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We developed a SARS-CoV-2 spike subunit vaccine formulation containing dual TLR ligand liposome adjuvant. The vaccine-induced robust systemic neutralizing antibodies and completely protected mice from a lethal challenge. Two immunizations protected against lung injury and cleared the virus from lungs upon challenge. The adjuvanted vaccine also elicited systemic and local anti-Spike IgA which can be an important feature for a COVID-19 vaccine.

The aim was to develop a fluorescent multiplex array for simultaneously measuring regulated food allergens using specific allergen protein molecules from peanut, tree nut, cow's milk, egg, soy, fish, shellfish, sesame, mustard and celery. Microspheres coupled to specific monoclonal antibodies were used for allergen detection, with purified allergens as reference standards.Standard curves for 17 allergens covered a 5-log dynamic range. Intra- and inter-assay acceptance criteria were within 70-130% recovery and a CV of ≤15%. Food reference materials contained high levels of their respective major allergens (2000-175,000 µg/g), Similar high allergen levels were found in 10 selected foods analysed using a 9-plex array. Egg, milk, peanut, hazelnut and walnut allergens were detectable in chocolate bars with incurred allergens at 3, 10, 30, and 100 ppm. The multiplex array is an efficient tool for measuring specific food allergens, with applications for risk assessment and standardization of therapeutic products for food allergy.

Rationale: Individuals with asthma may be at higher risk of becoming severely ill from COVID-19 which targets the respiratory tract and may cause asthma exacerbations. Serological testing can be useful to assess the true spread of COVID-19 and help protect vulnerable individuals. The SARS-CoV-2 spike and nucleocapsid proteins are the primary viral antigens against which antibodies are raised. We developed an efficient method for production of select SARS-CoV-2 proteins in E. coli to facilitate the development of diagnostics, research, and drug discovery. Methods: SARS-CoV-2 Spike-RBD, full-length Nucleocapsid, and Nucleocapsid RNA binding domain (BD), were expressed in E. coli under IPTG induction. The proteins were purified using multi-step chromatography techniques. The purity of the SARS-CoV-2 proteins was assessed by LC-MS/MS, and their IgG reactivity tested using COVID-19 positive patients’ sera by ELISA. Results: The Spike-RBD and Nucleocapsid proteins were expressed with high yields. The purified proteins had a relative abundance of >95% as assessed by LC-MS/MS with only trace contamination of host cell proteins. All three proteins showed high IgG reactivity (titers >1/10,000) against COVID-19 sera (n=50), with Spike-RBD and Nucleocapsid RNA-BD exhibiting the highest sensitivity. Conclusions: Production of high quality SARS-CoV-2 proteins is feasible in E.coli and the purified proteins will provide useful tools to study the immune responses involved in COVID-19 including antibody and T cell responses, epitope mapping, diagnostics and drug discovery.

The MepRAB operon in Staphylococcus aureus has been identified to play a role in drug resistance. Although the functions of MepA and MepR are known, little information is available on the function of MepB. Here we report the X-ray structure of MepB to 2.1 A revealing its structural similarity to the PD-(D/E)XK family of endonucleases. We further show that MepB binds DNA and RNA, with a higher affinity towards RNA and single stranded DNA than towards double stranded DNA. Notably, the PD-(D/E)XK catalytic active site residues are not conserved in MepB. MepB's association with a drug resistance operon suggests that it plays a role in responding to antimicrobials. This role is likely carried out through MepB's interactions with nucleic acids.

Crystal structures of factor (F) VIIa/soluble tissue factor (TF), obtained under high Mg(2+) (50mM Mg(2+)/5mM Ca(2+)), have three of seven Ca(2+) sites in the γ-carboxyglutamic acid (Gla) domain replaced by Mg(2+) at positions 1, 4, and 7. We now report structures under low Mg(2+) (2.5mM Mg(2+)/5mM Ca(2+)) as well as under high Ca(2+) (5mM Mg(2+)/45 mM Ca(2+)). Under low Mg(2+), four Ca(2+) and three Mg(2+) occupy the same positions as in high-Mg(2+) structures. Conversely, under low Mg(2+), reexamination of the structure of Gla domain of activated Protein C (APC) complexed with soluble endothelial Protein C receptor (sEPCR) has position 4 occupied by Ca(2+) and positions 1 and 7 by Mg(2+). Nonetheless, in direct binding experiments, Mg(2+) replaced three Ca(2+) sites in the unliganded Protein C or APC. Further, the high-Ca(2+) condition was necessary to replace Mg4 in the FVIIa/soluble TF structure. In biological studies, Mg(2+) enhanced phospholipid binding to FVIIa and APC at physiological Ca(2+). Additionally, Mg(2+) potentiated phospholipid-dependent activations of FIX and FX by FVIIa/TF and inactivation of activated factor V by APC. Since APC and FVIIa bind to sEPCR involving similar interactions, we conclude that under the low-Mg(2+) condition, sEPCR binding to APC-Gla (or FVIIa-Gla) replaces Mg4 by Ca4 with an attendant conformational change in the Gla domain ω-loop. Moreover, since phospholipid and sEPCR bind to FVIIa or APC via the ω-loop, we predict that phospholipid binding also induces the functional Ca4 conformation in this loop. Cumulatively, the data illustrate that Mg(2+) and Ca(2+) act in concert to promote coagulation and anticoagulation.

Protein engineering is an important tool for the design of proteins with novel and desirable features. Templates from the protein databank (PDB) are often used as initial models that can be modified to introduce new properties. We examine whether it is possible to reconnect a protein in a manner that generates a new topology yet preserves its structural integrity. Here, we describe the rethreading of dihydrofolate reductase (DHFR) from E. coli (wtDHFR). The rethreading process involved the removal of three native loops and the introduction of three new loops with alternate connections. The structure of the rethreaded DHFR (rDHFR-1) was determined to 1.6 Å, demonstrating the success of the rethreading process. Both wtDHFR and rDHFR-1 exhibited similar affinities towards methotrexate. However, rDHFR-1 showed no reducing activity towards dihydrofolate and exhibited about ~6-fold lower affinity towards NADPH than wtDHFR. This work demonstrates that protein rethreading can be a powerful tool for the design of a large array of proteins with novel structures and topologies and that by careful rearrangement of a protein sequence, the sequence to structure relationship can be expanded substantially.

We previously reported that the first epidermal growth factor-like (EGF1) domain in factor X (FX) or factor IX (FIX) plays an important role in the factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. To assess the role of gamma-carboxyglutamic acid (Gla) domains of FX and FIX in FVIIa/TF induced coagulation, we studied four new and two previously described replacement mutants: FX(PCGla) and FIX(PCGla) (Gla domain replaced with that of protein C), FX(PCEGF1) and FIX(PCEGF1) (EGF1 domain replaced with that of protein C), as well as FX(PCGla/EGF1) and FIX(PCGla/EGF1) (both Gla and EGF1 domains replaced with those of protein C). FVIIa/TF activation of each FX mutant and the corresponding reciprocal activation of FVII/TF by each FXa mutant were impaired. In contrast, FVIIa/TF activation of FIX(PCGla) was minimally affected, and the reciprocal activation of FVII/TF by FIXa(PCGla) was normal; however, both reactions were impaired for the FIX(PCEGF1) and FIX(PCGla/EGF1) mutants. Predictably, FXIa activation of FIX(PCEGF1) was normal, whereas it was impaired for the FIX(PCGla) and FIX(PCGla/EGF1) mutants. Molecular models reveal that alternate interactions exist for the Gla domain of protein C such that it is comparable with FIX but not FX in its binding to FVIIa/TF. Further, additional interactions exist for the EGF1 domain of FX, which are not possible for FIX. Importantly, a seven-residue insertion in the EGF1 domain of protein C prevents its interaction with FVIIa/TF. Cumulatively, our data provide a molecular framework demonstrating that the Gla and EGF1 domains of FX interact more strongly with FVIIa/TF than the corresponding domains in FIX.

Factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca2+ ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca2+ and Mg2+, and the Ca2+ sites in FVIIa that could be specifically occupied by Mg2+ are unknown. Furthermore, FVIIa contains a Na+ and two Zn2+ sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca2+, Mg2+, Na+, and Zn2+. The crystal diffracted to 1.8A resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca2+ and three bound Mg2+. The EGF1 domain contains one Ca2+ site, and the protease domain contains one Ca2+, one Na+, and two Zn2+ sites. 45Ca2+ binding in the presence/absence of Mg2+ to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly193 in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87A resolution. However, soaking benzamidine-FVIIa/sTF crystals with d-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, d-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.

Simultaneous replacement of Asp-94 with serine and Gly-98 with glutamate in rat alpha-parvalbumin creates a CD-site ligand array in the context of the EF-site binding loop. Previous work has shown that, relative to the wild-type CD site, this engineered site has markedly reduced Ca(2+) affinity. Seeking an explanation for this phenomenon, we have obtained the crystal structure of the alpha D94S/G98E variant. The Ca(2+) coordination within the engineered EF site of the 94/98E variant is nearly identical to that within the CD site, suggesting that the attenuated affinity of the EF site in 94/98E is not a consequence of suboptimal coordination geometry. We have also examined the divalent ion binding properties of the alpha 94/98E variant in both Na(+)- and K(+)-containing buffers. Although the Ca(2+) and Mg(2+) affinities are higher in K(+) solution, the increases are comparable to those observed for wild-type alpha. Consistent with that finding, the apparent Na(+) stoichiometry, estimated from stability studies conducted as a function of Na(+) concentration, is 1.0 +/- 0.1, identical to that of wild-type alpha. Thus, the reduced affinity for divalent ions is evidently not the result of heightened monovalent ion competition. The thermodynamic analysis indicates that the less favorable Gibbs free energy of binding reflects a substantial enthalpic penalty. Significantly, the crystal structure reveals a steric clash between Phe-57 and the C(gamma) atom of Glu-98. The consequent displacement of Phe-57 also produces a close contact with Ser-55. Thus, steric interference may be the source of the enthalpic penalty.

Association of the parvalbumin AB and CD-EF domains was examined in Hepes-buffered saline, pH 7.4, employing fragments from rat alpha and beta. All of the interactions require Ca(2+). In saturating Ca(2+), the alpha AB/alpha CD-EF (alpha/alpha) complex displays an association constant of (7.6 +/- 0.4) x 10(7) M(-1). Ca(2+)-binding data for a mixture of the alpha fragments are compatible with an identical two-site model, yielding an average binding constant of (8.5 +/- 0.2) x 10(5) M(-1). The beta/beta interaction is significantly weaker, exhibiting an association constant of (3.0 +/- 0.6) x 10(6) M(-1). The Ca(2+)-binding constants for beta/beta are likewise diminished, at (1.0 +/- 0.1) x 10(5) and (2.3 +/- 0.2) x 10(4) M(-1). The magnitude of the apparent DeltaDeltaG(degree)' for Ca(2+) binding by alpha/alpha and beta/beta, at 3.4 kcal/mol, approaches that measured for the intact proteins (3.6 kcal/mol) and is substantially larger than the 1.5 kcal/mol value previously measured for the isolated CD-EF domains. This result suggests that the AB domain can modulate the Ca(2+) affinities of the CD and EF sites. Interestingly, the heterologous alpha/beta complex displays a larger association constant [(6.6 +/- 0.4) x 10(6) M(-1)] than the homologous beta/beta complex and heightened Ca(2+) affinity [binding constants of (1.3 +/- 0.1) x 10(6) and (8.8 +/- 0.2) x 10(4) M(-1)]. By contrast, beta/alpha associates more weakly than alpha/alpha and exhibits sharply reduced affinity for Ca(2+). Thus, the interaction between the beta AB domain and beta CD-EF domain may act to attenuate Ca(2+) affinity in the intact protein.

The crystal structure of rat α-parvalbumin has been determined at 1.05 A resolution, using synchrotron data collected at Advanced Photon Source beamline 19-ID. After refinement with SHELX, employing anisotropic displacement parameters and riding hydrogen atoms, R = 0.132 and Rfree = 0.162. The average coordinate estimated standard deviations are 0.021 A and 0.038 A for backbone atoms and side-chain atoms, respectively. Besides providing a more precise view of the α-isoform than previously available, these data permit comparison with the 0.91 A structure determined for pike β-parvalbumin. Visualization of the anisotropic displacement parameters as thermal ellipsoids yields insight into the atomic motion within the Ca2+-binding sites. The asymmetric unit includes three parvalbumin (PV) molecules. Interestingly, the EF site in one displays uncharacteristic flexibility. The ellipsoids for Asp-92 are particularly large and non-spherical, and the shape of the Ca2+ ellipsoid implies significant vibrational motion perpendicular to the plane defined by the four y and z ligands. The relative dearth of crystal-packing interactions in this site suggests that the heightened flexibility may be the result of diminished intermolecular contacts. The implication is that, by impeding conformational mobility, crystal-packing forces may cause serious overestimation of EF-hand rigidity. The high quality of the data permitted 11 residues to be modeled in alternative side-chain conformations, including the two core residues, Ile-97 and Leu-105. The discrete disorder observed for Ile-97 may have functional ramifications, providing a mechanism for communicating binding status between the CD and EF binding loops and between the PV metal ion-binding domain and the N-terminal AB region.

Introduction of a fifth carboxylate into the ligand array of the CD site (via the combined S55D and E59D mutations) or the EF site (G98D) of rat alpha-parvalbumin substantially increases divalent ion affinity. This behavior, in conflict with that seen in model peptide systems, agrees with existing data for rat beta-parvalbumin [Henzl et al. (1996) Biochemistry 35, 5856-5869]. The complete analysis of the S55D/E59D double variant necessitated characterization of alpha E59D. Whereas the D59E mutation has minimal influence on beta CD site affinity, E59D has a major impact on the alpha CD site, lowering the apparent association constant by a factor of 14. The thermodynamic consequences of exchanging the rat alpha CD and EF site ligand arrays, which differ at the +z and -x coordination positions, were also examined. When the alpha CD array is imported into the EF site, it acquires a low-affinity phenotype, in agreement with previous findings for beta [Henzl et al. (1998) Biochemistry 37, 9101-9111]. However, when the EF ligand array is introduced into the alpha CD binding loop, it retains a high-affinity signature. This result, contrary to that observed in beta, suggests that the influence of the parvalbumin CD site environment supersedes the intrinsic behavior of the ligand array, a conclusion further supported by the disparate impact of the beta D59E and alpha E59D mutations.

Rat alpha- and beta-parvalbumins have distinct monovalent cation-binding properties [Henzl et al. (2000) Biochemistry 39, 5859-5867]. Beta binds two Na(+) or one K(+), and alpha binds one Na(+) and no K(+). Ca(2+) abolishes these binding events, suggesting that the monovalent ions occupy the EF-hand motifs. This study compares alpha and beta divalent ion affinities in Na(+) and K(+) solutions. Solvent cation identity seriously affects alpha. In Hepes-buffered NaCl, at 5 degrees C, the macroscopic Ca(2+)-binding constants are 2.6 x 10(8) and 6.4 x 10(7) M(-1) and the Mg(2+) constants, 1.8 x 10(4) and 4.3 x 10(3) M(-1). In Hepes-buffered KCl, the Ca(2+) values increase to 2.9 x 10(9) and 6.6 x 10(8) M(-1) and the Mg(2+) values to 2.2 x 10(5) and 3.7 x 10(4) M(-1). Monte Carlo simulation of alpha binding data-employing site-specific constants and explicitly considering Na(+) binding-yields a K(Na) of 630 M(-1) and indicates that divalent ion-binding is positively cooperative. NMR data suggest that the lone Na(+) ion occupies the CD loop. Solvent cation identity has a smaller impact on beta. In Na(+), the Ca(2+) constants for the EF and CD sites are 2.3 x 10(7) and 1.5 x 10(6) M(-1), respectively; the Mg(2+) constants are 9.2 x 10(3) and 1.7 x 10(2) M(-1). In K(+), these values shift to 3.1 x 10(7) and 3.8 x 10(6) M(-1) and the latter to 1.4 x 10(4) and 2.9 x 10(2) M(-1). These data suggest that parvalbumin divalent ion affinity, particularly that of rat alpha, can be significantly attenuated by increased intracellular Na(+) levels.

Despite its higher net charge and reduced opportunities for favorable tertiary interactions, Ca(2+)-free rat beta-parvalbumin is more stable than rat alpha-parvalbumin. Under conditions wherein alpha denatures at 45.8 degrees C, beta denatures at 53.6 degrees. The homologous chicken beta isoform known as CPV3 also exhibits heightened stability-prompting an inquiry into the stabilizing influence of Pro-21 and Pro-26. Individual P21A and P26A mutations lower the T(m) of rat beta by 3.2 degrees, decreasing conformational stability by 0.74 kcal/mol. Simultaneous replacement of Pro-21 and Pro-26 essentially abolishes the excess stability (DeltaT(m) = -7.6 degrees; DeltaDeltaG(conf) = -1.77 kcal/mol). Significantly, the P21A/P26A variant displays Ca(2+) affinity virtually indistinguishable from wild-type beta, implying that structural alterations in the AB domain do not necessarily influence the divalent ion affinity of the CD-EF domain. The consequences of introducing proline at positions 21 and 26 in rat alpha were also examined. Whereas the H26P mutation raises the T(m) by 5.6 degrees (DeltaDeltaG(conf) = 1.25 kcal/mol), A21P lowers the T(m) by 8.5 degrees (DeltaDeltaG(conf) = -1.9 kcal/mol). Replacement of Ala-21 by proline in an alpha AB/beta CD-EF chimera increases the T(m) by 5.8 degrees (DeltaDeltaG(conf) = 0.95 kcal/mol), implying that the destabilization of alpha by Pro-21 results from steric conflict with a residue in the CD-EF domain. Consistent with that hypothesis, the K80S mutation markedly stabilizes alpha A21P, yielding a protein with a T(m) 2.0 degrees higher than wild-type alpha. The observed differences in stability resulting from proline addition/removal are largely consistent with alterations in main-chain and side-chain conformational entropy.

The design of proteins that self-assemble into higher order architectures is of great interest due to their potential application in nanotechnology. Specifically, the self-assembly of proteins into ordered lattices is of special interest to the field of structural biology. Here we designed a 2 dimensional (2D) protein lattice using a fusion of a tandem repeat of three TelSAM domains (TTT) to the Ferric uptake regulator (FUR) domain. We determined the structure of the designed (TTT-FUR) fusion protein to 2.3 Å by X-ray crystallographic methods. In agreement with the design, a 2D lattice composed of TelSAM fibers interdigitated by the FUR domain was observed. As expected, the fusion of a tandem repeat of three TelSAM domains formed 21 screw axis, and the self-assembly of the ordered oligomer was under pH control. We demonstrated that the fusion of TTT to a domain having a 2-fold symmetry, such as the FUR domain, can produce an ordered 2D lattice. The TTT-FUR system combines features from the rotational symmetry matching approach with the oligomer driven crystallization method. This TTT-FUR fusion was amenable to X-ray crystallographic methods, and is a promising crystallization chaperone.

The structural biology of membrane proteins remains a challenging field, partly due to the difficulty in obtaining high-quality crystals. We developed the bicelle method as a tool to aid with the production of membrane protein crystals. Bicelles are bilayer discs that are formed by a mixture of a detergent and a lipid. They combine the ease of use of detergents with the benefits of a lipidic medium. Bicelles maintain membrane proteins in a bilayer milieu, which is more similar to their native environment than detergent micelles. At the same time, bicelles are liquid at certain temperatures and they can be integrated into standard crystallization techniques without the need for specialized equipment.

The structural biology of membrane proteins remains a challenging field, partly due to the difficulty in obtaining high-quality crystals. We developed the bicelle method as a tool to aid with the production of membrane protein crystals. Bicelles are bilayer discs that are formed by a mixture of a detergent and a lipid. They combine the ease of use of detergents with the benefits of a lipidic medium. Bicelles maintain membrane proteins in a bilayer milieu, which is more similar to their native environment than detergent micelles. At the same time, bicelles are liquid at certain temperatures and they can be integrated into standard crystallization techniques without the need for specialized equipment.

We previously reported that the first epidermal growth factor-like (EGF1) domain in factor X (FX) or factor IX (FIX) plays an important role in the factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. To assess the role of gamma-carboxyglutamic acid (Gla) domains of FX and FIX in FVIIa/TF induced coagulation, we studied four new and two previously described replacement mutants: FX(PCGla) and FIX(PCGla) (Gla domain replaced with that of protein C), FX(PCEGF1) and FIX(PCEGF1) (EGF1 domain replaced with that of protein C), as well as FX(PCGla/EGF1) and FIX(PCGla/EGF1) (both Gla and EGF1 domains replaced with those of protein C). FVIIa/TF activation of each FX mutant and the corresponding reciprocal activation of FVII/TF by each FXa mutant were impaired. In contrast, FVIIa/TF activation of FIX(PCGla) was minimally affected, and the reciprocal activation of FVII/TF by FIXa(PCGla) was normal; however, both reactions were impaired for the FIX(PCEGF1) and FIX(PCGla/EGF1) mutants. Predictably, FXIa activation of FIX(PCEGF1) was normal, whereas it was impaired for the FIX(PCGla) and FIX(PCGla/EGF1) mutants. Molecular models reveal that alternate interactions exist for the Gla domain of protein C such that it is comparable with FIX but not FX in its binding to FVIIa/TF. Further, additional interactions exist for the EGF1 domain of FX, which are not possible for FIX. Importantly, a seven-residue insertion in the EGF1 domain of protein C prevents its interaction with FVIIa/TF. Cumulatively, our data provide a molecular framework demonstrating that the Gla and EGF1 domains of FX interact more strongly with FVIIa/TF than the corresponding domains in FIX.

Factor VIIa (FVIIa) consists of a gamma-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca(2+) ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca(2+) and Mg(2+), and the Ca(2+) sites in FVIIa that could be specifically occupied by Mg(2+) are unknown. Furthermore, FVIIa contains a Na(+) and two Zn(2+) sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca(2+), Mg(2+), Na(+), and Zn(2+). The crystal diffracted to 1.8A resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca(2+) and three bound Mg(2+). The EGF1 domain contains one Ca(2+) site, and the protease domain contains one Ca(2+), one Na(+), and two Zn(2+) sites. (45)Ca(2+) binding in the presence/absence of Mg(2+) to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly(193) in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87A resolution. However, soaking benzamidine-FVIIa/sTF crystals with d-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, d-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.

Birds express three parvalbumins, one a isoform and two p isoforms. The latter are known as avian thymic hormone (ATH) and avian parvalbumin 3. Although both were discovered in thymus tissue, and presumably function in T-cell maturation, they have been detected in other tissue settings. We have conducted detailed Ca 2 + - and Mg2 + -binding studies on recombinant ATH and the C72S variant of CPV3, employing global analysis of isothermal titration calorimetry data. In Hepesbuffered saline, ATH binds Ca 2 + with apparent microscopic binding constants of 2.4 ′ 0.2 x 10 8 and 1.0 ′ 0.1 x 10 8 M - 1 . The corresponding values for CPV3-C72S are substantially lower, 4.5 ′ 0.5 x 10 7 and 2.4 ′ 0.2 x 10 7 M - 1 , a 1.9-kcal/mol difference in binding free energy. Thus, the β-parvalbumin lineage displays a spectrum of Ca 2 + -binding affinity, with ATH and the mammalian β isoform at the high-and low-affinity extremes and CPV3 in the middle. Interestingly, despite its decreased Ca 2 + affinity, CPV3-C72S exhibits increased affinity for Mg 2 + , relative to ATH. Whereas the latter displays Mg 2 + -binding constants of 2.2 ′ 0.2 x 10 4 and 1.2 ′ 0.1 x 10 4 M - 1 , CPV3-C72S yields values of 5.0 ′ 0.8 x 10 4 and 2.1 ′ 0.3 x 10 4 M - 1 .

The use of competitive isothermal titration calorimetry (ITC) to measure high-affinity binding constants has been largely restricted to systems with a single binding site or multiple identical sites. This study demonstrates the extension of this approach to proteins with two nonequivalent EF-hand Ca(2+)-binding sites--rat beta parvalbumin and the S55D/E59D variant of rat alpha parvalbumin. The method involves simultaneous (global) least-squares analysis of titrations with Ca(2+), with Mg(2+), with Ca(2+) in the presence of Mg(2+), and with Ca(2+) or Mg(2+) in the presence of a competitive chelator (EDTA or EGTA). The Ca(2+) and Mg(2+) binding constants obtained for rat beta agree well with estimates obtained by flow dialysis. Although the Ca(2+) affinity of alpha S55D/E59D is too high to measure by flow dialysis, it was amenable to analysis using the ITC-based approach. The combined S55D and E59D mutations increase the Ca(2+) and Mg(2+) affinities of the mutated binding site by factors of 14 and 26, respectively. This behavior is consistent with that seen previously for the rat beta S55D variant.

We have examined the metal ion-binding domains from rat alpha and beta parvalbumin. We find that the CD-EF fragments differ markedly in their tendency to self-associate. Whereas Ca(2+)-free alpha CD-EF is monomeric, the Ca(2+)-free beta peptide dimerizes weakly (K(2) = 2400 +/- 200 M(-1)). In buffer containing 1.0 mM Ca(2+), the apparent dimerization constant for beta CD-EF (191,000 +/- 29,000 M(-1)) is more than 50 times that of alpha (3400 +/- 200 M(-1)). Alpha CD-EF binds two Ca(2+) with positive cooperativity. Titration calorimetry data afford binding constants of 3.7(0.1) x 10(3) M(-1) and 8.6(0.2) x 10(4) M(-1). Beta CD-EF also binds two Ca(2+) cooperatively but with lower affinity. Equilibrium dialysis yields Adair constants of 4.2(0.1) x 10(3) and 6.1(0.2) x 10(3) M(-1). Significantly, the difference in Ca(2+) affinity is substantially smaller than that observed for the full-length proteins-suggesting that the AB domain can modulate divalent ion affinity. Analysis of beta calorimetry data requires explicit consideration of the self-association behavior. Data collected at low CD-EF concentration are consistent with preferential occupation of the EF site, dimerization of singly bound monomers, and cooperative filling of the CD sites. At higher concentrations, apo-protein dimerization can apparently precede cooperative occupation of the EF sites. In the presence of Ca(2+), alpha CD-EF exhibits higher thermal stability, consistent with its higher Ca(2+) affinity. However, the beta melting temperature shows greater concentration dependence, consistent with its greater tendency to dimerize. Neither fragment exhibits a sigmoidal melting curve in the Ca(2+)-free state, suggesting that the apo-peptides are disordered.

The mammalian genome encodes both alpha- and beta-parvalbumin isoforms. The rat beta-parvalbumin (aka "oncomodulin") is more stable than the alpha isoform at physiological pH and ionic strength, despite its substantially higher charge density and truncated C-terminal helix [Henzl, M. T., and Graham, J. S. (1999) FEBS Lett. 442, 241-245]. Reasoning that solvent interactions could contribute to this unexpected finding, we have examined the stabilities of the Ca(2+)-free alpha- and beta-parvalbumins as a function of Na(+) and K(+) concentration. Differential scanning calorimetry data suggest that, at physiological pH and ionic strength, the beta isoform binds roughly 2 equiv of Na(+) or a single equivalent of K(+) with moderate affinity. Under comparable conditions, the alpha isoform apparently binds just 1 equiv of Na(+) and essentially no K(+). Isothermal titration calorimetry experiments suggest that the bound monovalent ions occupy the EF-hand motifs. In 0.15 M K(+), at pH 7.4, the stability of the apo-beta-parvalbumin exceeds that of the alpha isoform by approximately 2.6 kcal/mol at 37 degrees C and by approximately 3.0 kcal/mol at 25 degrees C. The latter value represents a substantial fraction of the difference in Ca(2+)-binding free energies measured in vitro for the two proteins. Significantly, however, these results do not completely explain the paradoxical stability of the beta isoform, which maintains its higher melting temperature under all conditions examined.

Abstract 1172 Equilibrium dialysis experiments indicate that the Gla domain of coagulant protein factor (F) VIIa and of anticoagulant protein C (PC) each bind seven Ca2+ in the absence and four in the presence of physiologic Mg2+ (0.6 mM). The previous x-ray structure of FVIIa/soluble (s) tissue factor (TF) in the presence of 5 mM Ca2+/45 mM Mg2+ (5Ca/45Mg) (Bajaj et al., J Biol Chem, 281, 24873–24888, 2006) revealed that sites 2,3,5 and 6 were occupied by Ca2+ while sites 1, 4 and 7 contained Mg2+ (Tulinsky numbering, Biochemistry 31, 2554–2566, 1992). Since these concentrations of metal ions were supraphysiologic, we sought to obtain crystals under concentrations of Ca2+ and Mg2+ approaching those found in blood. We have solved the structures of FVIIa/sTF under three new conditions: 2.5 mM Ca2+/1.25 mM Mg2+ at 2.8 Å (2.5Ca/1.25Mg); 5 mM Ca2+/2.5 mM Mg2+ at 1.8 Å (5Ca/2.5Mg); and 45 mM Ca2+/5 mM Mg2+ at 1.7 Å (45Ca/5Mg). For 2.5Ca/1.25Mg, the Gla domain of FVIIa was disordered indicating insufficient concentrations of Ca2+/Mg2+ to fold the Gla domain. Interestingly, folding of the Ω-loop for 5Ca/2.5Mg was similar to that reported under 5Ca/45Mg, with identical positions for four Ca2+ and three Mg2+. In contrast, while the folding of the Ω-loop at 45Ca/5Mg was similar to that reported only in the presence of Ca2+ (Banner et al., Nature, 380, 41–46,1996); however, positions 1 and 7 contained Mg2+ (Fig. 1A). Thus, it would appear that four Ca2+ and three Mg2+ ions are bound to the circulating FVII/FVIIa. Moreover, circulating FVII/FVIIa will need high Ca2+ concentrations to have its position four switched from Mg2+ to Ca2+.Fig. 1.A) The superimposed structures of FVIIa Gla domains from 5Ca/2.5Mg (1.8 Å) and 45Ca/5Mg (1.7 Å) showing differences in the Ω-loops. B) The superimposed structures of Gla domain in FVIIa (45Ca/5Mg) and in 5Ca/5Mg PC-EPCR (1.6 Å) showing similar Ω-loop conformations.Fig. 1. A) The superimposed structures of FVIIa Gla domains from 5Ca/2.5Mg (1.8 Å) and 45Ca/5Mg (1.7 Å) showing differences in the Ω-loops. B) The superimposed structures of Gla domain in FVIIa (45Ca/5Mg) and in 5Ca/5Mg PC-EPCR (1.6 Å) showing similar Ω-loop conformations. All seven metal sites in the structure of PC-Gla complexed to endothelial protein C receptor (EPCR) had been refined with Ca2+ despite the presence of 5 mM Ca2+ and 5 mM Mg2+ in the crystallization buffer (Oganesyan et al., J Biol Chem, 277, 24851–24854, 2002). Based on our findings with VIIa/sTF, we revisited the PC-Gla/EPCR structure. We determined that positions 1 and 7 are Mg2+ sites while 2, 3, 4, 5 and 6 are Ca2+ sites in the PC-Gla/EPCR structure (Fig. 1B). This contrasts to four Ca2+ and three Mg2+ bound to VIIa/TF at 5Ca/2.5Mg. Since five Ca2+ and two Mg2+ were seen in VIIa/sTF at 45Ca/5Mg, it would appear that position 4 can accommodate either Ca2+ or Mg2+ depending upon conditions. We propose that at near physiologic Ca2+ and Mg2+, ligation of the Gla domain with EPCR and possibly phospholipid (PL) membranes facilitates substitution of the Mg2+ bound at the position four with Ca2+. This metal switch may be essential to achieve a favorable conformation of the Gla domain Ω-loop for optimal ligand of PL binding. Mg2+ also enhanced the Ca2+-dependent interaction of FVIIa or activated protein C (APC) to PL assessed by surface plasmon resonance. Binding of FVIIa and APC each was saturated by the physiological concentration of Ca2+ (1.1 mM) in the presence of physiological Mg2+ (0.6 mM). In contrast, only half-saturable binding was observed at the physiological concentration of Ca2+ in the absence of Mg2+. Further, 0.6 mM Mg2+ potentiated (∼2.5-fold) the PL-dependent activation of FX by FVIIa/TF at 1.1 mM Ca2+. Similarly, Mg2+ potentiated (∼3-fold) the activation of FX by FVIIa/TF assembled on the endotoxin-stimulated monocyte surface. PL-dependent inactivation of FVa by APC was also enhanced ∼3-fold by 0.6 mM Mg2+ at 1.1 mM Ca2+. At saturating Ca2+ (5 mM), the activation of FX by FVIIa/TF or the inactivation of FVa by APC was similar to that with 1.1 mM Ca2+/0.6 mM Mg2+. Thus Mg2+ at physiologic concentrations augments PL- or natural membrane-dependent coagulation and anticoagulation at the plasma concentration of Ca2+. We propose that vitamin K-dependent clotting and anti-clotting proteins circulate in blood with four Ca2+ ions bound. The remaining three (or more in FIX and FX) divalent metal binding sites in each Gla domain are occupied by Mg2+. The conformation of the Ω-loop in circulating vitamin K- dependent proteins is not favorable for binding to PL but it is achieved by switching Mg2+ at position four to Ca2+. Thus, the metal ion at position four regulates PL-dependent coagulation and anticoagulation reactions. Disclosures: No relevant conflicts of interest to declare.

During blood coagulation, the plasma zymogen factor IX (fIX) is converted to the active protease factor Ixaβ (fIXaβ). The severe bleeding disorder associated with deficiency of fIX (hemophilia B) attests to the importance of this protein in hemostasis. Conversion of fIX to fIXaβ requires two proteolytic cleavages after Arg145 and Arg180, releasing an activation peptide. This process is mediated by the proteases factor VIIa (fVIIa) and factor XIa (fXIa). FVIIa in complex with tissue factor initially cleaves fIX after Arg145 forming an intermediate, factor IXα (fIXα), which is then cleaved after Arg180 to form fIXaβ. Western blots of activation time courses demonstrate fIXα accumulation during this process, indicating cleavage at Arg180 is rate limiting. In contrast, little intermediate accumulation occurs during fIX activation by fXIa. Previously, we showed that fXIa also cleaves fIX initially after Arg145, generating fIXα (Smith et al., J. Biol. Chem.283;6696:2008). To account for the lack of intermediate accumulation, then, the subsequent cleavage after Arg180 must occur at least as rapidly as the initial cleavage. We examined the relative rates of conversion of fIX, fIXα, and the alternative intermediate factor IXaα (fIXaα - cleaved after Arg180) to fIXaβ by fXIa. FIXα or fIXaα were prepared from tritium-labeled fIX by incubation with fXIa-Pro192 (discussed below) or Russell’s Viper Venom protease, respectively, and purified by anion exchange chromatography. Conversion to fIXaβ was determined by measuring release of the tritiated activation peptide. FXIa converted fIX to fIXaβ with a kcat of 29.4 ± 0.4/min, a value reflecting cleavage at both activation sites. Kcat for conversion of fIXα and fIXaα to fIXaβ were 29.9 ± 0.5 and 30.0 ± 1.0/min, respectively. The rate of conversion of fIX to fIXα, estimated by measuring tritiated activation products separated by SDS-PAGE, was 30.0 ± 0.4/min. Recently, we showed that amino acid substitutions in fXIa for the conserved active site residue Gly193 (chymotrypsin numbering) decreased kcat for fIX activation 7–1000 fold, with residues with long branched side-chains having the greatest effect (Schmidt et al. Biochemistry47;1326:2008). Gly193 substitutions had a modestly larger detrimental effect (1.2–1.5 fold) on cleavage of fIX after Arg180 compared to Arg145 that was associated with varying degrees of fIXα accumulation. Similar effects were noted with substitutions for the adjacent residue Lys192. FXIa with Pro192 cleaved fIX after Arg180 >10-fold more slowly than after Arg145, generating fIXα with little subsequent conversion to fIXaβ. Cumulatively, these data support the premise that the rates for the two sequential reactions required for normal fIX activation by fXIa are comparable. Therefore, perturbations causing a greater effect on cleavage after Arg180 compared to Arg145, even if relatively small, result in fIXα accumulation. Initial recognition of fIX by fXIa involves substrate binding exosites distinct from the enzyme active site. At least one exosite appears to be located in the fXIa third apple (A3) domain, and may interact with an epitope on the fIX Ca2+-binding Gla-domain. The rate of fIX activation to fIXaβ by fXIa was significantly reduced in the presence of an antibody to the fXIa A3 domain or by mutations in the A3 domain. Similarly, rates of activation were decreased in the absence of Ca2+, in the presence of an antibody to the fIX Gla-domain, or when fIX with a decarboxylated Gla-domain was the substrate. In all cases, significant fIXα accumulation was noted in time courses, indicating that interfering with this particular substrate-exosite interaction has a significantly greater effect on cleavage after Arg180 than after Arg145. These findings raise the possibility that the exosite on fXIa A3 plays a larger role in conversion of fIXα to fIXaβ than in initial fIX conversion to fIXα, and are consistent with the possibility, recently proposed by Sinha et al. (Biochemistry46;9830:2007), that a second fIX binding exosite is present on fXIa.

Ca2+ is an obligatory factor for both the extrinsic and intrinsic pathways of coagulation. In majority of in vitro studies, investigators use saturating concentrations of Ca2+ (5 to 10 mM) for FVIIa/tissue factor (TF) activation of factor IX (FIX), and factor X (FX) (extrinsic coagulation), as well as for the activation of FIX by FXIa, FX by FIXa/FVIIIa, and prothrombin by FXa/FVa (intrinsic coagulation). However, the concentration of Ca2+ in plasma is only 1.1 mM, which is considerably below the saturating concentration needed for optimal coagulation. Importantly, plasma also contains 0.6 mM Mg2+ that could compensate for subsaturating concentrations of Ca2+ in promoting coagulation. Previous studies have attempted to clarify this concept in FIX, FX and prothrombin activation. However, these studies are sparse and in virtually all cases not detailed. We have systematically examined the role of plasma concentration of Mg2+ (in addition to the plasma concentration of Ca2+) in promoting all Ca2+ dependent steps of extrinsic and intrinsic coagulation and compared it with the saturating concentration of Ca2+. The Km (~ 100 nM) for activation of FIX by FXIa was similar in the presence of plasma concentrations of Ca2+/Mg2+ or 5 mM Ca2+. Furthermore, the Km and Vmax for the activation of FX and FIX by FVIIa/TF were essentially similar for both conditions. The Km and Vmax for the activation of FX by FVIIIa/FIXa ± phospholipid, and prothrombin by FXa/FVa ± phospholipid were also indistinguishable in these two different metal ion conditions. Notably, when only plasma concentrations of Ca2+ (1.1mM or 1.7mM) were used in all reactions mentioned above, coagulation proceeded at suboptimal rates. In further studies, we used Biacore to investigate the binding of FXIa and FVIII to FIXa, soluble TF to FVIIa, and FVa to FXa. Soluble TF, dansyl-Glu-Gly-Arg (dEGR)-IXa and dEGR-Xa were coupled to CM5 chips in the presence of 10 mM Ca2+. At 5 mM Ca2+, the binding of FXIa to dEGR-IXa was characterized by a Kd of ~40 nM, binding of FVIII to dEGR-IXa by a Kd of ~100 nM, and FVa to dEGR-Xa by a Kd of ~120 nM. In the presence of plasma concentrations of Ca2+ and Mg2+, binding constants were similar to those obtained in the presence of 5 mM saturating Ca2+ concentration. Additional 45Ca2+ binding studies using equilibrium dialysis and prothrombin fragment 1, dEGR-VIIa and decarboxylated dEGR-VIIa, FIX and decarboxylated FIX, and FX and decarboxylated FX, indicated that in the g-carboxyglutamic acid (Gla) domain, 2-3 Ca2+ binding sites (Shikimoto, et al., J. Biol. Chem. 278, 24090-24094, 2003; Wang, et al., Biochemistry42, 7959-7966, 2003; Bajaj, et al., J. Biol. Chem.281, 24873-24888, 2006) out of seven core divalent ion binding sites (Soriano-Garcia et al., Biochemistry31, 2554-2566, 1992) could be replaced by Mg2+. Conversely, Mg2+ could not displace the Ca2+ binding sites in the epidermal growth factor-like domain 1 (EGF1) and protease domains of FIX or FX. Overall these studies indicate that (1) saturating concentrations of Ca2+ used in in vitro investigations are valid representations of coagulation studies, except for that Mg2+ compensates for suboptimal concentrations of Ca2+ under physiological conditions; (2) two of the Ca2+-binding sites in the Gla domain (numbers 1 and 7, per Tulinsky numbering (Soriano-Garcia et al., Biochemistry31, 2554-2566, 1992)), and possibly a third site (number 4) are specific for Mg2+ under physiologic conditions; and (3) the Ca2+-binding sites in the EGF1 and protease domains are specific for Ca2+ and can not be occupied by Mg2+ under physiologic conditions. In conclusion, Ca2+ and Mg2+ act in concert to promote optimal coagulation under physiologic conditions. Mg2+ alone does not promote coagulation since it cannot bind to the Ca2+ specific sites in the Gla domain necessary for folding of the Gla domain omega loop.

Factor VII (FVII) consists of an N-terminal γ-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor-like (EGF1 and EGF2) domains and the C-terminal protease domain. Activation of FVII results in a two-chain FVIIa molecule consisting of a light chain (Gla-EGF1-EGF2 domains) and a heavy chain (protease domain) held together by a single disulfide bond. The complex of tissue factor (TF) and FVIIa activates FIX and FX during coagulation. FVIIa on its own is structurally more “zymogen-like” and when bound to TF it is more “active enzyme-like.” We obtained crystal structures of EGR-VIIa/soluble (s) TF (2.9 Å resolution), dansyl-EGR-VIIa/sTF (1.9 Å resolution) and benzamidine-VIIa/sTF (1.6 Å resolution). We also investigated the effect of TF binding on the S1, S2, and S3/S4 subsites (Schechter and Berger, BBRC, 27:157-162, 1967) in FVIIa. The affinity of variously inhibited FVIIa to sTF was also measured using Biacore technology. For obtaining second order inhibition rate constants, FVIIa ± soluble (s) TF was incubated with each inhibitor for various times, diluted several fold and assayed for the residual FVIIa activity. The second order rate constants were obtained by plotting the first order rate constants versus the inhibitor concentrations. These data are summarized in the table below. From these data it appears that all subsites are affected upon FVIIa binding to sTF. Since in the crystal structure of EGR-VIIa/sTF the P1 Arg residue is the only residue that makes contact with FVIIa, it follows that the S1 site is affected ~10-fold upon binding to sTF. Adding a dansyl group that partially occupies the S3/S4 position (1.9 Å structure) increases the second order rate constant 7-fold (2.41 versus 0.35) over that of EGR-ck. Moreover the addition of Pro (DFPR-ck) or Phe (DFFR-ck) residue occupying the S2 position increases the second order rate constant 357-fold and 1500- fold, respectively (125 and 525 versus 0.35). Thus, comparison of dEGR, DFPR, DFFR inhibition suggests that FVIIa prefers Phe at S2 and at S3/S4 positions, and that TF opens up the S1/S2/S3/S4 sites for substrate or inhibitor occupancy. These data are consistent with LTR (P3/P2/P1) residues in FX at its activation cleavage site as well as with LTR (P3/P2/P1) residues and FTR (P3/P2/P1) residues at the 145-146 and 180-181 FIX activation cleavage sites, respectively. Thus, these studies with chloromethylketone inhibitors have biologic relevance. For Biacore studies, sTF was amine coupled to a CM5 chip. The binding of unoccupied active site FVIIa in 5 mM calcium to sTF was characterized by a KD of 7 nM. Benzamidine (10 mM)-VIIa, p-aminobenzamidine (pAB, 1 mM)-VIIa, EGR-VIIa, dEGR-VIIa, DFPR-VIIa and DFFR-VIIa each bound to sTF with KD values ranging from 1- 2 nM. These affinity measurements indicate that the S1 site occupied FVIIa molecule (benzamidine-FVIIa, pAB-VIIa) has essentially the same conformation as the S1/S2/S3/S4 occupied FVIIa. This conclusion is consistent with similar crystal structures of variously inhibited FVIIa molecules complexed with sTF. The differential rates of incorporation of various chloromethylketone inhibitors could be due to the interaction of various residues (P1, P2, P3, P4) with the corresponding active subsites (S1/S2/S3/S4) of FVIIa. Additionally, the rate of incorporation of chloromethylketone inhibitors into FVIIa also involves the irreversible alkylation step, which could be faster for DFFR-ck and DFPR-ck. Once these inhibitors are incorporated, it appears that they induce the same conformation in FVIIa as achieved by S1 site occupancy alone. Thus S1 site occupancy in FVIIa induces the required conformation to modestly increase the affinity for TF. Second Order Rate Constants for Inhibition of FVIIa ± sTF with Various Chloromethylketone (ck) Inhibitors Inhibitor Minus sTF k (min−1 mM−1) Plus sTF k (min−1 mM−1) Fold Difference EGR-ck 0.04 0.35 8.8 dansyl EGR-ck 0.07 2.41 34.4 (D)FPR-ck 2.3 125 54.3 D)FFR-ck 5.6 525 93.8

In blood coagulation serine proteases, amino acid 192 (chymotrypsin numbering) influences substrate and inhibitor specificity, and also forms a peptide bond with Gly 193 , a critical part of the protease oxyanion hole. Residue 192 is either Glu or Gln in several serine proteases including thrombin, factor (F) Xa, FIXa, and activated protein C. However, it is Lys in factor FXIa and FVIIa. Both FXIa and FVIIa/tissue factor (TF) share FIX as their substrate. In addition, FVIIa/TF activates FX and is inhibited by TF pathway inhibitor (TFPI), whereas FXIa is inhibited by diisopropylfluorophosphate (DFP), p -aminobenzamidine ( p- AB), antithrombin and amyloid-b precursor protein Kunitz domain (AbPP). We investigated the importance of Lys 192 in FXIa and FVIIa for substrate/inhibitor specificity and oxyanion hole formation. Recombinant FXIa was prepared with Pro (FXIa K192P ), Glu (FXIa K192E ) or Gln (FXIa K192Q ) substitutions for Lys 192 . Rate constants for inhibition by DFP were similar for wild type FXIa (FXIa WT ), FXIa K192E and FXIa K192Q , but 80-fold slower for FXIa K192P . Only FXIa K192P differed in binding to p AB compared to FXIa WT . These data indicate that Pro 192 causes distortion of the oxyanion hole and S1 binding site. All mutants bound the FXIa substrate FIX normally. However, catalysis of FIX was impaired 400, 35, and 110-fold for FXIa K192P , FXIa K192E , and FXIa K192Q , respectively, while k 2 for inhibition by antithrombin was reduced 160, 15 and 16-fold. FXIa K192P bound to AbPP with 800-fold decreased affinity; while FXIa K192E and FXIa K192Q bound with 6-10-fold higher affinity. Modeling studies indicate that there is loss of the normal interaction between Lys 192 and the substrate/inhibitor P3’ residue in interactions between FXIa K192E and FXIa K192Q and factor IX and antithrombin. The K i for Glu and Gln mutant inhibition by AbPP is slightly better probably due to the ability of these two amino acids to make two H-bonds as compared to only one for Lys 192 . Lys 192 makes H-bond with the carbonyl O of Gly 12 in AbPP, whereas Glu and Gln at this position make H-bonds with the carbonyl O of Cys 14 and Ala 16 in AbPP. In FXIa K192P , the oxyanion hole is impaired due to a flip in the 192-193 peptide bond causing the amide N of Gly 193 to point away from the oxyanion hole, resulting in a severe impairment of all catalytic functions. Thus a non-proline residue 192 is essential for proper oxyanion hole formation in serine proteases, and Lys 192 in FXIa contributes significantly towards macromolecular substrate catalysis and inhibitor binding. In contrast to FXIa WT , the 192-193 peptide bond in FVIIa WT ± TF is already in a nonstandard conformation in which the amide N of Gly 193 points away, and the carbonyl O of the Lys 192 points into the oxyanion hole (Bajaj, et al., J. Biol. Chem.281, 24873-24888, 2006). The H-bond between the amide N of Gly 193 and the carbonyl side chain of Gln 143 , a residue unique to FVIIa, maintains this nonstandard conformation in FVIIa. Notably, upon substrate binding the 192-193 bond flips 180° such that the oxyanion hole is fully formed. Now the H-bond is formed between the amide N of Gly 193 and the carbonyl oxygen of the transition state intermediate. For this to occur, the H-bond between the amide N of Gly 193 and the carbonyl side chain of Gln 143 must first be broken. Previous studies have shown that changing Lys 192 to Glu or Gln in FVIIa severely impairs its biologic activity. However, the structural basis for this impairment is not understood. We performed molecular modeling studies which reveal that the side chain of the Glu 192 carboxyl group or of the Gln 192 carbonyl group makes strong H-bond with the side chain NH 2 group of Gln 143 ; this is not possible for Lys 192 . Thus in Glu 192 or Gln 192 mutant of FVIIa, it is energetically difficult to break the H-bond between the amide N of Gly 193 and the carbonyl side chain of Gln 143 because the H-bond involving the side chain of Glu 192 or Gln 192 and the Gln 143 must be simultaneously broken. This provides a structural rational for the impaired function of FVIIa K192E or FVIIa K192Q . To test this concept further, we prepared FVIIa in which Lys 192 was replaced with alanine. All properties of this alanine mutant were normal substantiating the proposed structural role of Lys 192 in FVIIa. This structural role is unique to FVIIa among all known serine proteases, as is the presence of residue Gln 143 that contributes to this phenomenon.