Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases

Nonribosomal peptide synthetases (NRPSs) are multimodular proteins capable of producing important peptide natural products. Using an assembly line process, the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS adenylation and PCP domains identified several conserved proline residues that are not found in standalone adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the adenylation‐PCP linker. The LPxP motif interacts with a region on the adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C‐terminal subdomain of the adenylation domain may coordinate movement of the PCP with the conformational change of the adenylation domain. Through this work, we extend the conserved A10 motif of the adenylation domain and identify residues that enable proper adenylation domain function. Proteins 2014; 82:2691–2702. © 2014 Wiley Periodicals, Inc.     

Structure of PA1221, a nonribosomal peptide synthetase containing adenylation and peptidyl carrier protein domains

Many bacteria use large modular enzymes for the synthesis of polyketide and peptide natural products. These multidomain enzymes contain integrated carrier domains that deliver bound substrates to multiple catalytic domains, requiring coordination of these chemical steps. Nonribosomal peptide synthetases (NRPSs) load amino acids onto carrier domains through the activity of an upstream adenylation domain. Our lab recently determined the structure of an engineered two-domain NRPS containing fused adenylation and carrier domains. This structure adopted a domain-swapped dimer that illustrated the interface between these two domains. To continue our investigation, we now examine PA1221, a natural two-domain protein from Pseudomonas aeruginosa. We have determined the amino acid specificity of this new enzyme and used domain specific mutations to demonstrate that loading the downstream carrier domain within a single protein molecule occurs more quickly than loading of a nonfused carrier domain intermolecularly. Finally, we have determined crystal structures of both apo- and holo-PA1221 proteins, the latter using a valine-adenosine vinylsulfonamide inhibitor that traps the adenylation domain-carrier domain interaction. The protein adopts an interface similar to that seen with the prior adenylation domain-carrier protein construct. A comparison of these structures with previous structures of multidomain NRPSs suggests that a large conformational change within the NRPS adenylation domains guides the carrier domain into the active site for thioester formation.     

The 1.75 A crystal structure of acetyl-CoA synthetase bound to adenosine-5'-propylphosphate and coenzyme A

Acetyl-coenzyme A synthetase catalyzes the two-step synthesis of acetyl-CoA from acetate, ATP, and CoA and belongs to a family of adenylate-forming enzymes that generate an acyl-AMP intermediate. This family includes other acyl- and aryl-CoA synthetases, firefly luciferase, and the adenylation domains of the modular nonribosomal peptide synthetases. We have determined the X-ray crystal structure of acetyl-CoA synthetase complexed with adenosine-5'-propylphosphate and CoA. The structure identifies the CoA binding pocket as well as a new conformation for members of this enzyme family in which the approximately 110-residue C-terminal domain exhibits a large rotation compared to structures of peptide synthetase adenylation domains. This domain movement presents a new set of residues to the active site and removes a conserved lysine residue that was previously shown to be important for catalysis of the adenylation half-reaction. Comparison of our structure with kinetic and structural data of closely related enzymes suggests that the members of the adenylate-forming family of enzymes may adopt two different orientations to catalyze the two half-reactions. Additionally, we provide a structural explanation for the recently shown control of enzyme activity by acetylation of an active site lysine.     

Crystal structure of DltA. Implications for the reaction mechanism of non-ribosomal peptide synthetase adenylation domains

DltA, the D-alanine:D-alanyl carrier protein ligase responsible for the initial step of lipoteichoic acid D-alanylation in Gram-positive bacteria, belongs to the adenylation domain superfamily, which also includes acetyl-CoA synthetase and the adenylation domains of non-ribosomal synthetases. The two-step reaction catalyzed by these enzymes (substrate adenylation followed by transfer to the reactive thiol group of CoA or the phosphopantheinyl prosthetic group of peptidyl carrier proteins) has been suggested to proceed via large scale rearrangements of structural domains within the enzyme. The structures of DltA reported here reveal the determinants for D-Ala substrate specificity and confirm that the peptidyl carrier protein-activating domains are able to adopt multiple conformational states, in this case corresponding to the thiolation reaction. Comparisons of available structures allow us to propose a mechanism whereby small perturbations of finely balanced metastable structural states would be able to direct an ordered formation of non-ribosomal synthetase products.     

Structural Basis for the ATP-dependent Configuration of Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase

o-Succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme targeted for development of novel antibiotics in the menaquinone biosynthesis. Using its crystal structures in a ligand-free form or in complex with nucleotides, a conserved pattern is identified in the interaction between ATP and adenylating enzymes, including acyl/aryl-CoA synthetases, adenylation domains of nonribosomal peptide synthetases, and luciferases. It involves tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site. In MenE catalysis, this ATP-enzyme interaction creates a new binding site for the carboxylate substrate, allowing revelation of the determinants of substrate specificities and in-line alignment of the two substrates for backside nucleophilic substitution reaction by molecular modeling. In addition, the ATP-enzyme interaction is suggested to play a crucial catalytic role by mutation of the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction has also clarified the positioning and catalytic role of a conserved lysine residue in stabilization of the transition state. These findings provide new insights into the adenylation half-reaction in the domain alteration catalytic mechanism of the adenylate-forming enzymes.     

Analysis of engineered multifunctional peptide synthetases. Enzymatic characterization of surfactin synthetase domains in hybrid bimodular systems.

The combinatorial reorganization of distinct modules of multimodular peptide synthetases is of increasing interest for the generation of new peptides with optimized bioactive properties. Each module is at least composed of enzymatic domains responsible for the adenylation, thioester formation, and condensation of an amino acid residue of the final peptide product. We analyzed various possible fusion sites for the recombination of peptide synthetases and evaluated the impact of different recombination strategies on the amino acid adenylation and acyl-thioester formation activities of peptide synthetase modules. Hybrid bimodular peptide synthetases were generated by recombination of the corresponding reading frames encoding for L-glutamic acid- and L-leucine-specific modules of surfactin synthetase SrfA-A at presumed inner- and intradomainic regions. We demonstrate that fusions at a previously postulated hinge region, dividing the amino acid adenylating domains of peptide synthetase modules into two subdomains, and at the highly conserved 4'-phosphopantetheine binding motif in acyl-thioester forming domains resulted in enzymatically active hybrid domains. By contrast, most manipulations in condensation domains like deletions, the complete exchange or the construction of chimeric domains considerably reduced or completely abolished the amino acid adenylation and thioester formation activity of the hybrid module.     

Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5

Chlamydia trachomatis is a bacterial pathogen responsible for one of the most prevalent sexually transmitted infections worldwide. Its unique development cycle has limited our understanding of its pathogenic mechanisms. However, CtHtrA has recently been identified as a potential C. trachomatis virulence factor. CtHtrA is a tightly regulated quality control protein with a monomeric structural unit comprised of a chymotrypsin-like protease domain and two PDZ domains. Activation of proteolytic activity relies on the C-terminus of the substrate allosterically binding to the PDZ1 domain, which triggers subsequent conformational change and oligomerization of the protein into 24-mers enabling proteolysis. This activation is mediated by a cascade of precise structural arrangements, but the specific CtHtrA residues and structural elements required to facilitate activation are unknown. Using in vitro analysis guided by homology modeling, we show that the mutation of residues Arg362 and Arg224, predicted to disrupt the interaction between the CtHtrA PDZ1 domain and loop L3, and between loop L3 and loop LD, respectively, are critical for the activation of proteolytic activity. We also demonstrate that mutation to residues Arg299 and Lys160, predicted to disrupt PDZ1 domain interactions with protease loop LC and strand β5, are also able to influence proteolysis, implying their involvement in the CtHtrA mechanism of activation. This is the first investigation of protease loop LC and strand β5 with respect to their potential interactions with the PDZ1 domain. Given their high level of conservation in bacterial HtrA, these structural elements may be equally significant in the activation mechanism of DegP and other HtrA family members.     

Solution structure of PCP, a prototype for the peptidyl carrier domains of modular peptide synthetases

BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are large modular enzymes responsible for the synthesis of a variety of microbial bioactive peptides. They consist of modules that each recognise and incorporate one specific amino acid into the peptide product. A module comprises several domains, which carry out the individual reaction steps. After activation by the adenylation domain, the amino acid substrate is covalently tethered to a 4'-phosphopantetheinyl cofactor of a peptidyl carrier domain (PCP) that passes the substrate to the reaction centres of the consecutive domains. RESULTS: The solution structure of PCP, a distinct peptidyl carrier protein derived from the equivalent domain of an NRPS, was solved using NMR techniques. PCP is a distorted four-helix bundle with an extended loop between the first two helices. Its overall fold resembles the topology of acyl carrier proteins (ACPs) from Escherichia coli fatty acid synthase and actinorhodin polyketide synthase from Streptomyces coelicolor; however, the surface polarity and the length and relative alignment of the helices are different. The conserved serine, which is the cofactor-binding site, has the same location as in the ACPs and is situated within a stretch of seven flexible residues. CONCLUSIONS: The structure of PCP reflects its character as a protein domain. The fold is well defined between residues 8 and 82 and the structural core of the PCP domain can now be defined as a region spanning 37 amino acids in both directions from the conserved serine. The flexibility of the post-translationally modified site might have implications for interactions with the cooperating proteins or NRPS domains.     

Role of a conserved arginine residue in linkers between the ketosynthase and acyltransferase domains of multimodular polyketide synthases

The role of interdomain linkers in modular polyketide synthases is poorly understood. Analysis of the 6-deoxyerythronolide B synthase (DEBS) has yielded a model in which chain elongation is governed by interactions between the acyl carrier protein domain and the ketosynthase domain plus an adjacent linker. Alanine scanning mutagenesis of the conserved residues of this linker in DEBS module 3 led to the identification of the R513A mutant with a markedly reduced rate of chain elongation. Limited proteolysis supported a structural role for this Arg. Our findings highlight the importance of domain-linker interactions in assembly line polyketide biosynthesis.     

Allosteric regulation of Hsp70 chaperones involves a conserved interdomain linker

The 70-kDa heat shock proteins (Hsp70) are essential members of the cellular chaperone machinery that assists protein-folding processes. To perform their functions Hsp70 chaperones toggle between two nucleotide-controlled conformational states. ATP binding to the ATPase domain triggers the transition to the low affinity state of the substrate-binding domain, while substrate binding to the substrate-binding domain in synergism with the action of a J-domain-containing cochaperone stimulates ATP hydrolysis and thereby transition to the high affinity state. Thus, ATPase and substrate-binding domains mutually affect each other through an allosteric control mechanism, the basis of which is largely unknown. In this study we identified two positively charged, surface-exposed residues in the ATPase domain and a negatively charged residue in the linker connecting both domains that are important for interdomain communication. Furthermore, we demonstrate that the linker alone is sufficient to stimulate the ATPase activity, an ability that is lost upon amino acid replacement. The linker therefore is most likely the lever that is wielded by the substrate-binding domain and the cochaperone onto the ATPase domain to induce a conformation favorable for ATP hydrolysis. Based on our results we propose a mechanism of interdomain communication.     

Structural domains of human apolipoprotein B-100. Differential accessibility to limited proteolysis of B-100 in low density and very low density lipoproteins

The structural domains of human apolipoprotein B-100 in low density lipoproteins (LDL) and the conformational changes of B-100 that accompany the conversion of very low density lipoproteins (VLDL) to LDL were investigated by limited proteolysis with 12 endoproteases of various specificities, and their cleavage sites were determined. In B-100 of LDL, we identified two peptide regions that are highly susceptible to proteolytic cleavage. One region encompassed about 40 amino acids (residues 1280-1320, designated as the NH2-terminal region) and the other about 100 amino acids (residues 3180-3280, designated as the COOH-terminal region). IN LDL, the cleavage sites in both susceptible regions of B-100 were readily accessible to limited proteolysis; but in VLDL, only sites in the COOH-terminal region were readily accessible. Moreover, B-100 in VLDL appeared less degraded than B-100 in LDL by all enzymes used. Reduction of disulfide bonds of B-100 in both LDL and VLDL before digestion by Staphylococcus aureus V8 protease and clostripain exposed additional cleavage sites and increased the rate of B-100 degradation, suggesting that disulfide bonds probably exert conformational constraints. These results indicate the presence of three principal structural domains in B-100 of LDL that are relatively resistant to limited proteolysis. These three domains are connected by the two susceptible peptide regions. Our results also demonstrate differential accessibility of cleavage sites in B-100 of LDL and VLDL to limited proteolysis. This differential accessibility suggests that substantial changes in the conformation or environment of B-100 accompany the conversion of VLDL to LDL.     

ε-Poly-l-Lysine Peptide Chain Length Regulated by the Linkers Connecting the Transmembrane Domains of ε-Poly-l-Lysine Synthetase

ε-Poly-l-lysine (ε-PL), consisting of 25 to 35 l-lysine residues with linkages between the α-carboxyl groups and ε-amino groups, is produced by Streptomyces albulus NBRC14147. ε-PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates ε-PL chain length diversity (25- to 35-mer), but the processes that control the chain length of ε-PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the ε-PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of ε-PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of ε-PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of ε-PL.     

Mapping of the vitronectin-binding site on the urokinase receptor: involvement of a coherent receptor interface consisting of residues from both domain I and the flanking interdomain linker region

The urokinase-type plasminogen activator receptor (uPAR) has been implicated as a modulator of several biochemical processes that are active during tumor invasion and metastasis, e.g. extracellular proteolysis, cell adhesion, and cell motility. The structural basis for the high affinity interaction between the urokinase-type plasminogen activator (uPA) and uPAR, which focuses cell surface-associated plasminogen activation in vivo, is now thoroughly characterized by site-directed mutagenesis studies and x-ray crystallography. In contrast, the structural basis for the interaction between uPAR and the extracellular matrix protein vitronectin, which is involved in the regulation of cell adhesion and motility, remains to be clarified. In this study, we have identified the functional epitope on uPAR that is responsible for its interaction with the full-length, extended form of vitronectin by using a comprehensive alanine-scanning library of purified single-site uPAR mutants (244 positions tested). Interestingly, the five residues identified as "hot spots" for vitronectin binding form a contiguous epitope consisting of two exposed loops connecting the central fourstranded beta-sheet in uPAR domain I (Trp(32), Arg(58), and Ile(63)) as well as a proximal region of the flexible linker peptide connecting uPAR domains I and II (Arg(91) and Tyr(92)). This binding topology provides the molecular basis for the observation that uPAR can form a ternary complex with uPA and vitronectin. Furthermore, it raises the intriguing possibility that the canonical receptor and inhibitor for uPA (uPAR and PAI-1) may have reached a convergent solution for binding to the somatomedin B domain of vitronectin.     

Biochemical and crystallographic analysis of substrate binding and conformational changes in acetyl-CoA synthetase

The adenylate-forming enzymes, including acyl-CoA synthetases, the adenylation domains of non-ribosomal peptide synthetases (NRPS), and firefly luciferase, perform two half-reactions in a ping-pong mechanism. We have proposed a domain alternation mechanism for these enzymes whereby, upon completion of the initial adenylation reaction, the C-terminal domain of these enzymes undergoes a 140 degrees rotation to perform the second thioester-forming half-reaction. Structural and kinetic data of mutant enzymes support this hypothesis. We present here mutations to Salmonella enterica acetyl-CoA synthetase (Acs) and test the ability of the enzymes to catalyze the complete reaction and the adenylation half-reaction. Substitution of Lys609 with alanine results in an enzyme that is unable to catalyze the adenylate reaction, while the Gly524 to leucine substitution is unable to catalyze the complete reaction yet catalyzes the adenylation half-reaction with activity comparable to the wild-type enzyme. The positions of these two residues, which are located on the mobile C-terminal domain, strongly support the domain alternation hypothesis. We also present steady-state kinetic data of putative substrate-binding residues and demonstrate that no single residue plays a dominant role in dictating CoA binding. We have also created two mutations in the active site to alter the acyl substrate specificity. Finally, the crystallographic structures of wild-type Acs and mutants R194A, R584A, R584E, K609A, and V386A are presented to support the biochemical analysis.     

The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains.

The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His6-tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PPi exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The Km values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3' end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.     

Phosphorylation driven motions in the COOH-terminal Src kinase,CSK, revealed through enhanced hydrogen-deuterium exchange and mass spectrometry (DXMS)

Previous kinetic studies demonstrated that nucleotide-derived conformational changes regulate function in the COOH-terminal Src kinase. We have employed enhanced methods of hydrogen-deuterium exchange-mass spectrometry (DXMS) to probe conformational changes on CSK in the absence and presence of nucleotides and thereby provide a structural framework for understanding phosphorylation-driven conformational changes. High quality peptic fragments covering approximately 63% of the entire CSK polypeptide were isolated using DXMS. Time-dependent deuterium incorporation into these probes was monitored to identify short peptide segments that exchange differentially with solvent. Regions expected to lie in loops exchange rapidly, whereas other regions expected to lie in stable secondary structure exchange slowly with solvent implying that CSK adopts a modular structure. The ATP analog, AMPPNP, protects probes in the active site and distal regions in the large and small lobes of the kinase domain, the SH2 domain, and the linker connecting the SH2 and kinase domains. The product ADP protects similar regions of the protein but the extent of protection varies markedly in several crucial areas. These areas correspond to the activation loop and helix G in the kinase domain and several inter-domain regions. These results imply that delivery of the gamma phosphate group of ATP induces unique local and long-range conformational changes in CSK that may influence regulatory motions in the catalytic pathway.     

Substrate-Induced Conformational Changes of the Tyrocidine Synthetase 1 Adenylation Domain Probed by Intrinsic Trp Fluorescence

Nonribosomal peptide synthetases (NRPS) are multifunctional proteins that catalyze the synthesis of the peptide products with enormous biological potential. The process of biosynthesis starts with the adenylation (A) domain, which during the catalytic cycle undergoes extensive structural rearrangements. In this paper, we present the first study of the tyrocidine synthetase 1 A-domain (TycA-A) fluorescence properties. The TycA-A protein contains five potentially fluorescent Trp residues at positions 227, 301, 323, 376 and 406. The contribution of each Trp to the TycA-A emission was determined using protein variants bearing single Trp to Phe substitutions. The accessibility of the Trp side chains during adenylation showed that only W227 is affected by substrate binding. The protein variant containing solely fluorescent W227 residue was constructed and further used as a probe to explore the binding effect of different non-cognate amino acid substrates. The results indicate a different accessibility of W227 residue in the presence of non-cognate amino acids, which might offer an explanation for the higher aminoacyl-adenenylate leakage. Overall, our results suggest that intrinsic tryptophan fluorescence could be used as a method to probe the effect of substrate binding on the local structure in NRPS adenylation domains.     

Structural basis for phosphoserine-proline recognition by group IV WW domains

Pin1 contains an N-terminal WW domain and a C-terminal peptidyl-prolyl cis-trans isomerase (PPIase) domain connected by a flexible linker. To address the energetic and structural basis for WW domain recognition of phosphoserine (P.Ser)/phosphothreonine (P. Thr)- proline containing proteins, we report the energetic and structural analysis of a Pin1-phosphopeptide complex. The X-ray crystal structure of Pin1 bound to a doubly phosphorylated peptide (Tyr-P.Ser-Pro-Thr-P.Ser-Pro-Ser) representing a heptad repeat of the RNA polymerase II large subunit's C-terminal domain (CTD), reveals the residues involved in the recognition of a single P.Ser side chain, the rings of two prolines, and the backbone of the CTD peptide. The side chains of neighboring Arg and Ser residues along with a backbone amide contribute to recognition of P.Ser. The lack of widespread conservation of the Arg and Ser residues responsible for P.Ser recognition in the WW domain family suggests that only a subset of WW domains can bind P.Ser-Pro in a similar fashion to that of Pin1.     

Molecular characterization of the principal substrate binding site of the ubiquitous folding catalyst protein disulfide isomerase

Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI). The effectiveness of PDI as a catalyst of native disulfide bond formation in folding polypeptides depends on the ability to catalyze disulfide-dithiol exchange, to bind non-native proteins, and to trigger conformational changes in the bound substrate, allowing access to buried cysteine residues. It is known that the b' domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates. Here we use homology modeling to define more precisely the boundaries of the b' domain and show the existence of an intradomain linker between the b' and a' domains. We have expressed the recombinant b' domain thus defined; the stability and conformational properties of the recombinant product confirm the validity of the domain boundaries. We have modeled the tertiary structure of the b' domain and identified the primary substrate binding site within it. Mutations within this site, expressed both in the isolated domain and in full-length PDI, greatly reduce the binding affinity for small peptide substrates, with the greatest effect being I272W, a mutation that appears to have no structural effect.     

Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study

Background

Human blood coagulation factor VIII (fVIII) is a large plasma glycoprotein with sequential domain arrangement in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A1, A2 and A3 domains are interconnected by long linker peptides (a1, a2 and a3) that possess the activation sites. Proteolysis of fVIII zymogen by thrombin or factor Xa results in the generation of the activated form (fVIIIa) which serves as a critical co-factor for factor IXa (fIXa) enzyme in the intrinsic coagulation pathway.

Results

In our efforts to elucidate the structural differences between fVIII and fVIIIa, we developed the solution structural models of both forms, starting from an incomplete 3.7 Å X-ray crystal structure of fVIII zymogen, using explicit solvent MD simulations. The full assembly of B-domainless single-chain fVIII was built between the A1-A2 (Ala1-Arg740) and A3-C1-C2 (Ser1669-Tyr2332) domains. The structural dynamics of fVIII and fVIIIa, simulated for over 70 ns of time scale, enabled us to evaluate the integral motions of the multi-domain assembly of the co-factor and the possible coordination pattern of the functionally important calcium and copper ion binding in the protein.

Conclusions

MD simulations predicted that the acidic linker peptide (a1) between the A1 and A2 domains is largely flexible and appears to mask the exposure of putative fIXa enzyme binding loop (Tyr555-Asp569) region in the A2 domain. The simulation of fVIIIa, generated from the zymogen structure, predicted that the linker peptide (a1) undergoes significant conformational reorganization upon activation by relocating completely to the A1-domain. The conformational transition led to the exposure of the Tyr555-Asp569 loop and the surrounding region in the A2 domain. While the proposed linker peptide conformation is predictive in nature and warrants further experimental validation, the observed conformational differences between the zymogen and activated forms may explain and support the large body of experimental data that implicated the critical importance of the cleavage of the peptide bond between the Arg372 and Ser373 residues for the full co-factor activity of fVIII.