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.

A factor VIIIa-mimetic bispecific antibody, Mim8, ameliorates bleeding upon severe vascular challenge in hemophilia A mice.

Hemophilia A is a bleeding disorder resulting from deficient factor VIII (FVIII), which normally functions as a cofactor to activated factor IX (FIXa) that facilitates activation of factor X (FX). To mimic this property in a bispecific antibody format, a screening was conducted to identify functional pairs of anti-FIXa and anti-FX antibodies, followed by optimization of functional and biophysical properties. The resulting bispecific antibody (Mim8) assembled efficiently with FIXa and FX on membranes, and supported activation with an apparent equilibrium dissociation constant of 16 nM. Binding affinity with FIXa and FX in solution was much lower, with equilibrium dissociation constant values for FIXa and FX of 2.3 and 1.5 µM, respectively. In addition, the activity of Mim8 was dependent on stimulatory activity contributed by the anti-FIXa arm, which enhanced the proteolytic activity of FIXa by 4 orders of magnitude. In hemophilia A plasma and whole blood, Mim8 normalized thrombin generation and clot formation, with potencies 13 and 18 times higher than a sequence-identical analogue of emicizumab. A similar potency difference was observed in a tail vein transection model in hemophilia A mice, whereas reduction of bleeding in a severe tail-clip model was observed only for Mim8. Furthermore, the pharmacokinetic parameters of Mim8 were investigated and a half-life of 14 days shown in cynomolgus monkeys. In conclusion, Mim8 is an activated FVIII mimetic with a potent and efficacious hemostatic effect based on preclinical data.

19F NMR reveals the conformational properties of free thrombin and its zymogen precursor prethrombin-2.

The conformational properties of trypsin-like proteases and their zymogen forms remain controversial because of a lack of sufficient information on their free forms. Specifically, it is unclear whether the free protease is zymogen-like and shifts to its mature form upon a ligand-induced fit or exists in multiple conformations in equilibrium from which the ligand selects the optimal fit via conformational selection. Here we report the results of 19F NMR measurements that reveal the conformational properties of a protease and its zymogen precursor in the free form. Using the trypsin-like, clotting protease thrombin as a relevant model system, we show that its conformation is quite different from that of its direct zymogen precursor prethrombin-2 and more similar to that of its fully active Na+-bound form. The results cast doubts on recent hypotheses that free thrombin is zymogen-like and transitions to protease-like forms upon ligand binding. Rather, they validate the scenario emerged from previous findings of X-ray crystallography and rapid kinetics supporting a pre-existing equilibrium between open (E) and closed (E*) forms of the active site. In this scenario, prethrombin-2 is more dynamic and exists predominantly in the E* form, whereas thrombin is more rigid and exists predominantly in the E form. Ligand binding to thrombin takes place exclusively in the E form without significant changes in the overall conformation. In summary, these results disclose the structural architecture of the free forms of thrombin and prethrombin-2, consistent with an E*-E equilibrium and providing no evidence that free thrombin is zymogen-like.

Tissue Distribution and Receptor Activation by Somapacitan, a Long Acting Growth Hormone Derivative.

Somapacitan is a long-acting, once-weekly, albumin-binding growth hormone (GH) derivative. The reversible albumin-binding properties leads to prolonged circulation half-life. Here, we investigated and compared somapacitan with human GH on downstream receptor signaling in primary hepatocytes and hepatocellular models and using isothermal titration calorimetry to characterize receptor binding of somapacitan in the presence or absence of human serum albumin (HSA). With non-invasive fluorescence imaging we quantitatively visualize and compare the temporal distribution and examine the tissue-specific growth hormone receptor (GHR) activation at distribution sites. We found that signaling kinetics were slightly more rapid and intense for GH compared with somapacitan. Receptor binding isotherms were characterized by a high and a low affinity interaction site with or without HSA. Using in vivo optical imaging we found prolonged systemically biodistribution of somapacitan compared with GH, which correlated with plasma pharmacokinetics. Ex vivo mouse organ analysis revealed that the temporal fluorescent intensity in livers dosed with somapacitan was significantly increased compared with GH-dosed livers and correlated with the degree of downstream GHR activation. Finally, we show that fluorescent-labeled analogs distributed to the hypertrophic zone in the epiphysis of proximal tibia of hypophysectomized rats and that somapacitan and GH activate the GHR signaling in epiphyseal tissues.

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.

Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor.

The Glucagon-like peptide-1 receptor (GLP-1R) is a member of the class B G protein-coupled receptor (GPCR) family and a well-established target for the treatment of type 2 diabetes. The N-terminal extracellular domain (ECD) of GLP-1R is important for GLP-1 binding and the crystal structure of the GLP-1/ECD complex was reported previously. The first structure of a class B GPCR transmembrane (TM) domain was solved recently, but the full length receptor structure is still not well understood. Here we describe the molecular details of antibody-mediated antagonism of the GLP-1R using both in vitro pharmacology and x-ray crystallography. We showed that the antibody Fab fragment (Fab 3F52) blocked the GLP-1 binding site of the ECD directly and thereby acts as a competitive antagonist of native GLP-1. Interestingly, Fab 3F52 also blocked a short peptide agonist believed to engage primarily the transmembrane and extracellular loop region of GLP-1R, whereas functionality of an allosteric small-molecule agonist was not inhibited. This study has implications for the structural understanding of the GLP-1R and related class B GPCRs, which is important for the development of new and improved therapeutics targeting these receptors.

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.

Structural basis of thrombin-protease-activated receptor interactions.

Aggregation of platelets is an essential step in the formation of a stable blood clot during vascular injury. The trypsin-like protease thrombin acts as the dominant agonist of platelet activation on engagement of protease-activated receptors (PARs). Important details on the molecular aspects of thrombin-PAR interactions have been revealed recently by structural biology. In the case of human platelets, PAR1 engages thrombin via an extended surface of recognition encompassing the active site and exosite I. In the case of murine platelets, PAR4 binds to the active site in a conformation that leaves exosite I free for interaction with cofactors like PAR3. Human PAR4 mimics the murine receptor binding mechanism for residues upstream of the scissile bond. This information is consistent with existing functional data and provides a solid background for future structural and mutagenesis studies of PAR interaction with thrombin and related proteases.

Crystallographic and kinetic evidence of allostery in a trypsin-like protease.

Protein allostery is based on the existence of multiple conformations in equilibrium linked to distinct functional properties. Although evidence of allosteric transitions is relatively easy to identify by functional studies, structural detection of a pre-existing equilibrium between alternative conformations remains challenging even for textbook examples of allosteric proteins. Kinetic studies show that the trypsin-like protease thrombin exists in equilibrium between two conformations where the active site is either collapsed (E*) or accessible to substrate (E). However, structural demonstration that the two conformations exist in the same enzyme construct free of ligands has remained elusive. Here we report the crystal structure of the thrombin mutant N143P in the E form, which complements the recently reported structure in the E* form, and both the E and E* forms of the thrombin mutant Y225P. The side chain of W215 moves 10.9 Å between the two forms, causing a displacement of 6.6 Å of the entire 215-217 segment into the active site that in turn opens or closes access to the primary specificity pocket. Rapid kinetic measurements of p-aminobenzamidine binding to the active site confirm the existence of the E*-E equilibrium in solution for wild-type and the mutants N143P and Y225P. These findings provide unequivocal proof of the allosteric nature of thrombin and lend strong support to the recent proposal that the E*-E equilibrium is a key property of the trypsin fold.

Engineering thrombin for selective specificity toward protein C and PAR1.

Thrombin elicits functional responses critical to blood homeostasis by interacting with diverse physiological substrates. Ala-scanning mutagenesis of 97 residues covering 53% of the solvent accessible surface area of the enzyme identifies Trp(215) as the single most important determinant of thrombin specificity. Saturation mutagenesis of Trp(215) produces constructs featuring k(cat)/K(m) values for the hydrolysis of fibrinogen, protease-activated receptor PAR1, and protein C that span five orders of magnitude. Importantly, the effect of Trp(215) replacement is context dependent. Mutant W215E is 10-fold more specific for protein C than fibrinogen and PAR1, which represents a striking shift in specificity relative to wild-type that is 100-fold more specific for fibrinogen and PAR1 than protein C. However, when the W215E mutation is combined with deletion of nine residues in the autolysis loop, which by itself shifts the specificity of the enzyme from fibrinogen and PAR1 to protein C, the resulting construct features significant activity only toward PAR1. These findings demonstrate that thrombin can be re-engineered for selective specificity toward protein C and PAR1. Mutations of Trp(215) provide important reagents for dissecting the multiple functional roles of thrombin in the blood and for clinical applications.

Crystal structure of thrombin bound to the uncleaved extracellular fragment of PAR1.

Abundant structural information exists on how thrombin recognizes ligands at the active site or at exosites separate from the active site region, but remarkably little is known about how thrombin recognizes substrates that bridge both the active site and exosite I. The case of the protease-activated receptor PAR1 is particularly relevant in view of the plethora of biological effects associated with its activation by thrombin. Here, we present the 1.8 A resolution structure of thrombin S195A in complex with a 30-residue long uncleaved extracellular fragment of PAR1 that documents for the first time a productive binding mode bridging the active site and exosite I. The structure reveals two unexpected features of the thrombin-PAR1 interaction. The acidic P3 residue of PAR1, Asp(39), does not hinder binding to the active site and actually makes favorable interactions with Gly(219) of thrombin. The tethered ligand domain shows a considerable degree of disorder even when bound to thrombin. The results fill a significant gap in our understanding of the molecular mechanisms of recognition by thrombin in ways that are relevant to other physiological substrates.

Mutant N143P reveals how Na+ activates thrombin.

The molecular mechanism of thrombin activation by Na(+) remains elusive. Its kinetic formulation requires extension of the classical Botts-Morales theory for the action of a modifier on an enzyme to correctly account for the contribution of the E*, E, and E:Na(+) forms. The extended scheme establishes that analysis of k(cat) unequivocally identifies allosteric transduction of Na(+) binding into enhanced catalytic activity. The thrombin mutant N143P features no Na(+)-dependent enhancement of k(cat) yet binds Na(+) with an affinity comparable to that of wild type. Crystal structures of the mutant in the presence and absence of Na(+) confirm that Pro(143) abrogates the important H-bond between the backbone N atom of residue 143 and the carbonyl O atom of Glu(192), which in turn controls the orientation of the Glu(192)-Gly(193) peptide bond and the correct architecture of the oxyanion hole. We conclude that Na(+) activates thrombin by securing the correct orientation of the Glu(192)-Gly(193) peptide bond, which is likely flipped in the absence of cation. Absolute conservation of the 143-192 H-bond in trypsin-like proteases and the importance of the oxyanion hole in protease function suggest that this mechanism of Na(+) activation is present in all Na(+)-activated trypsin-like proteases.

Mechanism of the anticoagulant activity of thrombin mutant W215A/E217A.

The thrombin mutant W215A/E217A (WE) is a potent anticoagulant both in vitro and in vivo. Previous x-ray structural studies have shown that WE assumes a partially collapsed conformation that is similar to the inactive E* form, which explains its drastically reduced activity toward substrate. Whether this collapsed conformation is genuine, rather than the result of crystal packing or the mutation introduced in the critical 215-217 beta-strand, and whether binding of thrombomodulin to exosite I can allosterically shift the E* form to the active E form to restore activity toward protein C are issues of considerable mechanistic importance to improve the design of an anticoagulant thrombin mutant for therapeutic applications. Here we present four crystal structures of WE in the human and murine forms that confirm the collapsed conformation reported previously under different experimental conditions and crystal packing. We also present structures of human and murine WE bound to exosite I with a fragment of the platelet receptor PAR1, which is unable to shift WE to the E form. These structural findings, along with kinetic and calorimetry data, indicate that WE is strongly stabilized in the E* form and explain why binding of ligands to exosite I has only a modest effect on the E*-E equilibrium for this mutant. The E* --> E transition requires the combined binding of thrombomodulin and protein C and restores activity of the mutant WE in the anticoagulant pathway.

Stabilization of the E* form turns thrombin into an anticoagulant.

Previous studies have shown that deletion of nine residues in the autolysis loop of thrombin produces a mutant with an anticoagulant propensity of potential clinical relevance, but the molecular origin of the effect has remained unresolved. The x-ray crystal structure of this mutant solved in the free form at 1.55 A resolution reveals an inactive conformation that is practically identical (root mean square deviation of 0.154 A) to the recently identified E* form. The side chain of Trp(215) collapses into the active site by shifting > 10 A from its position in the active E form, and the oxyanion hole is disrupted by a flip of the Glu(192)-Gly(193) peptide bond. This finding confirms the existence of the inactive form E* in essentially the same incarnation as first identified in the structure of the thrombin mutant D102N. In addition, it demonstrates that the anticoagulant profile often caused by a mutation of the thrombin scaffold finds its likely molecular origin in the stabilization of the inactive E* form that is selectively shifted to the active E form upon thrombomodulin and protein C binding.

Structural identification of the pathway of long-range communication in an allosteric enzyme.

Allostery is a common mechanism of regulation of enzyme activity and specificity, and its signatures are readily identified from functional studies. For many allosteric systems, structural evidence exists of long-range communication among protein domains, but rarely has this communication been traced to a detailed pathway. The thrombin mutant D102N is stabilized in a self-inhibited conformation where access to the active site is occluded by a collapse of the entire 215-219 beta-strand. Binding of a fragment of the protease activated receptor PAR1 to exosite I, 30-A away from the active site region, causes a large conformational change that corrects the position of the 215-219 beta-strand and restores access to the active site. The crystal structure of the thrombin-PAR1 complex, solved at 2.2-A resolution, reveals the details of this long-range allosteric communication in terms of a network of polar interactions.