Molecular dynamics simulations reveal that apo‐HisJ can sample a closed conformation

The Escherichia coli histidine binding protein HisJ is a type II periplasmic binding protein (PBP) that preferentially binds histidine and interacts with its cytoplasmic membrane ABC transporter, HisQMP₂, to initiate histidine transport. HisJ is a bilobal protein where the larger Domain 1 is connected to the smaller Domain 2 via two linking strands. Type II PBPs are thought to undergo “Venus flytrap” movements where the protein is able to reversibly transition from an open to a closed conformation. To explore the accessibility of the closed conformation to the apo state of the protein, we performed a set of all‐atom molecular dynamics simulations of HisJ starting from four different conformations: apo‐open, apo‐closed, apo‐semiopen, and holo‐closed. The simulations reveal that the closed conformation is less dynamic than the open one. HisJ experienced closing motions and explored semiopen conformations that reverted to closed forms resembling those found in the holo‐closed state. Essential dynamics analysis of the simulations identified domain closing/opening and twisting as main motions. The formation of specific inter‐hinge strand and interdomain polar interactions contributed to the adoption of the closed apo‐conformations although they are up to 2.5‐fold less prevalent compared with the holo‐closed simulations. The overall sampling of the closed form by apo‐HisJ provides a rationale for the binding of unliganded PBPs with their cytoplasmic membrane ABC transporters. Proteins 2014; 82:386–398. © 2013 Wiley Periodicals, Inc.     

Investigation of the mechanism of domain closure in citrate synthase by molecular dynamics simulation

Six, 2 ns molecular dynamics simulations have been performed on the homodimeric enzyme citrate synthase. In three, both monomers were started from the open, unliganded X-ray conformation. In the remaining three, both monomers started from a closed, liganded X-ray conformation, with the ligands removed. Projecting the motion from the simulations onto the experimental domain motion revealed that the free-energy profile is rather flat around the open conformation, with steep sides. The most closed conformations correspond to hinge-bending angles of 12-14 compared to the 20 degrees that occurs upon the binding of oxaloacetate. It is also found that the open, unliganded X-ray conformation is situated at the edge of the steep rise in free energy, although conformations that are about 5 degrees more open were sampled. A rigid-body essential dynamics analysis of the combined open trajectories has shown that domain motions in the direction of the closed X-ray conformation are compatible with the natural domain motion of the unliganded protein, which has just two main degrees of freedom. The simulations starting from the closed conformation suggest a free-energy profile with a small barrier in going from the closed to open conformation. A combined essential dynamics and hinge-bending analysis of a trajectory that spontaneously converts from the closed to open state shows an almost exact correspondence to the experimental transition that occurs upon ligand binding. The simulations support the conclusion from an earlier analysis of the experimental transition that the beta-hairpin acts as a mechanical hinge by attaching the small domain to the large domain through a conserved main-chain hydrogen bond and salt-bridges, and allowing rotation to occur via its two flexible termini. The results point to a mechanism of domain closure in citrate synthase that has analogy to the process of closing a door.     

Molecular dynamics simulations of Factor Xa: insight into conformational transition of its binding subsites

Protein flexibility and conformational diversity is well known to be a key characteristic of the function of many proteins. Human blood coagulation proteins have multiple substrates, and various protein-protein interactions are required for the smooth functioning of the coagulation cascade to maintain blood hemostasis. To address how a protein may cope with multiple interactions with its structurally diverse substrates and the accompanied structural changes that may drive these changes, we studied human Factor X. We employed 20 ns of molecular dynamics (MD) and steered molecular dynamics (SMD) simulations on two different conformational forms of Factor X, open and closed, and observed an interchangeable conformational transition from one to another. This work also demonstrates the roles of various aromatic residues involved in aromatic-aromatic interactions, which make this dynamic transition possible.     

MD simulations reveal alternate conformations of the oxyanion hole in the Zika virus NS2B/NS3 protease

Recent crystallography studies have shown that the binding site oxyanion hole plays an important role in inhibitor binding, but can exist in two conformations (active/inactive). We have undertaken molecular dynamics (MD) calculations to better understand oxyanion hole dynamics and thermodynamics. We find that the Zika virus (ZIKV) NS2B/NS3 protease maintains a stable closed conformation over multiple 100-ns conventional MD simulations in both the presence and absence of inhibitors. The S1, S2, and S3 pockets are stable as well. However, in two of eight simulations, the A132-G133 peptide bond in the binding pocket of S1' spontaneously flips to form a 3₁₀-helix that corresponds to the inactive conformation of the oxyanion hole, and then maintains this conformation until the end of the 100-ns conventional MD simulations without inversion of the flip. This conformational change affects the S1' pocket in ZIKV NS2B/NS3 protease active site, which is important for small molecule binding. The simulation results provide evidence at the atomic level that the inactive conformation of the oxyanion hole is more favored energetically when no specific interactions are formed between substrate/inhibitor and oxyanion hole residues. Interestingly, however, transition between the active and inactive conformation of the oxyanion hole can be observed by boosting the valley potential in accelerated MD simulations. This supports a proposed induced-fit mechanism of ZIKV NS2B/NS3 protease from computational methods and provides useful direction to enhance inhibitor binding predictions in structure-based drug design.     

Conformational dynamics of threonine 195 and the S1 subsite in functional trypsin variants

Replacing the catalytic serine in trypsin with threonine (S195T variant) leads to a nearly complete loss of catalytic activity, which can be partially restored by eliminating the C42-C58 disulfide bond. The 0.69 μs of combined explicit solvent molecular dynamics (MD) simulations revealed continuous rearrangement of T195 with different conformational preferences between five trypsin variants tested. Among three conformational families observed for the T195 residue, one showed the T195 hydroxyl in a conformation analogous to that of the serine residue in wild-type trypsin, positioning the hydroxyl oxygen atom for attack on the carbonyl carbon of the peptide substrate. MD simulations demonstrated that this conformation was more populated for the C42A/C58V/S195T and C42A/C58A/S195T triple variants than for the catalytically inactive S195T variant and correlated with restored enzymatic activities for triple variants. In addition, observation of the increased motion of the S214-G219 segment in the S195T substituted variants suggested an existence of open and closed conformations for the substrate binding pocket. The closed conformation precludes access to the S1 binding site and could further reduce enzymatic activities for triple variants. Double variants with intact serine residues (C42A/C58A/S195 and C42A/C58V/S195) also showed interchange between closed and open conformations for the S214-G219 segment, but to a lesser extent than the triple variants. The increased conformational flexibility of the S1 subsite, which was not observed for the wild-type, correlated with reduced enzymatic activities and suggested a possible mode of substrate regulation for the trypsin variants tested.     

A molecular dynamics study of the 41-56 beta-hairpin from B1 domain of protein G.

The structural and dynamical behavior of the 41-56 beta-hairpin from the protein G B1 domain (GB1) has been studied at different temperatures using molecular dynamics (MD) simulations in an aqueous environment. The purpose of these simulations is to establish the stability of this hairpin in view of its possible role as a nucleation site for protein folding. The conformation of the peptide in the crystallographic structure of the protein GB1 (native conformation) was lost in all simulations. The new equilibrium conformations are stable for several nanoseconds at 300K (>10 ns), 350 K (>6.5 ns), and even at 450 K (up to 2.5 ns). The new structures have very similar hairpin-like conformations with properties in agreement with available experimental nuclear Overhauser effect (NOE) data. The stability of the structure in the hydrophobic core region during the simulations is consistent with the experimental data and provides further evidence for the role played by hydrophobic interactions in hairpin structures. Essential dynamics analysis shows that the dynamics of the peptide at different temperatures spans basically the same essential subspace. The main equilibrium motions in this subspace involve large fluctuations of the residues in the turn and ends regions. Of the six interchain hydrogen bonds, the inner four remain stable during the simulations. The space spanned by the first two eigenvectors, as sampled at 450 K, includes almost all of the 47 different hairpin structures found in the database. Finally, analysis of the hydration of the 300 K average conformations shows that the hydration sites observed in the native conformation are still well hydrated in the equilibrium MD ensemble.     

Binding of human blood-coagulation Factors IXa and X to phospholipid membranes.

A simple centrifugation technique has been developed to study the interaction of human coagulation Factors IXa and X with phospholipid membranes. In the presence of Ca2+, equimolar phosphatidylserine/phosphatidylcholine membranes form tight complexes with Factor X (KD = 2.8 X 10(-8) M); the KD is independent of the phospholipid concentration. Binding sites are available for about 2 mmol of Factor X/mol of phospholipid. Factor IXa has a slightly higher affinity for the phospholipid membrane (KD = 1.2 X 10(-8)M), and competes with Factor X for binding. The experimentally observed competition between Factor X and Factor IXa is in agreement with a model that describes the binding of two distinct ligands to a single class of independent binding sites.     

Probing the interaction between vesicular stomatitis virus and phosphatidylserine

The entry of enveloped animal viruses into their host cells always depends on membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion between the viral envelope and the endosomal membrane at the acidic environment of this compartment. In this work, we evaluated VSV interactions with membranes of different phospholipid compositions, at neutral and acidic pH, using atomic force microscopy (AFM) operating in the force spectroscopy mode, isothermal calorimetry (ITC) and molecular dynamics simulation. We found that the binding forces differed dramatically depending on the membrane phospholipid composition, revealing a high specificity of G protein binding to membranes containing phosphatidylserine (PS). In a previous work, we showed that the sequence corresponding amino acid 164 of VSV G protein was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Here, we used this sequence to explore VSV–PS interaction using ITC. We found that peptide binding to membranes was exothermic, suggesting the participation of electrostatic interactions. Peptide–membrane interaction at pH 7.5 was shown to be specific to PS and dependent on the presence of His residues in the fusion peptide. The application of the simplified continuum Gouy–Chapman theory to our system predicted a pH of 5.0 at membrane surface, suggesting that the His residues should be protonated when located close to the membrane. Molecular dynamics simulations suggested that the peptide interacts with the lipid bilayer through its N-terminal residues, especially Val¹⁴⁵ and His¹⁴⁸. Fabiana A.Carneiro and Pedro A. Lapido-Loureiro contributed equally to this work An erratum to this article can be found at     

The Interaction of Vinculin with Actin

Vinculin can interact with F-actin both in recruitment of actin filaments to the growing focal adhesions and also in capping of actin filaments to regulate actin dynamics. Using molecular dynamics, both interactions are simulated using different vinculin conformations. Vinculin is simulated either with only its vinculin tail domain (Vt), with all residues in its closed conformation, with all residues in an open I conformation, and with all residues in an open II conformation. The open I conformation results from movement of domain 1 away from Vt; the open II conformation results from complete dissociation of Vt from the vinculin head domains. Simulation of vinculin binding along the actin filament showed that Vt alone can bind along the actin filaments, that vinculin in its closed conformation cannot bind along the actin filaments, and that vinculin in its open I conformation can bind along the actin filaments. The simulations confirm that movement of domain 1 away from Vt in formation of vinculin 1 is sufficient for allowing Vt to bind along the actin filament. Simulation of Vt capping actin filaments probe six possible bound structures and suggest that vinculin would cap actin filaments by interacting with both S1 and S3 of the barbed-end, using the surface of Vt normally occluded by D4 and nearby vinculin head domain residues. Simulation of D4 separation from Vt after D1 separation formed the open II conformation. Binding of open II vinculin to the barbed-end suggests this conformation allows for vinculin capping. Three binding sites on F-actin are suggested as regions that could link to vinculin. Vinculin is suggested to function as a variable switch at the focal adhesions. The conformation of vinculin and the precise F-actin binding conformation is dependent on the level of mechanical load on the focal adhesion.     

Protonation state of Asp30 exerts crucial influence over surface loop rearrangements responsible for NO release in nitrophorin 4

pK(a) values of ionizable residues were calculated for the crystal structures describing the pH and NO binding dependant conformations of nitrophorin 4, a pH sensitive NO carrier heme protein. Comparison of resultant H-bonding patterns allowed the identification of the amino acids that take part in signaling pH change. We carried out MD simulations to show that the protonation state of Asp30, buried in the closed conformation, is crucial for maintaining the tight packed conformation of the closed form of the complex - presenting a model for the functional decrease of NO binding affinity of nitrophorins at physiological pH.     

Ca(2+) and membrane binding to annexin 3 modulate the structure and dynamics of its N terminus and domain III

Annexin 3 (ANX A3) represents approximately 1% of the total protein of human neutrophils and promotes tight contact between membranes of isolated specific granules in vitro leading to their aggregation. Like for other annexins, the primary molecular events of the action of this protein is likely its binding to negatively charged phospholipid membranes in a Ca(2+)-dependent manner, via Ca(2+)-binding sites located on the convex side of the highly conserved core of the molecule. The conformation and dynamics of domain III can be affected by this process, as it was shown for other members of the family. The 20 amino-acid, N-terminal segment of the protein also could be affected and also might play a role in the modulation of its binding to the membranes. The structure and dynamics of these two regions were investigated by fluorescence of the two tryptophan residues of the protein (respectively, W190 in domain III and W5 in the N-terminal segment) in the wild type and in single-tryptophan mutants. By contrast to ANX A5, which shows a closed conformation and a buried W187 residue in the absence of Ca(2+), domain III of ANX A3 exhibits an open conformation and a widely solvent-accessible W190 residue in the same conditions. This is in agreement with the three-dimensional structure of the ANX A3-E231A mutant lacking the bidentate Ca(2+) ligand in domain III. Ca(2+) in the millimolar concentration range provokes nevertheless a large mobility increase of the W190 residue, while interaction with the membranes reduces it slightly. In the N-terminal region, the W5 residue, inserted in the central pore of the protein, is weakly accessible to the solvent and less mobile than W190. Its amplitude of rotation increases upon binding of Ca(2+) and returns to its original value when interacting with membranes. Ca(2+) concentration for half binding of the W5A mutant to negatively charged membranes is approximately 0.5 mM while it increases to approximately 1 mM for the ANX A3 wild type and to approximately 3 mM for the W190 ANX A3 mutant. In addition to the expected perturbation of the W190 environment at the contact surface between the protein and the membrane bilayer, binding of the protein to Ca(2+) and to membranes modulates the flexibility of the ANX A3 hinge region at the opposite of this interface and might affect its membrane permeabilizing properties.     

Atomic view of calcium-induced clustering of phosphatidylserine in mixed lipid bilayers

Membranes play key regulatory roles in biological processes, with bilayer composition exerting marked effects on binding affinities and catalytic activities of a number of membrane-associated proteins. In particular, proteins involved in diverse processes such as vesicle fusion, intracellular signaling cascades, and blood coagulation interact specifically with anionic lipids such as phosphatidylserine (PS) in the presence of Ca(2+) ions. While Ca(2+) is suspected to induce PS clustering in mixed phospholipid bilayers, the detailed structural effects of this ion on anionic lipids are not established. In this study, combining magic angle spinning (MAS) solid-state NMR (SSNMR) measurements of isotopically labeled serine headgroups in mixed lipid bilayers with molecular dynamics (MD) simulations of PS lipid bilayers in the presence of different counterions, we provide site-resolved insights into the effects of Ca(2+) on the structure and dynamics of lipid bilayers. Ca(2+)-induced conformational changes of PS in mixed bilayers are observed in both liposomes and Nanodiscs, a nanoscale membrane mimetic of bilayer patches. Site-resolved multidimensional correlation SSNMR spectra of bilayers containing (13)C,(15)N-labeled PS demonstrate that Ca(2+) ions promote two major PS headgroup conformations, which are well resolved in two-dimensional (13)C-(13)C, (15)N-(13)C, and (31)P-(13)C spectra. The results of MD simulations performed on PS lipid bilayers in the presence or absence of Ca(2+) provide an atomic view of the conformational effects underlying the observed spectra.     

Molecular dynamics-based investigation of InhA substrate binding loop for diverse biological activity of direct InhA inhibitors

The closed conformation of substrate binding loop (SBL) is considered significant for biological activity of direct InhA inhibitors (DIIs). However, exact interactions of SBL with inhibitors are not characterized yet to emphasize over SBL conformations. The seven InhA-DII complexes are analyzed using molecular dynamics simulation to deduce the mechanism for closed and open conformation of SBL. MMGBSA binding energy calculations and decompositions help to identify Ala198, Met199, Ile202, Val203, Ile215, and Leu218 in SBL region as the key residues. The interactions of DIIs with SBL residues particularly Ile202, Val203, Ile215, and Leu218 are found considerable for closed SBL conformation. This difference is accounted for closed state of SBL in 2X23, and open/moderately open state in other complexes. This study substantiates the loop ordering property of DIIs as the basis for high-affinity InhA inhibitors under the molecular recognition phenomena. This property can be used as a parameter to identify potential DIIs using virtual screening approaches.     

The role of oligomerization and cooperative regulation in protein function: the case of tryptophan synthase

The oligomerization/co-localization of protein complexes and their cooperative regulation in protein function is a key feature in many biological systems. The synergistic regulation in different subunits often enhances the functional properties of the multi-enzyme complex. The present study used molecular dynamics and Brownian dynamics simulations to study the effects of allostery, oligomerization and intermediate channeling on enhancing the protein function of tryptophan synthase (TRPS). TRPS uses a set of α/β-dimeric units to catalyze the last two steps of L-tryptophan biosynthesis, and the rate is remarkably slower in the isolated monomers. Our work shows that without their binding partner, the isolated monomers are stable and more rigid. The substrates can form fairly stable interactions with the protein in both forms when the protein reaches the final ligand-bound conformations. Our simulations also revealed that the α/β-dimeric unit stabilizes the substrate-protein conformation in the ligand binding process, which lowers the conformation transition barrier and helps the protein conformations shift from an open/inactive form to a closed/active form. Brownian dynamics simulations with a coarse-grained model illustrate how protein conformations affect substrate channeling. The results highlight the complex roles of protein oligomerization and the fine balance between rigidity and dynamics in protein function.     

Conformational equilibria and free energy profiles for the allosteric transition of the ribose-binding protein

The ribose-binding protein (RBP) is a sugar-binding bacterial periplasmic protein whose function is associated with a large allosteric conformational change from an open to a closed conformation upon binding to ribose. The crystal structures of RBP in open and closed conformations have been solved. It has been hypothesized that the open and closed conformations exist in a dynamic equilibrium in solution, and that sugar binding shifts the population from open conformations to closed conformations. Here, we study by computer simulations the thermodynamic changes that accompany this conformational change, and model the structural changes that accompany the allosteric transition, using umbrella sampling molecular dynamics and the weighted histogram analysis method. The open state is comprised of a diverse ensemble of conformations; the open ribose-free X-ray crystal conformations being representative of this ensemble. The unligated open form of RBP is stabilized by conformational entropy. The simulations predict detectable populations of closed ribose-free conformations in solution. Additional interdomain hydrogen bonds stabilize this state. The predicted shift in equilibrium from the open to the closed state on binding to ribose is in agreement with experiments. This is driven by the energetic stabilization of the closed conformation due to ribose-protein interactions. We also observe a significant population of a hitherto unobserved ribose-bound partially open state. We believe that this state is the one that has been suggested to play a role in the transfer of ribose to the membrane-bound permease complex.     

The role of phosphorylation on the structure and dynamics of phospholamban: a model from molecular simulations

Phospholamban (PLB) is a small membrane protein that regulates the activity of the calcium ATP-ase in the cardiac, slow-twitch, and smooth muscle sarcoplasmic reticulum through the reversible phosphorylation of Ser16. We present here a comparative molecular dynamics study of unmodified and phosphorylated PLB immersed in a phospholipid membrane. The study has been performed under different ionic strength conditions, using the NMR structures of two PLB variants determined in mixed organic solvent and dodecylphosphocholine micelles. The simulations indicate that all PLB forms studied display a highly dynamic behavior of the N-terminal cytoplasmic moiety, with a decrease of its helical content in the phosphorylated forms. The cytoplasmic domain undergoes large collective motions sampling conformations parallel as well as perpendicular to the membrane surface in all the simulations. The transmembrane domain retains a tightly folded helical conformation with a small tilt with respect to the membrane plane probably induced by the presence of Asn30 and Asn34 within the hydrophobic environment. Furthermore, the phosphoric group on Ser16 establishes transient electrostatic interactions with the phospholipid heads. We propose a model in which phosphorylation diminishes the probability of interactions of PLB with residues near Lys400 in the SERCA pump, thus relieving its inhibition.     

Molecular Dynamics Simulation Studies of dTTP Binding and Catalysis Mediated by YhdE Dimerization

YhdE is a Maf-like (multicopy associated filamentation) protein that primarily acts as dTTPase to hydrolyze dTTP into dTMP and two phosphate molecules in cell metabolism pathway. Two crystal structures of YhdE have been previously determined, representing the open and closed active site conformations, respectively. Based on the structures, we have carried out molecular dynamics simulations and free energy calculations to investigate dTTP binding to and hydrolysis by YhdE. Our results suggest that YhdE closed state is structurally more compact than its open state at room temperature. YhdE open state is a favorable conformation for dTTP binding and closed state is a structurally favorable conformation for catalytic reaction. This observation is supported by the structure of YhdE homolog in complex with a nucleotide analog. Free energy calculations reveal that YhdE dimerization occurs preferentially in dTTP binding and is favorable for successive cooperative reaction. The key residues R11, R12 and K80, are found to contribute to the substrate stabilization. Further, YhdE dimerization and binding of dTTP induce the cooperative effect through a direct allosteric communication network in YhdE from the dTTP binding sites in the catalytic center to the intermolecular β-strand in YhdE dimer.     

Modeling of factor XIII activation peptide (28-41) V34L mutant bound to thrombin

Factor XIII (FXIII) is a transglutaminase involved in blood coagulation. The enzyme is activated by thrombin cleaving the peptide bond R(37)-G(38). A common mutation V34L found in FXIII has been correlated with protection from myocardial infarction. Also FXIII V34L is activated more quickly than the wild type. In the present study, FXIII (28-41) V34L mutant peptide bound to thrombin has been modeled and molecular dynamics simulations were carried out using Insight II. An average structure was calculated after simulation. The structure showed significant difference from the crystal structure of the wild type FXIII (28-37) peptide bound to thrombin. In the crystal structure the peptide adopts a folded conformation in such a way that the hydrophobic side chains of V(29) and V(34) occupy the apolar binding site of thrombin. The modeled V34L peptide adopts a significantly different conformation and only the bulkier L(34) occupies the apolar binding site while V(29) side chain is exposed to the bulk solvent. Hence, this may speed up the release of FXIII from thrombin after its activation.     

Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis

We have previously determined that the C2-domain of human factor V (residues 2037-2196) is required for expression of cofactor activity and binding to phosphatidylserine (PS)-containing membranes. Naturally occurring factor V inhibitors and a monoclonal antibody (HV-1) recognized epitopes in the amino terminus of the C2-domain (residues 2037-2087) and blocked PS binding. We have now investigated the function of individual amino acids within the C2-domain using charge to alanine mutagenesis. Charged residues located within the C2-domain were changed to alanine in clusters of 1-3 mutations per construct. In addition, mutants W2063A, W2064A, (W2063, W2064)A, and L2116A were constructed as well. The resultant 30 mutants were expressed in COS cells using a B-domain deleted factor V construct (rHFV des B). All mutants were expressed efficiently based on the polyclonal antibody ELISA. The charged residues, Arg(2074), Asp(2098), Arg(2171), Arg(2174), and Glu(2189) are required for maintaining the structural integrity of the C2-domain of factor V. Four of these residues (Arg(2074), Asp(2098), Arg(2171), and Arg(2174)) correspond to positions in the factor VIII C-type domains that have been identified as point mutations in patients with hemophilia A. The epitope for the inhibitory monoclonal antibody HV-1 has been localized to Lys(2060) through Glu(2069) in the factor V C2-domain. The epitope for the inhibitory monoclonal antibody 6A5 is composed of amino acids His(2128) through Lys(2137). The PS-binding site in the factor V C2-domain includes amino acid residues Trp(2063) and Trp(2064). This site overlaps with the epitope for monoclonal antibody HV-1. These factor V C2-domain mutants should provide valuable tools for further defining the molecular interactions responsible for factor V binding to phospholipid membranes.     

The phosphatidylserine binding site of the factor Va C2 domain accounts for membrane binding but does not contribute to the assembly or activity of a human factor Xa-factor Va complex

Factors V(a) and X(a) (FV(a) and FX(a), respectively) assemble on phosphatidylserine (PS)-containing platelet membranes to form the essential "prothrombinase" complex of blood coagulation. The C-terminal domain (C2) of FV(a) (residues 2037-2196 in human FV(a)) contains a soluble phosphatidylserine (C6PS) binding pocket flanked by a pair of tryptophan residues, Trp(2063) and Trp(2064). Mutating these tryptophans abolishes FV(a) membrane binding. To address both the roles of these tryptophans in C6PS or membrane binding and the role of the C2 domain lipid binding site in regulation of FV(a) cofactor activity, we expressed W(2063,2064)A mutants of the recombinant C2 domain (rFV(a2)-C2) and of a B domain-deleted factor V light isoform (rFV(a2)) in Hi-5 and COS cells, respectively. Intrinsic fluorescence showed that wild-type rFV(a2)-C2 binds to C6PS and to 20% PS/PC membranes with apparent K(d) values of 2.8 microM and 9 nM, respectively, while mutant rFV(a2)-C2 does not. Equilibrium dialysis confirmed that mutant rFV(a2)-C2 does not bind to C6PS. Mutant rFV(a2) binds to C6PS (K(d) approximately 37 microM) with an affinity comparable to that of wild-type rFV(a2) (K(d) approximately 20 microM), although it does not bind to PS/PC membranes to which wild-type rFV(a2) binds with native affinity (K(d) approximately 3 nM). Both wild-type and mutant rFV(a2) bind to active site-labeled FX(a) (DEGR-X(a)) in the presence of 400 microM C6PS with native affinity (K(d) approximately 3-4 nM) to produce a solution rFV(a2)-FX(a) complex of native activity. We conclude that (1) the C2 domain PS site provides all but approximately 1 kT of the free energy of FV(a) membrane binding, (2) tryptophans lining the C2 lipid binding pocket are critical to C6PS and membrane binding and insert into the bilayer interface during membrane binding, (3) occupancy of the C2 lipid binding pocket is not necessary for C6PS-induced formation of the FX(a)-FV(a) complex or its activity, but (4) another PS site on FV(a) does have a regulatory role.