Deciphering specificity and cross-reactivity in tachykinin NK1 and NK2 receptors.

The tachykinin receptors neurokinin 1 (NK1R) and neurokinin 2 (NK2R) are G protein-coupled receptors that bind preferentially to the natural peptide ligands substance P and neurokinin A, respectively, and have been targets for drug development. Despite sharing a common C-terminal sequence of Phe-X-Gly-Leu-Met-NH2 that helps direct biological function, the peptide ligands exhibit some degree of cross-reactivity toward each other's non-natural receptor. Here, we investigate the detailed structure-activity relationships of the ligand-bound receptor complexes that underlie both potent activation by the natural ligand and cross-reactivity. We find that the specificity and cross-reactivity of the peptide ligands can be explained by the interactions between the amino acids preceding the FxGLM consensus motif of the bound peptide ligand and two regions of the receptor: the ß-hairpin of the extracellular loop 2 (ECL2) and a N-terminal segment leading into transmembrane helix 1. Positively charged sidechains of the ECL2 (R177 of NK1R and K180 of NK2R) are seen to play a vital role in the interaction. The N-terminal positions 1 to 3 of the peptide ligand are entirely dispensable. Mutated and chimeric receptor and ligand constructs neatly swap around ligand specificity as expected, validating the structure-activity hypotheses presented. These findings will help in developing improved agonists or antagonists for NK1R and NK2R.

Mechanistic basis of GPCR activation explored by ensemble refinement of crystallographic structures.

G protein-coupled receptors (GPCRs) are important drug targets characterized by a canonical seven transmembrane (TM) helix architecture. Recent advances in X-ray crystallography and cryo-EM have resulted in a wealth of GPCR structures that have been used in drug design and formed the basis for mechanistic activation hypotheses. Here, ensemble refinement (ER) of crystallographic structures is applied to explore the impact of binding of agonists and antagonist/inverse agonists to selected structures of cannabinoid receptor 1 (CB1R), ß2 adrenergic receptor (ß2 AR), and A2A adenosine receptor (A2A AR). To assess the conformational flexibility and its role in GPCR activation, hydrogen bond (H-bond) networks are analyzed by calculating and comparing H-bond propensities. Mapping pairwise propensity differences between agonist- and inverse agonist/antagonist-bound structures for CB1R and ß2 AR shows that agonist binding destabilizes H-bonds in the intracellular parts of TM 5-7, forming the G protein binding cavity, while H-bonds of the extracellular segment of TMs surrounding the orthosteric site are conversely stabilized. Certain class A GPCRs, for example, A2A AR, bind an allosteric sodium ion that negatively modulates agonist binding. The impact of sodium-excluding mutants (D522.50 N, S913.39 A) of A2A AR on agonist binding is examined by applying ER analysis to structures of wildtype and the two mutants in complex with a full agonist. While S913.39 A exhibits normal activity, D522.50 N quenches the downstream signaling. The mainchain H-bond pattern of the latter is stabilized in the intracellular part of TM 7 containing the NPxxY motif, indicating that an induced rigidity of the mutation prevents conformational selection of G proteins resulting in receptor inactivation.

A systematic approach for evaluating the role of surface-exposed loops in trypsin-like serine proteases applied to the 170 loop in coagulation factor VIIa.

Proteases play a major role in many vital physiological processes. Trypsin-like serine proteases (TLPs), in particular, are paramount in proteolytic cascade systems such as blood coagulation and complement activation. The structural topology of TLPs is highly conserved, with the trypsin fold comprising two ß-barrels connected by a number of variable surface-exposed loops that provide a surprising capacity for functional diversity and substrate specificity. To expand our understanding of the roles these loops play in substrate and co-factor interactions, we employ a systematic methodology akin to the natural truncations and insertions observed through evolution of TLPs. The approach explores a larger deletion space than classical random or directed mutagenesis. Using FVIIa as a model system, deletions of 1-7 amino acids through the surface exposed 170 loop, a vital allosteric regulator, was introduced. All variants were extensively evaluated by established functional assays and computational loop modelling with Rosetta. The approach revealed detailed structural and functional insights recapitulation and expanding on the main findings in relation to 170 loop functions elucidated over several decades using more cumbersome crystallization and single deletion/mutation methodologies. The larger deletion space was key in capturing the most active variant, which unexpectedly had a six-amino acid truncation. This variant would have remained undiscovered if only 2-3 deletions were considered, supporting the usefulness of the methodology in general protease engineering approaches. Our findings shed further light on the complex role that surface-exposed loops play in TLP function and supports the important role of loop length in the regulation and fine-tunning of enzymatic function throughout evolution.

Conformational Plasticity-Rigidity Axis of the Coagulation Factor VII Zymogen Elucidated by Atomistic Simulations of the N-Terminally Truncated Factor VIIa Protease Domain.

The vast majority of coagulation factor VII (FVII), a trypsin-like protease, circulates as the inactive zymogen. Activated FVII (FVIIa) is formed upon proteolytic activation of FVII, where it remains in a zymogen-like state and it is fully activated only when bound to tissue factor (TF). The catalytic domains of trypsin-like proteases adopt strikingly similar structures in their fully active forms. However, the dynamics and structures of the available corresponding zymogens reveal remarkable conformational plasticity of the protease domain prior to activation in many cases. Exactly how ligands and cofactors modulate the conformational dynamics and function of these proteases is not entirely understood. Here, we employ atomistic simulations of FVIIa (and variants hereof, including a TF-independent variant and N-terminally truncated variants) to provide fundamental insights with atomistic resolution into the plasticity-rigidity interplay of the protease domain conformations that appears to govern the functional response to proteolytic and allosteric activation. We argue that these findings are relevant to the FVII zymogen, whose structure has remained elusive despite substantial efforts. Our results shed light on the nature of FVII and demonstrate how conformational dynamics has played a crucial role in the evolutionary adaptation of regulatory mechanisms that were not present in the ancestral trypsin. Exploiting this knowledge could lead to engineering of protease variants for use as next-generation hemostatic therapeutics.

Beating tissue factor at its own game: Design and properties of a soluble tissue factor-independent coagulation factor VIIa.

Two decades of research have uncovered the mechanism by which the complex of tissue factor (TF) and the plasma serine protease factor VIIa (FVIIa) mediates the initiation of blood coagulation. Membrane-anchored TF directly interacts with substrates and induces allosteric effects in the protease domain of FVIIa. These properties are also recapitulated by the soluble ectodomain of TF (sTF). At least two interdependent allosteric activation pathways originate at the FVIIa:sTF interface are proposed to enhance FVIIa activity upon sTF binding. Here, we sought to engineer an sTF-independent FVIIa variant by stabilizing both proposed pathways, with one pathway terminating at segment 215-217 in the activation domain and the other pathway terminating at the N terminus insertion site. To stabilize segment 215-217, we replaced the flexible 170 loop of FVIIa with the more rigid 170 loop from trypsin and combined it with an L163V substitution (FVIIa-VYT). The FVIIa-VYT variant exhibited 60-fold higher amidolytic activity than FVIIa, and displayed similar FX activation and antithrombin inhibition kinetics to the FVIIa.sTF complex. The sTF-independent activity of FVIIa-VYT was partly mediated by an increase in the N terminus insertion and, as shown by X-ray crystallography, partly by Tyr-172 inserting into a cavity in the activation domain stabilizing the S1 substrate-binding pocket. The combination with L163V likely drove additional changes in a delicate hydrogen-bonding network that further stabilized S1-S3 sites. In summary, we report the first FVIIa variant that is catalytically independent of sTF and provide evidence supporting the existence of two TF-mediated allosteric activation pathways.

Allostery in Coagulation Factor VIIa Revealed by Ensemble Refinement of Crystallographic Structures.

A critical step in injury-induced initiation of blood coagulation is the formation of the complex between the trypsin-like protease coagulation factor VIIa (FVIIa) and its cofactor tissue factor (TF), which converts FVIIa from an intrinsically poor enzyme to an active protease capable of activating zymogens of downstream coagulation proteases. Unlike its constitutively active ancestor trypsin, FVIIa is allosterically activated (by TF). Here, ensemble refinement of crystallographic structures, which uses multiple copies of the entire structure as a means of representing structural flexibility, is applied to explore the impacts of inhibitor binding to trypsin and FVIIa, as well as cofactor binding to FVIIa. To assess the conformational flexibility and its role in allosteric pathways in these proteases, main-chain hydrogen bond networks are analyzed by calculating the hydrogen-bond propensity. Mapping pairwise propensity differences between relevant structures shows that binding of the inhibitor benzamidine to trypsin has a minor influence on the protease flexibility. For FVIIa, in contrast, the protease domain is "locked" into the catalytically competent trypsin-like configuration upon benzamidine binding as indicated by the stabilization of key structural features: the nonprime binding cleft and the oxyanion hole are stabilized, and the effect propagates from the active site region to the calcium-binding site and to the vicinity of the disulphide bridge connecting with the light chain. TF binding to FVIIa furthermore results in stabilization of the 170 loop, which in turn propagates an allosteric signal from the TF-binding region to the active site. Analyses of disulphide bridge energy and flexibility reflect the striking stability difference between the unregulated enzyme and the allosterically activated form after inhibitor or cofactor binding. The ensemble refinement analyses show directly, for the first time to our knowledge, whole-domain structural footprints of TF-induced allosteric networks present in x-ray crystallographic structures of FVIIa, which previously only have been hypothesized or indirectly inferred.

Engineering of a membrane-triggered activity switch in coagulation factor VIIa.

Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa.

Releasing the brakes in coagulation Factor IXa by co-operative maturation of the substrate-binding site.

Coagulation Factor IX is positioned at the merging point of the intrinsic and extrinsic blood coagulation cascades. Factor IXa (activated Factor IX) serves as the trigger for amplification of coagulation through formation of the so-called Xase complex, which is a ternary complex of Factor IXa, its substrate Factor X and the cofactor Factor VIIIa on the surface of activated platelets. Within the Xase complex the substrate turnover by Factor IXa is enhanced 200000-fold; however, the mechanistic and structural basis for this dramatic enhancement remains only partly understood. A multifaceted approach using enzymatic, biophysical and crystallographic methods to evaluate a key set of activity-enhanced Factor IXa variants has demonstrated a delicately balanced bidirectional network. Essential molecular interactions across multiple regions of the Factor IXa molecule co-operate in the maturation of the active site. This maturation is specifically facilitated by long-range communication through the Ile(212)-Ile(213) motif unique to Factor IXa and a flexibility of the 170-loop that is further dependent on the conformation in the Cys(168)-Cys(182) disulfide bond. Ultimately, the network consists of compensatory brakes (Val(16) and Ile(213)) and accelerators (Tyr(99) and Phe(174)) that together allow for a subtle fine-tuning of enzymatic activity.

Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity.

The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215-217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215-217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity.

Membrane Interaction of the Factor VIIIa Discoidin Domains in Atomistic Detail.

A recently developed membrane-mimetic model was applied to study membrane interaction and binding of the two anchoring C2-like discoidin domains of human coagulation factor VIIIa (FVIIIa), the C1 and C2 domains. Both individual domains, FVIII C1 and FVIII C2, were observed to bind the phospholipid membrane by partial or full insertion of their extruding loops (the spikes). However, the two domains adopted different molecular orientations in their membrane-bound states; FVIII C2 roughly was positioned normal to the membrane plane, while FVIII C1 displayed a multitude of tilted orientations. The results indicate that FVIII C1 may be important in modulating the orientation of the FVIIIa molecule to optimize the interaction with FIXa, which is anchored to the membrane via its γ-carboxyglutamic acid-rich (Gla) domain. Additionally, a structural change was observed in FVIII C1 in the coiled main chain leading the first spike. A tight interaction with one lipid per domain, similar to what has been suggested for the homologous FVa C2, is characterized. Finally, we rationalize known FVIII antibody epitopes and the scarcity of documented hemophilic missense mutations related to improper membrane binding of FVIIIa, based on the prevalent nonspecificity of ionic interactions in the simulated membrane-bound states of FVIII C1 and FVIII C2.

Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising "Hot-Spot" Lysine Residues.

Lysine residues are implicated in driving the ligand binding to the LDL receptor family. However, it has remained unclear how specificity is regulated. Using coagulation factor VIII as a model ligand, we now study the contribution of individual lysine residues in the interaction with the largest member of the LDL receptor family, low-density lipoprotein receptor-related protein (LRP1). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and SPR interaction analysis on a library of lysine replacement variants as two independent approaches, we demonstrate that the interaction between factor VIII (FVIII) and LRP1 occurs over an extended surface containing multiple lysine residues. None of the individual lysine residues account completely for LRP1 binding, suggesting an additive binding model. Together with structural docking studies, our data suggest that FVIII interacts with LRP1 via an extended surface of multiple lysine residues that starts at the bottom of the C1 domain and winds around the FVIII molecule.

Evidence for restricted reactivity of ADAMDEC1 with protein substrates and endogenous inhibitors.

ADAMDEC1 is a proteolytically active metzincin metalloprotease displaying rare active site architecture with a zinc-binding Asp residue (Asp-362). We previously demonstrated that substitution of Asp-362 for a His residue, thereby reconstituting the canonical metzincin zinc-binding environment with three His zinc ligands, increases the proteolytic activity. The protease also has an atypically short domain structure with an odd number of Cys residues in the metalloprotease domain. Here, we investigated how these rare structural features in the ADAMDEC1 metalloprotease domain impact the proteolytic activity, the substrate specificity, and the effect of inhibitors. We identified carboxymethylated transferrin (Cm-Tf) as a new ADAMDEC1 substrate and determined the primary and secondary cleavage sites, which suggests a strong preference for Leu in the P1' position. Cys(392), present in humans but only partially conserved within sequenced ADAMDEC1 orthologs, was found to be unpaired, and substitution of Cys(392) for a Ser increased the reactivity with α2-macroglobulin but not with casein or Cm-Tf. Substitution of Asp(362) for His resulted in a general increase in proteolytic activity and a change in substrate specificity was observed with Cm-Tf. ADAMDEC1 was inhibited by the small molecule inhibitor batimastat but not by tissue inhibitor of metalloproteases (TIMP)-1, TIMP-2, or the N-terminal inhibitory domain of TIMP-3 (N-TIMP-3). However, N-TIMP-3 displayed profound inhibitory activity against the D362H variants with a reconstituted consensus metzincin zinc-binding environment. We hypothesize that these unique features of ADAMDEC1 may have evolved to escape from inhibition by endogenous metalloprotease inhibitors.

Sites involved in intra- and interdomain allostery associated with the activation of factor VIIa pinpointed by hydrogen-deuterium exchange and electron transfer dissociation mass spectrometry.

Factor VIIa (FVIIa) is a trypsin-like protease that plays an important role in initiating blood coagulation. Very limited structural information is available for the free, inactive form of FVIIa that circulates in the blood prior to vascular injury and the molecular details of its activity enhancement remain elusive. Here we have applied hydrogen/deuterium exchange mass spectrometry coupled to electron transfer dissociation to pinpoint individual residues in the heavy chain of FVIIa whose conformation and/or local interaction pattern changes when the enzyme transitions to the active form, as induced either by its cofactor tissue factor or a covalent active site inhibitor. Identified regulatory residues are situated at key sites across one continuous surface of the protease domain spanning the TF-binding helix across the activation pocket to the calcium binding site and are embedded in elements of secondary structure and at the base of flexible loops. Thus these residues are optimally positioned to mediate crosstalk between functional sites in FVIIa, particularly the cofactor binding site and the active site. Our results unambiguously show that the conformational allosteric activation signal extends to the EGF1 domain in the light chain of FVIIa, underscoring a remarkable intra- and interdomain allosteric regulation of this trypsin-like protease.

The length of the linker between the epidermal growth factor-like domains in factor VIIa is critical for a productive interaction with tissue factor.

Formation of the factor VIIa (FVIIa)-tissue factor (TF) complex triggers the blood coagulation cascade. Using a structure-based rationale, we investigated how the length of the linker region between the two epidermal growth factor (EGF)-like domains in FVIIa influences TF binding and the allosteric activity enhancement, as well as the interplay between the γ-carboxyglutamic acid (Gla)-containing and protease domains. Removal of two residues from the native linker was compatible with normal cofactor binding and accompanying stimulation of the enzymatic activity, as was extension by two (Gly-Ser) residues. In sharp contrast, truncation by three or four residues abolished the TF-mediated stabilization of the active conformation of FVIIa and abrogated TF-induced activity enhancement. In addition, FVIIa variants with short linkers associated 80-fold slower with soluble TF (sTF) as compared with wild-type FVIIa, resulting in a corresponding increase in the equilibrium dissociation constant. Molecular modeling suggested that the shortest FVIIa variants would have to be forced into a tense and energetically unfavorable conformation in order to be able to interact productively with TF, explaining our experimental observations. We also found a correlation between linker length and the residual intrinsic enzymatic activity of Ca(2+)-free FVIIa; stepwise truncation resulting in gradually higher activity with des(83-86)-FVIIa reaching the level of Gla-domainless FVIIa. The linker appears to determine the average distance between the negatively charged Gla domain and a structural element in the protease domain, presumably of opposite charge, and proximity has a negative impact on apo-FVIIa activity.

ADAMDEC1 is a metzincin metalloprotease with dampened proteolytic activity.

ADAMDEC1 (Decysin-1) is a putative ADAM (a disintegrin and metalloprotease)-like metalloprotease with an unknown physiological role, selectively expressed in mature dendritic cells and macrophages. When compared with other members of the ADAM family, ADAMDEC1 displays some unusual features. It lacks the auxiliary cysteine-rich, EGF, and transmembrane domains, as well as the cytoplasmic tail. The active site of ADAMDEC1 is unique by being the only mammalian ADAM protease with a non-histidine zinc ligand, having an aspartic acid residue instead. Here we demonstrate that ADAMDEC1, despite these unique features, functions as an active metalloprotease. Thus, ADAMDEC1 is secreted as a mature, glycosylated, and proteolytically active metalloprotease, capable of cleaving macromolecular substrates. In the recombinant form, three of the four potential N-linked glycosylation sites are modified by carbohydrate attachment. Substitution of basic residues at the predicted proprotein convertase cleavage site blocks proprotein processing, revealing both specific ADAMDEC1-dependent and specific ADAMDEC1-independent cleavage of the prodomain. The pro-form of ADAMDEC1 does not have proteolytic activity, demonstrating that the prodomain of ADAMDEC1, like in other members of the ADAM family, confers catalytic latency. Interestingly, the proteolytic activity of mature ADAMDEC1 can be significantly enhanced when a canonical ADAM active site with three zinc-coordinating histidine residues is introduced.

Vatreptacog alfa from conception to clinical proof of concept.

Vatreptacog alfa is a genetically engineered variant of recombinant factor VIIa (rFVIIa) containing three amino acid changes. Aspartic acid, valine, and glutamine residues replace valine, glutamic acid, and methionine at positions 158, 296, and 298, respectively. These substitutions result in considerable enhancement of the intrinsic (tissue factor-independent) capability to activate factor X and the downstream hemostatic events are consequently augmented. The beneficial effects of vatreptacog alfa have been demonstrated in numerous in vitro systems attempting to mimic hemophilia and corroborated in in vivo models. Vatreptacog alfa has successfully passed through phase 1 and 2 clinical trials and the molecule is currently being explored in phase 3 clinical trial for the treatment of bleedings in hemophilia patients with inhibitors. This article describes the proposed mechanism behind the increased activity and action of vatreptacog alfa and reviews available data, which suggest that vatreptacog alfa could be a valuable addition to the existing portfolio of treatment options for hemophilia patients with inhibitors.

Engineering of substrate selectivity for tissue factor.factor VIIa complex signaling through protease-activated receptor 2.

The complex of factor VIIa (FVIIa) with tissue factor (TF) triggers coagulation by recognizing its macromolecular substrate factors IX (FIX) and X (FX) predominantly through extended exosite interactions. In addition, TF mediates unique cell-signaling properties in cancer, angiogenesis, and inflammation that involve proteolytic cleavage of protease-activated receptor 2 (PAR2). PAR2 is cleaved by FVIIa in the binary TF.FVIIa complex and by FXa in the ternary TF.FVIIa.FXa complex, but physiological roles of these signaling complexes are incompletely understood. In a screen of FVIIa protease domain mutants, three variants (Q40A, Q143N, and T151S) activated macromolecular coagulation substrates and supported signaling of the ternary TF.FVIIa-Xa complex normally but were severely impaired in binary TF.FVIIa.PAR2 signaling. The residues identified were located in the model-predicted S2' pocket of FVIIa, and complementary PAR2 P2' Leu-38 replacements demonstrated that the P2' side chain was indeed crucial for PAR2 cleavage by TF.FVIIa. In addition, PAR2 was activated more efficiently by FVIIa T99Y, consistent with further contributions from the S2 subsite. The P2 residue preference of FVIIa and FXa predicted additional PAR2 mutants that were efficiently activated by TF.FVIIa but resistant to cleavage by the alternative PAR2 activator FXa. Thus, contrary to the paradigm of exosite-assisted cleavage of PAR1 by thrombin, the cofactor-associated protease FVIIa recognizes PAR2 predominantly by catalytic cleft interactions. Furthermore, the delineated molecular details of this substrate interaction enabled protein engineering of protease-selective PAR2 receptors that will aid further studies to dissect the roles of TF signaling complexes in vivo.

Current status on tissue factor activation of factor VIIa.

Free factor VIIa displays a zymogen-like behavior with low intrinsic activity. Formation of a complex between factor VIIa and tissue factor is necessary to enhance the procoagulant activity of factor VIIa, not only by providing membrane localization, substrate exosites and positioning the active site at an appropriate distance above the surface but also by allosteric enhancement of the enzymatic activity, and this event signals initiation of blood coagulation. The interaction is of high affinity and all the domains are engaged at the interface. The crosstalk between the protease domain of factor VIIa, in particular residue Met-306, and the N-terminal domain of tissue factor provides the starting point for the allosteric activation of factor VIIa. The pathway(s) of conformational transitions in factor VIIa ensuing tissue factor binding has not been entirely mapped. The present paper is a brief compilation of our current knowledge of the allosteric mechanism by which tissue factor induces and stabilizes the active conformation of factor VIIa.

Activation loop 3 and the 170 loop interact in the active conformation of coagulation factor VIIa.

The initiation of blood coagulation involves tissue factor (TF)-induced allosteric activation of factor VIIa (FVIIa), which circulates in a zymogen-like state. In addition, the (most) active conformation of FVIIa presumably relies on a number of intramolecular interactions. We have characterized the role of Gly372(223) in FVIIa, which is the sole residue in activation loop 3 that is capable of forming backbone hydrogen bonds with the unusually long 170 loop and with activation loop 2, by studying the effects of replacement with Ala [G372(223)A]. G372A-FVIIa, both in the free and TF-bound form, exhibited reduced cleavage of factor X (FX) and of peptidyl substrates, and had increased K(m) values compared with wild-type FVIIa. Inhibition of G372A-FVIIa.sTF by p-aminobenzamidine was characterized by a seven-fold higher K(i) than obtained with FVIIa.sTF. Crystallographic and modelling data suggest that the most active conformation of FVIIa depends on the backbone hydrogen bond between Gly372(223) and Arg315(170C) in the 170 loop. Despite the reduced activity and inhibitor susceptibility, native and active site-inhibited G372A-FVIIa bound sTF with the same affinity as the corresponding forms of FVIIa, and burial of the N-terminus of the protease domain increased similarly upon sTF binding to G372A-FVIIa and FVIIa. Thus Gly372(223) in FVIIa appears to play a critical role in maturation of the S1 pocket and adjacent subsites, but does not appear to be of importance for TF binding and the ensuing allostery.

Mechanism of the Ca2+-induced enhancement of the intrinsic factor VIIa activity.

The intrinsic activity of coagulation factor VIIa (FVIIa) is dependent on Ca(2+) binding to a loop (residues 210-220) in the protease domain. Structural analysis revealed that Ca(2+) may enhance the activity by attenuating electrostatic repulsion of Glu(296) and/or by facilitating interactions between the loop and Lys(161) in the N-terminal tail. In support of the first mechanism, the mutations E296V and D212N resulted in similar, about 2-fold, enhancements of the amidolytic activity. Moreover, mutation of the Lys(161)-interactive residue Asp(217) or Asp(219) to Ala reduced the amidolytic activity by 40-50%, whereas the K161A mutation resulted in 80% reduction. Hence one of these Asp residues in the Ca(2+)-binding loop appears to suffice for some residual interaction with Lys(161), whereas the more severe effect upon replacement of Lys(161) is due to abrogation of the interaction with the N-terminal tail. However, Ca(2+) attenuation of the repulsion between Asp(212) and Glu(296) keeps the activity above that of apoFVIIa. Altogether, our data suggest that repulsion involving Asp(212) in the Ca(2+)-binding loop suppresses FVIIa activity and that optimal activity requires a favorable interaction between the Ca(2+)-binding loop and the N-terminal tail. Crystal structures of tissue factor-bound FVIIa(D212N) and FVIIa(V158D/E296V/M298Q) revealed altered hydrogen bond networks, resembling those in factor Xa and thrombin, after introduction of the D212N and E296V mutations plausibly responsible for tethering the N-terminal tail to the activation domain. The charge repulsion between the Ca(2+)-binding loop and the activation domain appeared to be either relieved by charge removal and new hydrogen bonds (D212N) or abolished (E296V). We propose that Ca(2+) stimulates the intrinsic FVIIa activity by a combination of charge neutralization and loop stabilization.