Unfractionated heparin (UFH), the standard anticoagulant for cardiopulmonary bypass (CPB) surgery, carries a risk of post-operative bleeding and is potentially harmful in patients with ...heparin-induced thrombocytopenia-associated antibodies. To improve the activity of an alternative anticoagulant, the RNA aptamer 11F7t, we solved X-ray crystal structures of the aptamer bound to factor Xa (FXa). The finding that 11F7t did not bind the catalytic site suggested that it could complement small-molecule FXa inhibitors. We demonstrate that combinations of 11F7t and catalytic-site FXa inhibitors enhance anticoagulation in purified reaction mixtures and plasma. Aptamer-drug combinations prevented clot formation as effectively as UFH in human blood circulated in an extracorporeal oxygenator circuit that mimicked CPB, while avoiding side effects of UFH. An antidote could promptly neutralize the anticoagulant effects of both FXa inhibitors. Our results suggest that drugs and aptamers with shared targets can be combined to exert more specific and potent effects than either agent alone.
Coagulation factor V (FV) circulates as an inactive procofactor with a domain organization of A1-A2-B-A3-C1-C2. Factor Va (FVa), the active cofactor, is produced in steps essential for rapid thrombin ...formation, by the proteolytic excision of the B domain which resolves the molecule into a heterodimer (A1-A2/A3-C1-C2). Removal of the B domain imbues the resulting FVa with the ability to bind factor Xa (Xa) on a membrane surface to assemble prothrombinase and greatly enhance the rate of thrombin formation. A recombinant variant of human factor V (HFVDT) with a shortened B domain exhibits constitutive cofactor activity. Cofactor activity even without proteolysis arises from its lack of a conserved basic region (BR) located in the large B domain of FV. Exogenously added BR peptide binds tightly to HFVDT in a Ca2+-dependent fashion and restores procofactor-like properties. The BR is proposed to restrict Xa binding and cofactor function by interacting with an acidic region at the C terminus of the B domain (AR2) and likely also an acidic sequence at the C terminus of the A2 domain (AR1). These two sequences are ~800 residues apart in FV with no structural information to explain how AR1 and AR2 might cooperate to engage the BR in the central portion of the B domain to autoinhibit FV. The available structures of an inactivated form of bovine factor Va (BFVai), of a FV ortholog from Pseudonaja textilis (FVPtex) and a lower resolution structure of B domainless human factor VIII (HFVIII) shed no light on this problem. We obtained diffraction quality crystals of HFVDT complexed with a single chain antibody (scFvE10) directed to FVa. Crystals were not obtained in the absence of scFvE10. The crystals diffracted to a resolution of 2.8 Å and the structure was solved by molecular replacement. The refined structure shows high similarity to BFVai, FVPtex and HFVIII. Insufficient electron density precluded the placement of scFvE10 in the modeled structure. The three homologous A domains in HFVDT adopt a typical cupredoxin-fold with the A domains arranged in a pseudo-three-fold axis of symmetry. The two C domains are cylindrical and oriented side-by-side to form the base of the A domain rosette. These features are equivalent to those seen in structures of BFVai, HFVIII and FVPtex. Two bound calcium ions are evident, one in A1 and the other in the A3 domain. The most important feature newly revealed in the structure of HFVDT is the close spatial proximity of AR1 and AR2 at the outer edge of the A domain rosette at the 3 o'clock position in the standard orientation. These acidic regions form adjacently positioned surfaces in spite of being bisected by the long primary sequence of the intervening B domain. Our observations provide the first structural evidence that the two distinct acidic regions come together in space to provide an extended surface. This provides a plausible explanation for how the BR in the middle of the B domain may bind to both AR1 and AR2 to restrict cofactor function in FV. The need for this extended but bipartite acidic surface, to which the BR may bind, also provides a plausible explanation for how proteolytic cleavage at position 1545 at the C terminus of AR2 destabilizes BR binding and results in cofactor formation. The Ca2+-stabilized loop in the A3 domain abuts the bisegmental acidic cluster potentially explaining why BR binding to HFVDT is strongly dependent on Ca2+. In the structure of FVPtex bound to snake venom factor X, AR1 extends away from the body of the cofactor to make intimate contacts with factor X. If this is mirrored in human prothrombinase, then our findings provide a structure-based model to phrase the long-standing procofactor activation paradox. BR binding to the AR1/AR2 extended surface ties up surfaces necessary for Xa binding and restricts cofactor activity. Proteolytic processing of the B domain and probably most importantly following AR2 destabilizes the BR/AR1/AR2 complex to free up surfaces including AR1 necessary to support Xa binding. This model reconciles the biochemical evidence with structural findings to provide new insights into the role played by the BR/AR1/AR2 complex in restricting Xa binding and cofactor function in FV. It provides a platform to further explore mechanistic details of FV and FVa function and for the development of novel strategies to modulate their functions to regulate thrombin formation for therapeutic gain.
Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding; Spark Therapeutics: Other: Scientific advisory board.
Receptor tyrosine kinases (RTKs) orchestrate embryonic development and tissue homeostasis by controlling key cellular processes like growth and proliferation. Consequently, the vast majority of the ...58 RTKs in humans are linked to cancer or metabolic disorders. For instance, RTKs represent known oncogenic drivers (such as EGFR in non‐small cell lung cancer), contribute to cancer hallmarks (such as VEGFR in pro‐tumor angiogenesis), and mediate resistance to therapeutics or hormones (such as the Insulin Receptor in diabetes). Classically, RTKs transduce signals through various modes of ligand‐induced receptor dimerization and subsequent tyrosine kinase‐mediated signaling; however, ~10% of all metazoan RTKs are pseudokinases—that is, they lack the capability to catalyze phosphotransfer. Our objective is to ascertain how RTK pseudokinases relay extracellular cues without kinase activity and whether they can be targeted therapeutically. Using structural, biophysical, and cell signaling approaches, we investigated this class of RTKs to shed light on potential non‐catalytic signaling mechanisms. We identified structural elements conserved between the pseudokinases and their kinase relatives, found their pseudokinase domains to be conformationally dynamic, and uncovered a putative route to drugging these receptors via “conformational disrupting” small molecules. Our findings suggest that RTK pseudokinases could serve as viable drug targets and set the scene for future dissection of signaling pathways that have long been considered enigmatic.
Bacteriophage lambda has for many years been a model system for understanding mechanisms of gene regulation. A 'genetic switch' enables the phage to transition from lysogenic growth to lytic ...development when triggered by specific environmental conditions. The key component of the switch is the cI repressor, which binds to two sets of three operator sites on the lambda chromosome that are separated by about 2,400 base pairs (bp). A hallmark of the lambda system is the pairwise cooperativity of repressor binding. In the absence of detailed structural information, it has been difficult to understand fully how repressor molecules establish the cooperativity complex. Here we present the X-ray crystal structure of the intact lambda cI repressor dimer bound to a DNA operator site. The structure of the repressor, determined by multiple isomorphous replacement methods, reveals an unusual overall architecture that allows it to adopt a conformation that appears to facilitate pairwise cooperative binding to adjacent operator sites.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The prothrombinase complex assembles through high affinity interactions between factor Xa and factor Va on the surface of membranes exposing phosphatidylserine. Assembly of prothrombinase allows for ...the highly accelerated formation of thrombin localized at the injury site. Because the rate of thrombin formation by factor Xa alone is profoundly increased upon its incorporation into prothrombinase, disrupting the proteinase-cofactor interaction could represent an effective therapeutic strategy for the treatment of thrombosis. However, the extended interface between Xa and Va, likely involving shallow surfaces of the interacting proteins, limits the available strategies for the development of ligands that interfere with prothrombinase assembly with high affinity. The RNA aptamer, 11F7t, was selected in an unbiased SELEX screen using human factor Xa as a target and identified as a potent anticoagulant. Detailed functional characterization revealed that 11F7t binds both X and Xa with high affinity (~1 nM) and competes for the interaction between Xa and Va without occluding active site function of the proteinase. Competititive inhibition of the assembly of prothrombinase likely accounts for a major fraction of its anticoagulant effects. However, additional inhibitory effects of 11F7t on coagulation also probably arise from its ability to partially inhibit X activation by the intrinsic Xase and the inhibition of factor VIII activation by Xa. Here we report the crystal structure of 11F7t complexed with desGla-Xa containing the catalytic Ser195 replaced with Ala at 2.5 Å resolution and solved by molecular replacement. In the Xa model, the first 30 residues expected in the light chain, 5 residues in the autolysis loop and 7 residues at the C terminus were not defined by electron density. In 11F7t, 25 of 38 nucleotides could be unambiguously modeled. The tertiary fold of 11F7t presents an extended molecular surface for interactions with desGla-Xa and buries 1384 Å2 of solvent accessible surface area in the interacting species. The RNA aptamer exclusively binds the proteinase domain with contacts over a broad surface area that includes residues Leu59-Lys62, Val87-Arg93, Phe101, Lys236, Trp237 and Arg240 but not the catalytic site. Some of these residues have been implicated in heparin binding and have also been proposed to participate in Va binding based on modest changes in function upon mutagenesis. Even though 11F7t does not make any contacts in the 165 helix previously proposed to be a key region for the interaction with Va, aptamer binding is sufficient to disrupt the high affinity cofactor-proteinase interaction. The structure also reveals that most of the aptamer–protein contact involves nucleotides that constitute a base-paired stem (formed between G3-C6 and 31G-34C) and loop (C7-G11) of 11F7t with these interactions mostly specified by the nucleotide bases rather than the phosphate backbone which might be expected for a protein surface already implicated in binding a polyanion such as heparin or an acidic peptide from the C terminus of the A2 domain of Va. The regions of desGla-Xa occluded by the aptamer point to an extended surface that plays an important role in mediating interactions with factor Va within prothrombinase and suggests commonalities between this binding mode and the way that factor X might bind to factor VIIIa as a substrate within intrinsic Xase or the way that factor Xa might engage factor VIII as an enzyme.
No relevant conflicts of interest to declare.
Factor V, the inactive precursor to factor Va, has a domain organization of A1-A2-B-A3-C1-C2. Factor Va is formed by the proteolytic excision of the central B domain, which resolves the molecule into ...a heterodimer (A1-A2/A3-C1-C2). Removal of the B domain enables the cofactor to engage factor Xa on phosphatidylserine-containing membranes, assemble prothrombinase and greatly enhance the rate of thrombin formation. Recent studies have shown a key role for a basic region (BR), which lies approximately in the center of the B domain, in enforcing procofactor properties in human factor V (hFV). Exogenously added recombinant BR fragments can bind with high affinity to a cofactor-like variant of human hFV (hFVDT), in which a large central portion of the B domain has been deleted, interfere with Xa binding and restore procofactor-like properties. Biochemical evidence suggests that BR binding results from its interaction with an acidic region (AR2) at the C terminus of the B domain and likely also an acidic sequence (AR1) at the C terminus of the A2 domain. Our recent crystal structure of hFVDT provided the first structural evidence that AR1 and AR2, ~800 residues apart in the primary structure of hFV, are positioned adjacent to each other and could plausibly form an extended surface for high affinity BR binding to reconstitute a tripartite procofactor-regulatory region (AR1/BR/AR2). However, the lack of BR in hFVDT precluded independent structural verification of this possibility. In a computational approach, we created a molecular model for the 58 residue BR peptide. The top scoring three-dimensional models of the 58 residue BR peptide showed a helix-loop arrangement, contrary to the general belief that the B domain lacks structured regions. The best scoring BR peptide model was used for ab initio docking studies using the crystal structure of hFVDT to predict possible binding sites using PIPER and ClusPro. The most highly represented and statistically probable solutions showed the BR peptide in intimate contact with juxtaposed surfaces provided by AR1 and AR2. Interestingly, the docked BR peptide contacted regions in AR1 and on the A2 domain implicated in FXa binding in the structure of Pseudonaja textilis FV bound to snake venom factor X. Computational predictions were tested using hydrogen-deuterium exchange detected by protein fragmentation and mass spectroscopy (HDX). Proteolytic fragmentation of hFVDT and fragment detection by LC-MS was optimized to cover >95% of its 1514 residues with an average redundancy of 4.27 peptides/residue. Only 4 or 5 segments of ~10-15 residue length were not covered. Addition of the BR peptide had minor effects on amide proton exchange over the bulk of the molecule. However, BR peptide binding was accompanied by reductions in amide proton exchange rates of ~7-30-fold in immediately adjacent regions of hFVDT corresponding to sequences within A2 (626-634), AR1 (658-695), AR2 (872-881) and A3 (983-995). BR peptide binding to hFVDT is accompanied by perturbations in these spatially adjacent regions covering a small fraction of the surface area at approximately the 3 o’clock position with the molecule in the standard orientation. The marked agreement between the HDX findings and the computational docking studies supports our proposal that the BR engages an extended surface contributed by AR1 and AR2 to form a tripartite procofactor-regulatory region. The interaction of BR with AR1 and a small region in A2, both implicated in binding Xa, potentially explains how the BR might restrict Xa binding to the procofactor. Destabilization of BR binding by proteolysis at the C terminus of AR2 is envisioned to result in cofactor formation by releasing the BR and revealing sites responsible for binding Xa. Our findings provide a structural explanation for the long standing puzzle of factor V activation and pave the way for further definition of mechanistic details of procofactor and cofactor function. They have implications for how interactions with TFPIα through the basic region at its C-terminus might regulate FV(a). They also reveal previously unanticipated strategies to modulate functions of hFV and hFVa for therapeutic gain.
Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding; Bayer: Consultancy; Novo Nordisk: Research Funding; sparK: Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Krishnaswamy:Portola: Research Funding; Janssen: Consultancy, Research Funding.
Factor Va binds to membranes exposing phosphatidylserine (PS) with nanomolar affinity. This high affinity interaction plays a vital role in the assembly of membrane-bound prothrombinase thereby ...supporting robust thrombin formation at the site of vascular damage. Although the importance of the C1 and C2 domains of FVa in membrane binding has long been recognized, mechanistic details are incompletely understood. Unlike human prothrombinase, Pseudonaja textilis (common brown snake, P. tex) has evolved a membrane-independent form of prothrombinase composed of a FVa-like protein (VPtex) tightly bound to a FXa-like protein in solution. Our structural advances with FVPtex provide new tools to address major unresolved questions related to membrane binding by FVa. The high resolution (1.95Å) structure of VPtex resembles previously published structures of inactivated bovine FVa and human FVIII of lower resolution with all the structural features considered critical for membrane binding by FVa. However, VPtex bound to PS-containing membranes with very poor affinity (Kd > 1 µM). Substitution of 9 residues in the C1 and C2 domains of VPtex with residues present in the hemostatic form of FV present in the plasma of the snake yielded a derivative (VPtexC1C2) that bound to PS-containing membranes with nanomolar affinity equivalent to hFV. Interestingly, the newly acquired function of membrane binding in VPtexC1C2 does not affect its ability to function in solution, suggesting that membrane binding and solution-phase function are controlled independently. Variants containing substitutions in the individual C domains (VPtexC1 and VPtexC2) exhibited intermediate affinities (Kd=100 nM and Kd=50 nM) for binding to PS-containing membranes. However, the binding energy contributions from the individual C-domains did not additively explain the affinity of VPtexC1C2 for membranes. The large connection energy (-8.7 kcal/mole) implies substantial energetic expenditure, possibly through a conformational rearrangement, upon membrane binding. This correlates well with higher thermal factors observed in the C1 and C2 domains of structures of VPtexC1C2 and VPtexC2 as compared to VPtex. It is also supported by rapid kinetic studies illustrating equivalence in the bimolecular association rate constants for human Va and VPtex variants regardless of their membrane affinity. Thus, high affinity membrane binding results from large decreases in the dissociation rate constant expected from a conformational change that allows the protein to adopt a new stable membrane-bound configuration. A second explanation for the lack of an obvious correlation between x-ray structures of VPtexC1C2, VPtexC2 and VPtex and their affinity for membranes lies in the possibility that their solution-phase conformations differ. We explored this using small angle x-ray scattering (SAXS) of VPtex, VPtexC2 and VPtexC1C2 in solution. The low resolution SAXS envelope for VPtex could be accounted for by minor shifts in the individual domains, particularly in C1 and C2, as seen in the crystal structure. However, the SAXS envelopes for membrane binding variants (VPtexC2 and VPtexC1C2) showed major shape changes in the C-domains. Rigid body modeling revealed an increasingly extended end-on arrangement, rather than a side-by-side configuration, of the C1 and C2 domains seen in the x-ray structure and in the SAXS envelope for FVPtex. The C2-domain was found to extend away from the base of the C1 domain along the long axis of the molecule correlating major structural differences in these VPtex variants with increasing affinity for membranes. Accordingly, the spatial disposition of the C1 and C2 domains in VPtexC2 appears intermediate to their arrangement in VPtex and VPtexC1C2.These findings contrast to the arrangement seen in the crystal structures of all factor V forms, where the C-domains are arranged side-by-side, probably due to limitations imposed by crystal packing. Our SAXS studies provide clear evidence of an unforeseen framework of C-domains associated with the ability of factor V forms to bind to membranes with high affinity. The findings reveal new mechanistic insights into the structural correlates of the membrane binding function of factor V.
Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding.
Abstract 3363
Proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 depends on its non-catalytic domain (or exosite) interactions. These exosites, particularly the residues between Tyr659 ...and Tyr665 and their adjacent residues Arg568 and Phe592 in the ADAMTS13 spacer domain, are the major targets of anti-ADAMTS13 autoantibodies in patients with acquired TTP. In an effort to further determine the potential exosite interactions between ADAMTS13 and VWF, we carried out crystal screening of a recombinant full-length ADAMTS13 purified from stably transfected Chinese hamster ovary cells. After over 900 conditions were tested, we obtained multiple well-formed and diffractable crystals. Upon X-ray diffraction, we discovered that only a proteolytic fragment of full-length ADAMTS13 (Met432-Arg670) had been consistently crystallized. Here, we report the structure of this non-catalytic ADAMTS13 fragment at 2.2Å. The fragment encompasses 8 residues from the first TSP-1 repeat, an entire cysteine-rich domain (CA and CB), and a majority of the spacer domain. While recombinant ADAMTS13 in the present study is fully glycosylated, the overall structure agrees very well with the analogous portion of the ADAMTS13-DTCS structure previously reported (RMSD = 0.02Å, 1817 atoms, 232 residues). A region within the CA domain does vary slightly from that in the ADAMTS13-DTCS (RMSD = 0.08Å, 379 atoms, 51 residues). As with the ADAMTS13-DTCS structure, our structure shows 3 intermolecular disulfide bonds in CA, in addition to 2 free cysteine residues. In this region, our structure shows differences in the disulfide bonding pairing, and a slight shift in the α4 helix from Gly479-Cys483. Specifically, our structure reveals an intermolecular disulfide bond between the residues Cys483 and Cys527 and Cys508 and Cys522. These disulfide pair patterns are different from those reported in the ADAMTS13-DTCS structure, in which disulfide bonds are formed between the residues Cys483 and Cys522 (4.6Å apart in our structure) and the residues Cys508 and Cys527 (3.8Å apart in our structure). Our results indicate that, despite the difference in conditions under which crystals of ADAMTS13 fragments are formed, the overall structure of the Cys-rich and spacer domains are quite similar. The difference in the disulfide bond pattering may provide a novel insight into the mechanism regarding the ADAMTS13 exosite interaction with VWF. Our ongoing work is aimed to determine the complex formation between ADAMTS13 and its substrate or autoantibodies.
Rottensteiner:Baxter Innovations GmbH: Employment.
Abstract 375
Poisonous snakes frequently harbor activators of mammalian coagulation as part of the toxin repertoire in their venom. The venom of Pseudonaja textilis (Ptex, common brown snake) ...contains an efficient activator of human prothrombin comprised of a Xa-like protein tightly bound to a Va-like protein. The constituents of this complex exhibit high sequence homology to the corresponding activated coagulation factors in mammalian blood. Factors Xa and Va are produced in blood upon proteolytic activation of their precursors, complex with each other in membrane-dependent reactions to form prothrombinase and catalyze thrombin formation at the site of vascular damage. In contrast, the venom proteins are constitutively active, form a complex in solution and can efficiently catalyze prothrombin activation in the absence of membranes. These properties likely drive the disseminated and consumptive coagulopathy associated with evenomation by P. textilis. The Va-like component (Ptex-Va) of P. textilis venom is a single chain glycoprotein of 1430 residues with 53% identity to human factor V (hV) and a common A1-A2-B-A3-C1-C2 domain organization. The B-domain of Ptex-Va is significantly shorter than its counterpart in hV (46 vs. 836 residues). We now report a high resolution x-ray structure of recombinant Ptex-Va collected at 1.9 Å resolution and solved by molecular replacement. The resulting structure closely mimics those seen at lower resolution for inactivated bovine factor Va lacking the A2 domain and full length B-domainless human factor VIII (hVIII). Each A domain is formed by two cupredoxin-like β barrels with the A domains arranged in a pseudo-three-fold axis of symmetry. The two C domains are roughly cylindrical and oriented side-by-side to form a pedestal for the A1-A2-A3 rosette. The A3 domain makes extensive contacts with the C1 and A2 domains. The structure also reveals a disulfide bond unique to Ptex-Va, linking Cys642 in the A2 domain with Cys1002 in the A3. These features likely account for the high stability of the molecule even after proteolytic processing of the B domain and/or cleavage between the A1 and A2 domains. Although the C2 domain is significantly more disordered than the other domains, both C1 and C2 each contain protruding loops at their base with hydrophobic residues pointing outward. These structural features replicate those found in inactivated bovine Va and hVIII considered critical for membrane binding by the hemostatic cofactors. Surprisingly, despite the presence of these structures, light scattering measurements revealed negligible binding of Ptex-Va to synthetic membranes composed of phosphatidylcholine and phosphatidylserine with an estimated 103-fold weaker affinity than that of hV. We reasoned that this unexpected property of the venom protein, not conducive to regulated coagulation, was unlikely to be replicated in factor V from the plasma of the snake. Sequence alignment of Ptex-Va with factor V from snake plasma revealed 11 differences in the 328 residues of the C1 and C2 domains. Of these, 9 were located on the distal region of these domains, occupying a band approximately 7 Å thick across the molecule. Mutagenesis of Ptex-Va to introduce these 9 substitutions followed by its expression and purification yielded a derivative that bound to membranes with high affinity in a manner equivalent to hV. This striking gain in function sheds new and unexpected light on the structural determinants of high affinity membrane binding in Ptex-Va and by extension, its homologues hV and hVIII. Our high resolution structure of this hV-like species with a series of unusual properties provides a unique platform to address major but unresolved questions related to the structural correlates of hV function. It also reveals the basis for molecular mimicry whereby a cofactor essential for regulated blood coagulation has served as a scaffold for the evolution of a potent toxin by simultaneous loss in the ability to bind membranes and a gain in the ability to bind its proteinase with high affinity in a membrane-independent fashion.
No relevant conflicts of interest to declare.
Abstract only
Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes. Inappropriate ...activation of these receptors is a key feature of many human cancers, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signaling, called Argos, was identified in Drosophila. Argos functions by directly binding to (and sequestering) growth factor ligands that activate EGFR. Here we describe the 1.6Å‐resolution crystal structure of Argos bound to Spitz, an EGFR ligand. Contrary to expectations and previous reports, Argos itself does not contain an EGF‐like domain. Instead, a trio of closely related domains (resembling a three‐finger toxin fold) form a clamp‐like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF domain of Spitz. The three‐domain clamp of Argos resembles the human urokinase‐type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF‐like module of uPA. Our results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues.
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