ER folding chaperone calnexin and the neurexins, a family of non-lectin proteins expressed on neurons. The CRD comprises one concave and one convex beta-sheet packed into a beta-sandwich. The ligand binding site resides in a negatively charged cleft formed by conserved residues. A large surface patch of conserved residues with a putative role in protein-protein interactions and oligomerization lies on the opposite side of the ligand binding site. Together with previous functional data, the structure defines a new and expanding class of calcium-dependent animal lectins and provides a starting point for the understanding of glycoprotein sorting between the ER and the Golgi.