Solution structure and backbone dynamics of the AF-6 PDZ domain/Bcr peptide complex

The human AF-6, a scaffold protein between cell membrane-associated proteins and the actin cytoskeleton, plays an important role in special cell-cell junctions and signal transduction. It can be phosphorylated by the protein kinase Bcr, which allows efficient binding of the C terminus of Bcr to the PDZ domain of AF-6 and consequently enhances the binding affinity of AF-6 to Ras. Formation of the AF-6, Bcr, and Ras ternary complex results in down-regulation of the Ras-mediated signal transduction pathway. To better understand the molecular basis for the recognition of the AF-6 PDZ domain and Bcr, we solve the solution structure of the AF-6 PDZ domain complexed with the C-terminal peptide of Bcr and explore the interactions between them in detail. Compared with previously reported structures, the complex exhibits a noncanonical binding mode of PDZ/peptide. Owing to the distinct residues involved in the AF-6 PDZ domain and Bcr peptide interaction, the interaction mode does not adapt to the existing classification rules that have been put forward, based on the ligand or the PDZ domain specificity. Furthermore, the PDZ domain of AF-6 can bind to the C terminus of Bcr efficiently after phosphorylation of AF-6 by the Bcr kinase. The phosphorylation may induce a conformational change of AF-6, which makes the binding surface on the PDZ domain accessible to Bcr for efficient binding. This study not only characterizes the structural details of the AF-6 PDZ/Bcr peptide complex, but also provides a potential target for future drug design and disease therapy.     

Solution structure of a Nedd4 WW domain-ENaC peptide complex

Nedd4 is a ubiquitin protein ligase composed of a C2 domain, three (or four) WW domains and a ubiquitin ligase Hect domain. Nedd4 was demonstrated to bind the epithelial sodium channel (alphabetagammaENaC), by association of its WW domains with PY motifs (XPPXY) present in each ENaC subunit, and to regulate the cell surface stability of the channel. The PY motif of betaENaC is deleted or mutated in Liddle syndrome, a hereditary form of hypertension caused by elevated ENaC activity. Here we report the solution structure of the third WW domain of Nedd4 complexed to the PY motif-containing region of betaENaC (TLPIPGTPPPNYDSL, referred to as betaP2). A polyproline type II helical conformation is adopted by the PPPN sequence. Unexpectedly, the C-terminal sequence YDSL forms a helical turn and both the tyrosine and the C-terminal leucine contact the WW domain. This is unlike other proline-rich peptides complexed to WW domains, which bind in an extended conformation and lack molecular interactions with residues C-terminal to the tyrosine or the structurally equivalent residue in non-PY motif WW domain targets. The Nedd4 WW domain-ENaC betaP2 peptide structure expands our understanding of the mechanisms involved in WW domain-ligand recognition and the molecular basis of Liddle syndrome.     

Solution structure of a phage-derived peptide antagonist in complex with vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mediator of angiogenesis and vasculogenesis. VEGF is involved pathologically in cancer, proliferative retinopathy and rheumatoid arthritis, and as such represents an important therapeutic target. Three classes of disulfide-constrained peptides that antagonize binding of the VEGF dimer to its receptors, KDR and Flt-1, were identified previously using phage display methods. NMR studies of a representative peptide from the most potent class of these peptide antagonists, v107 (GGNECDAIRMWEWECFERL), were undertaken to characterize its interactions with VEGF. v107 has no defined structure free in solution, but binding to VEGF induces folding of the peptide. The solution structure of the VEGF receptor-binding domain-v107 complex was determined using 3940 (1970 per VEGF monomer) internuclear distance and 476 (238 per VEGF monomer) dihedral angle restraints derived from NMR data obtained using samples containing either (13)C/(15)N-labeled protein plus excess unlabeled peptide or (13)C/(15)N-labeled peptide plus excess unlabeled protein. Residual dipolar coupling restraints supplemented the structure determination of the complex and were found to increase significantly both the global precision of VEGF in the complex and the agreement with available crystal structures of VEGF. The calculated ensemble of structures is of high precision and is in excellent agreement with the experimental restraints. v107 has a turn-helix conformation with hydrophobic residues partitioned to one face of the peptide and polar or charged residues at the other face. Contacts between two v107 peptides and the VEGF dimer are mediated by primarily hydrophobic side-chain interactions. The v107-binding site on VEGF overlaps partially with the binding site of KDR and is similar to that for domain 2 of Flt-1. The structure of the VEGF-v107 complex provides new insight into how binding to VEGF can be achieved that may be useful for the design of small molecule antagonists.     

Solution structure of the voltage-gated Tim23 channel in complex with a mitochondrial presequence peptide

Dear Editor, Most mitochondrial proteins are synthesized in the cytosol and transported into various mitochondrial subcompartments in a process that is mediated...     

Ternary complex formation of pVHL, elongin B and elongin C visualized in living cells by a fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique

The tumor suppressor von Hippel-Lindau (VHL) gene product forms a complex with elongin B and elongin C, and acts as a recognition subunit of a ubiquitin E3 ligase. Interactions between components in the complex were investigated in living cells by fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM). Elongin B-cerulean or cerulean-elongin B was coexpressed with elongin C-citrine or citrine-elongin C in CHO-K1 cells. FRET signals were examined by measuring a change in the fluorescence lifetime of donors and by monitoring a corresponding fluorescence rise of acceptors. Clear FRET signals between elongin B and elongin C were observed in all combinations, except for the combination of elongin B-cerulean and citrine-elongin C. Although similar experiments to examine interaction between pVHL30 and elongin C linked to cerulean or citrine were performed, FRET signals were rarely observed among all the combinations. However, the signal was greatly increased by coexpression of elongin B. These results, together with results of coimmunoprecipitation experiment using pVHL, elongin C and elongin B, suggest that a conformational change of elongin C and/or pVHL was induced by binding of elongin B. The conformational change of elongin C was investigated by measuring changes in the intramolecular FRET signal of elongin C linked to cerulean and citrine at its N- and C-terminus, respectively. A strong FRET signal was observed in the absence of elongin B, and this signal was modestly increased by coexpression of elongin B, demonstrating that a conformation change of elongin C was induced by the binding of elongin B.     

Solution structure of a hydrocarbon stapled peptide inhibitor in complex with monomeric C-terminal domain of HIV-1 capsid

The human immunodeficiency virus type 1 (HIV-1) capsid protein plays a critical role in virus core particle assembly and is an important target for novel therapeutic strategies. In a previous study, we characterized the binding affinity of a hydrocarbon stapled helical peptide, NYAD-1, for the capsid protein (K(d) approximately 1 mum) and demonstrated its ability to penetrate the cell membrane (Zhang, H., Zhao, Q., Bhattacharya, S., Waheed, A. A., Tong, X., Hong, A., Heck, S., Goger, M., Cowburn, D., Freed, E. O., and Debnath, A. K. (2008) J. Mol. Biol. 378, 565-580). In cell-based assays, NYAD-1 colocalized with the Gag polyprotein during traffic to the plasma membrane and disrupted the formation of mature and immature virus particles in vitro systems. Here, we complement the cellular and biochemical data with structural characterization of the interactions between the capsid and a soluble peptide analogue, NYAD-13. Solution NMR methods were used to determine a high resolution structure of the complex between the inhibitor and a monomeric form of the C-terminal domain of the capsid protein (mCA-CTD). The intermolecular interactions are mediated by the packing of hydrophobic side chains at the buried interface and unperturbed by the presence of the olefinic chain on the solvent-exposed surface of the peptide. The results of the structural analysis provide valuable insight into the determinants for high affinity and selective inhibitors for HIV-1 particle assembly.     

Solution structure and dynamics of ribonuclease Sa.

We have used NMR methods to characterize the structure and dynamics of ribonuclease Sa in solution. The solution structure of RNase Sa was obtained using the distance constraints provided by 2,276 NOEs and the C6-C96 disulfide bond. The 40 resulting structures are well determined; their mean pairwise RMSD is 0.76 A (backbone) and 1.26 A (heavy atoms). The solution structures are similar to previously determined crystal structures, especially in the secondary structure, but exhibit new features: the loop composed of Pro 45 to Ser 48 adopts distinct conformations and the rings of tyrosines 51, 52, and 55 have reduced flipping rates. Amide protons with greatly reduced exchange rates are found predominantly in interior beta-strands and the alpha-helix, but also in the external 3/10 helix and edge beta-strand linked by the disulfide bond. Analysis of (15)N relaxation experiments (R1, R2, and NOE) at 600 MHz revealed five segments, consisting of residues 1-5, 28-31, 46-50, 60-65, 74-77, retaining flexibility in solution. The change in conformation entropy for RNase SA folding is smaller than previously believed, since the native protein is more flexible in solution than in a crystal.     

Solution structure and dynamics of a prototypical chordin-like cysteine-rich repeat (von Willebrand Factor type C module) from collagen IIA

Chordin-like cysteine-rich (CR) repeats (also referred to as von Willebrand factor type C (VWC) modules) have been identified in approximately 200 extracellular matrix proteins. These repeats, named on the basis of amino acid conservation of 10 cysteine residues, have been shown to bind members of the transforming growth factor-beta (TGF-beta) superfamily and are proposed to regulate growth factor signaling. Here we describe the intramolecular disulfide bonding, solution structure, and dynamics of a prototypical chordin-like CR repeat from procollagen IIA (CR(ColIIA)), which has been previously shown to bind TGF-beta1 and bone morphogenetic protein-2. The CR(ColIIA) structure manifests a two sub-domain architecture tethered by a flexible linkage. Initial structures were calculated using RosettaNMR, a de novo prediction method, and final structure calculations were performed using CANDID within CYANA. The N-terminal region contains mainly beta-sheet and the C-terminal region is more irregular with the fold constrained by disulfide bonds. Mobility between the N- and C-terminal sub-domains on a fast timescale was confirmed using NMR relaxation measurements. We speculate that the mobility between the two sub-domains may decrease upon ligand binding. Structure and sequence comparisons have revealed an evolutionary relationship between the N-terminal sub-domain of the CR module and the fibronectin type 1 domain, suggesting that these domains share a common ancestry. Based on the previously reported mapping of fibronectin binding sites for vascular endothelial growth factor to regions containing fibronectin type 1 domains, we discuss the possibility that this structural homology might also have functional relevance.     

Solution structure and dynamics of the single-chain hepatitis C virus NS3 protease NS4A cofactor complex

The backbone assignments, secondary structure, topology, and dynamics of the single-chain hepatitis C virus NS3 protease NS4A cofactor complex have been determined by NMR spectroscopy. Residues I34 to S181 of NS3 and the central three residues of the NS4A cofactor were assigned and the secondary structure was verified for these residues. In several X-ray structures of NS4A-bound NS3 protease, residues 1 to 28 are stabilized by crystal packing, which allows for the formation of the A0 strand and alpha0 helix. In solution, these N-terminal residues are largely unassigned and no evidence of a well-structured A0 strand or alpha0 helix was detected. NOEs between residues in the E1-F1 loop (containing D81) and the alpha1 helix (containing H57) together with the detection of a D81-H57 hydrogen bond indicate that in solution the catalytic triad (D81, H57, S139) of the protease is better ordered in the presence of the NS4A cofactor. This is consistent with the earlier crystallographic results and may explain the observed increase in catalytic activity of the enzyme due to NS4A binding. A model-free analysis of our relaxation data indicates substantial exchange rates for residues V51-D81, which comprise the upper part of the N-terminal beta-barrel. A comparison of chemical-shift differences between NS3 protease and the NS3 protease-NS4A complex shows extensive chemical-shift changes for residues V51-D81 indicating that non-local structural changes occur upon NS4A binding to the NS3 protease that are propagated well beyond the protease-cofactor interaction site. This is consistent with crystallographic data that reveal large structural rearrangements of the strand and loop regions formed by residues V51-D81 as a result of NS4A binding. The coincidence of large exchange rates for the NS3 protease-NS4A complex with chemical-shift differences due to NS4A binding suggests that residues V51-D81 of the NS3 protease NS4A complex are in slow exchange with a NS4A-free conformation of NS3 protease.     

Solution structure of a let-7 miRNA:lin-41 mRNA complex from C. elegans

let-7 microRNA (miRNA) regulates heterochronic genes in developmental timing of the nematode Caenorhabditis elegans. Binding of miRNA to messenger RNA (mRNA) and structural features of the complex are crucial for gene silencing. We herein present the NMR solution structure of a model mimicking the interaction of let-7 miRNA with its complementary site (LCS 2) in the 3′ untranslated region (3′-UTR) of the lin-41 mRNA. A structural study was performed by NMR spectroscopy using NOE restraints, torsion angle restraints and residual dipolar couplings. The 33-nt RNA construct folds into a stem–loop structure that features two stem regions which are separated by an asymmetric internal loop. One of the stems comprises a GU wobble base pair, which does not alter its overall A-form RNA conformation. The asymmetric internal loop adopts a single, well-defined structure in which three uracils form a base triple, while two adenines form a base pair. The 3D structure of the construct gives insight into the structural aspects of interactions between let-7 miRNA and lin-41 mRNA.     

Solution structure of the luzopeptin-DNA complex.

The luzopeptin-d(C-A-T-G) complex (1 drug/duplex) has been generated in aqueous solution and its structure characterized by a combined application of two-dimensional NMR experiments and molecular dynamics calculations. One equivalent of luzopeptin binds to the self-complementary tetranucleotide duplex with the 2-fold symmetry of the antitumor agent and the DNA oligomer retained on complex formation. We have assigned the exchangeable and nonexchangeable proton resonances of luzopeptin and the d(C-A-T-G) duplex in the complex and identified the intermolecular proton-proton NOEs that define the alignment of the antitumor agent at its binding site in duplex DNA. The analysis was greatly aided by a large number of intermolecular NOEs involving exchangeable protons on both the luzopeptin and the DNA in the complex. The molecular dynamics calculations were guided by 140 intramolecular nucleic acid distance constraints, 74 intramolecular luzopeptin distance constraints, and 96 intermolecular distance constraints between luzopeptin and the nucleic acid protons in the complex. The quinoline rings of luzopeptin bisintercalate at d(C-A).d(T-G) steps in the d(C-A-T-G) duplex and sandwich two Watson-Crick A.T base pairs between the bisintercalation site. The long axis of the quinoline rings are collinear with the long axis of the flanking Watson-Crick C1.G4 and A2.T3 base pairs such that the OCH3-6 group is directed toward the C1-A2 step and the OH-3 group is directed toward the T3-G4 step in the complex. The quinoline chromophore stacks with purines on both strands, with the quinoline A ring stacked on A2 and the quinoline B ring stacked on G4 in the complex. The C1.G4 and A2.T3 base pairs that flank the intercalation sites are parallel to each other with partial overlap of T3 and G4 in the T3-G4 step but no overlap of C1 and A2 in the C1-A2 step in the complex. The cyclic depsipeptide ring of luzopeptin is positioned in the minor groove of the d(C-A-T-G) duplex with the oligopeptide and oligonucleotide chains running antiparallel to each other. The cyclic depsipeptide backbone of luzopeptin exhibits cis peptide bonds at Pyr-Gly and Gly-Sar steps in the luzopeptin-d(C-A-T-G) complex in solution, in contrast to all trans peptide bonds for free luzopeptin in the crystalline state.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the secondary structure of calmodulin in complex with a calmodulin-binding domain peptide.

The interaction between calcium-saturated chicken calmodulin and a peptide corresponding to the calmodulin-binding domain of the chicken smooth muscle myosin light chain kinase has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. Extensive 1H and 15N resonance assignments of calmodulin in the complex have been obtained from the analysis of two- and three-dimensional nuclear magnetic resonance spectra. The assignment of calmodulin in the complex was facilitated by the use of selective labeling of the protein with alpha-15N-labeled valine, alanine, lysine, leucine, and glycine. These provided reference points during the main-chain-directed analysis of three-dimensional spectra of complexes prepared with uniformly 15N-labeled calmodulin. The pattern of nuclear Overhauser effects (NOE) seen among main-chain amide NH, C alpha H, and C beta H hydrogens indicates that the secondary structure of the globular domains of calmodulin in the complex closely corresponds to that observed in the calcium-saturated state of the protein in the absence of bound peptide. However, the backbone conformation of residues 76-84 adopts an extended chain conformation upon binding of the peptide in contrast to its helical conformation in the absence of peptide. A sufficient number of NOEs between the globular domains of calmodulin and the bound peptide have been found to indicate that the N- and C-terminal regions of the peptide interact with the C- and N-terminal domains of calmodulin, respectively. The significance of these results are discussed in terms of recently proposed models for the structure of calmodulin-peptide complexes.     

Solution structure of a novel tryptophan-rich peptide with bidirectional antimicrobial activity

Trp-rich antimicrobial peptides play important roles in the host innate defense mechanisms of many plants, insects, and mammals. A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH(2), designated Pac-525, was found to possess improved activity against both gram-positive and -negative bacteria. We have determined that the solution structures of Pac-525 bound to membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The SDS micelle-bound structure of Pac-525 adopts an alpha-helical segment at residues Trp2, Arg3, and Arg4. The positively charged residues are clustered together to form a hydrophilic patch. The three hydrophobic residues Trp2, Val6, and Ile9 form a hydrophobic core. The surface electrostatic potential map indicates the three tryptophan indole rings are packed against the peptide backbone and form an amphipathic structure. Moreover, the reverse sequence of Pac-525, Ac-IWRVWRRWK-NH(2), designated Pac-525(rev), also demonstrates similar antimicrobial activity and structure in membrane-mimetic micelles and vesicles. A variety of biophysical and biochemical methods, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the antimicrobial activity of Pac-525 may be due to interactions with bacterial membranes.     

Solution structure of a tRNA with a large variable region: yeast tRNASer

Different chemical reagents were used to study the tertiary structure of yeast tRNASer, a tRNA with a large variable region: ethylnitrosourea, which alkylates the phosphate groups; dimethylsulphate, which methylates N-7 of guanosine and N-3 of cytosine; and diethylpyrocarbonate, which modifies N-7 of adenine. The non-reactivity of N-3 of cytidine 47:1, 47:6, 47:7 and 47:8 and the reactivity of cytidine 47:3 confirms the existence of a variable stem of four base-pairs and a short variable loop of three residues. For the N-7 positions in purines, accessible residues are G1, G10, Gm18, G19, G30, I34, G35, A36, i6A37, G45, G47, G47:5, G47:9 and G73. The protection of N-7 atoms of residues G9, G15, A21, A22 and G47:9 reflects the tertiary folding. Strong phosphate protection was observed for P8 to P11, P20:1 to P22, P48 to P50 and for P59 and P60. A model was built on a PS300 graphic system on the basis of these data and its stereochemistry refined. While trying to keep most tertiary interactions, we adapted the tertiary folding of the known structures of tRNAAsp and tRNAPhe to the present sequence and solution data. The resulting model has the variable arm not far from the plane of the common L-shaped structure. A generalization of this model to other tRNAs with large variable regions is discussed.     

Crystal structure of a membrane stomatin-specific protease in complex with a substrate peptide

Membrane-bound proteases are involved in various regulatory functions. A previous report indicated that the N-terminal region of PH1510p (1510-N) from the hyperthermophilic archaeon Pyrococcus horikoshii is a serine protease with a catalytic Ser-Lys dyad (Ser97 and Lys138) and specifically cleaves the C-terminal hydrophobic region of the p-stomatin PH1511p. In humans, an absence of stomatin is associated with a form of hemolytic anemia known as hereditary stomatocytosis. Here, the crystal structure of 1510-N K138A in complex with a peptide substrate was determined at 2.25 ? resolution. In the structure, a 1510-N dimer binds to one peptide. The six central residues (VIVLML) of the peptide are hydrophobic and in a pseudopalindromic structure and therefore favorably fit into the hydrophobic active tunnel of the 1510-N dimer, although 1510-N degrades the substrate at only one point. A comparison with unliganded 1510-N K138A revealed that the binding of the substrate causes a large rotational and translational displacement between protomers and produces a tunnel suitable for binding the peptide. When the peptide binds, the flexible L2 loop of one protomer forms β-strands, whereas that of the other protomer remains in a loop form, indicating that one protomer binds to the peptide more tightly than the other protomer. The Ala138 residues of the two protomers are located very close together (the distance between the two Cβ atoms is 3.6 ?). Thus, in wild-type 1510-N, the close positioning of the catalytic Ser97 and Lys138 residues may be induced by electrostatic repulsion of the two Lys138 side chains of the protomers.     

Solution structure of calmodulin and its complex with a myosin light chain kinase fragment.

The solution structure of Ca2+ ligated calmodulin and of its complex with a 26-residue peptide fragment of skeletal muscle myosin light chain kinase (skMLCK) have been investigated by multi-dimensional NMR. In the absence of peptide, the two globular domains of calmodulin adopt the same structure as observed in the crystalline form. The so-called 'central helix' which is observed in the crystalline state is disrupted in solution. 15N relaxation studies show that residues Asp78 through Ser81, located near the middle of this 'central helix', form a very flexible link between the two globular domains. In the presence of skMLCK target peptide, the peptide-protein complex adopts a globular ellipsoidal shape. The helical peptide is located in a hydrophobic channel that goes through the center of the complex and makes an angle of approximately 45 degrees with the long axis of the ellipsoid.     

Solution structure of ileal lipid binding protein in complex with glycocholate.

Ileal lipid binding protein (ILBP) is a cytosolic lipid-binding protein that binds both bile acids and fatty acids. We have determined the solution structure of porcine ILBP in complex with glycocholate by homonuclear and heteronuclear two-dimensional NMR spectroscopy. The conformation of the protein-ligand complex was determined by restrained energy minimization and simulated annealing calculations after docking the glycocholate ligand into the protein structure. The overall tertiary structure of ILBP is highly analogous to the three-dimensional structures of several other intracellular lipid binding proteins (LBPs). Like the apo-structure, the bile-acid complex of ILBP is composed of 10 anti-parallel beta-strands that form a water-filled clam-shell structure, and two short alpha-helices. Chemical shift data indicated that the bile acid ligand is bound inside the protein cavity. Furthermore, 13C-edited heteronuclear single-quantum correlation-NOESY experiments showed NOE contacts between several aromatic residues located in the proposed bile acid portal region and the 13C-labeled ligand. A single bile acid molecule is bound inside the protein, with the steroid moiety penetrating deep into the water-accessible internal cavity, such that ring A is located right above the plane of the Trp49 indole ring. The carboxylate tail of the ligand is protruding from the proposed bile acid portal into the surrounding aqueous solution. The body of the steroid moiety is oriented with the nonpolar face in contact with the mostly hydrophobic residues of beta-strands C, D and E, while the polar face shows contacts with the side-chains of Tyr97, His99, Glu110 and Arg121 in beta-strands H, I and J. Thus, the conformational arrangement of the ligand complex suggests that the binding affinity of ILBP for bile acid molecules is based mainly on strong hydrophobic interactions inside the protein cavity. Furthermore, this binding mode explains how ILBP can transport unconjugated and conjugated bile acids.     

Solution structure of a platelet receptor peptide bound to bovine alpha-thrombin.

NMR experiments were carried out to study the interaction of thrombin with a synthetic peptide, ESKATNATLDPR, derived from the newly-identified platelet receptor for thrombin [Vu, T.-K. H., Hung, D. T., Wheaton, V. I., & Coughlin, S. R. (1991) Cell 64, 1057-1068]. On the basis of the observation of the thrombin-induced line broadening and transferred NOEs, binding of the peptide was found to be located exclusively within residues LDPR of the proteolytic cleavage site LDPR/S essential for receptor activation by thrombin. Measurement of transferred NOEs and molecular modeling indicate that the side chain of the Asp(P3) residue may form a hydrogen bond with thrombin and, by doing so, it is brought near a positively-charged thrombin residue Arg(221A), thereby partially neutralizing the negative charge of an Asp residue at this site of protein substrates. The hydrophobic side chains of residues Leu(P4) and Pro(P2) reside on the same side of the peptide backbone as indicated by transferred NOEs and were found by modeling to fit into a hydrophobic cage around the thrombin active site. These results suggest that the interaction of thrombin with protein substrates such as prothrombin, protein C, protein S, the platelet receptor, and the A alpha- and B beta-chains of fibrinogen all follow the same canonical binding mode in that the substrate forms an antiparallel beta-strand with thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)