Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands.
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ABSTRACT:
SUBMITTER: Troisi R
PROVIDER: S-EPMC8509272 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Substrates and cofactors of the serine protease thrombin (IIa) employ two anion binding exosites (ABE-I and -II) to aid in binding. On the surface of platelets resides the GpIbalpha/beta-GpIX-GpV membrane-bound receptor complex. IIa's ABE-II is proposed to interact with an anionic portion of GpIbalpha which enhances IIa cleavage of PAR-1 and subsequent activation of platelets. In this work, one-dimensional (1D) and two-dimensional (2D) NMR, analytical ultracentrifugation (AUC), and hydrogen-deuterium exchange (HDX) coupled with MALDI-TOF MS were performed to further characterize the features of binding to IIa's ABEs. The described work builds upon investigations performed in a prior study with fibrin(ogen)'s gamma' peptide and IIa [Sabo, T. M., Farrell, D. H., and Maurer, M. C. (2006) Biochemistry 45, 7434-7445]. 1D line broadening NMR (1H and 31P) and 2D trNOESY NMR studies indicate that GpIbalpha residues D274-E285 interact extensively with the IIa surface in an extended conformation. AUC demonstrates that both GpIbalpha (269-286) and gamma' (410-427) peptides interact with IIa with a 1:1 stoichiometry. When the HDX results are compared to those for the ABE-I targeting peptide hirudin (54-65), the data imply that GpIbalpha (269-286), GpIbalpha (1-290), and gamma' (410-427) are indeed directed to ABE-II. The ABE-II binding fragments reduce HDX for sites distant from the interface, suggesting long-range conformational effects. These studies illustrate that GpIbalpha and gamma' target ABE-II with similar consequences on IIa dynamics, albeit with differing structural features.
| S-EPMC2842903 | biostudies-literature
Project description:The gamma-loop of thrombin is a flexible, surface-accessible loop in free thrombin that appears to be one of several sites participating in the interaction of the enzyme with macromolecular substrates and inhibitors. Using limited proteolysis and intrinsic fluorescence measurements, we have studied changes in thrombin structure induced by small, site-specific ligands. Binding of a C-terminal peptide of hirudin to the anion-binding exosite of thrombin induced a structural change in the gamma-loop, which caused a 6-fold reduction in the susceptibility of the enzyme to limited proteolysis by elastase and chymotrypsin. Binding of several active site-specific thrombin inhibitors conferred an even greater protection from proteolysis at the gamma-loop. For example, the covalent complex of thrombin with D-Phe-Pro-Arg-CH2Cl was 95-fold less susceptible to cleavage by chymotrypsin than the free enzyme. Furthermore, binding of either exosite or active-site probes induced a common intrinsic fluorescence change in thrombin (a fractional increase of 0.13). These results are surprising because crystallographic studies indicate that direct contact between the bound probes and relevant residues of the gamma-loop is very unlikely. Thus we have identified an allosteric interaction that couples the active site of thrombin to the gamma-loop. An interaction of this nature may be one way in which thrombomodulin modulates the reactivity of thrombin.
| S-EPMC1132332 | biostudies-other
Project description:Thrombin participates in procoagulation, anticoagulation, and platelet activation. This enzyme contains anion binding exosites, ABE I and ABE II, which attract regulatory biomolecules. As prothrombin is activated to thrombin, pro-ABE I is converted into mature ABE I. Unexpectedly, certain ligands can bind to pro-ABE I specifically. Moreover, knowledge of changes in conformation and affinity that occur at the individual residue level as pro-ABE I is converted to ABE I is lacking. Such changes are transient and were not captured by crystallography. Therefore, we employed nuclear magnetic resonance (NMR) titrations to monitor development of ABE I using peptides based on protease-activated receptor 3 (PAR3). Proton line broadening NMR revealed that PAR3 (44-56) and more weakly binding PAR3G (44-56) could already interact with pro-ABE I on prothrombin. 1H-15N heteronuclear single-quantum coherence NMR titrations were then used to probe binding of individual 15N-labeled PAR3G residues (F47, E48, L52, and D54). PAR3G E48 and D54 could interact electrostatically with prothrombin and tightened upon thrombin maturation. The higher affinity for PAR3G D54 suggests the region surrounding thrombin R77a is better oriented to bind D54 than the interaction between PAR3G E48 and thrombin R75. Aromatic PAR3G F47 and aliphatic L52 both reported on significant changes in the chemical environment upon conversion of prothrombin to thrombin. The ABE I region surrounding the 30s loop was more affected than the hydrophobic pocket (F34, L65, and I82). Our NMR titrations demonstrate that PAR3 residues document structural rearrangements occurring during exosite maturation that are missed by reported X-ray crystal structures.
| S-EPMC6076386 | biostudies-literature
Project description:Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.
| S-EPMC3107735 | biostudies-literature
Project description:INTRODUCTION:Thrombin is a primary target of most anticoagulants. Yet, thrombin's dual and opposing role in pro- as well as anti- coagulant processes imposes considerable challenges in discovering finely tuned regulators that maintain homeostasis, rather than disproportionately changing the equilibrium to one side. In this connection, we have been studying exosite 2-mediated allosteric modulation of thrombin activity using synthetic agents called low molecular weight lignins (LMWLs). Although the aromatic scaffold of LMWLs is completely different from the polysaccharidic scaffold of heparin, the presence of multiple negatively charged groups on both ligands induces binding to exosite 2 of thrombin. This work characterizes the nature of interactions between LMWLs and thrombin to understand the energetic cooperativity between exosite 2 and active site of thrombin. MATERIALS AND METHODS:The thermodynamics of thrombin-LMWL complexes was studied using spectrofluorimetric titrations as a function of ionic strength and temperature of the buffer. The contributions of enthalpy and entropy to binding were evaluated using classic thermodynamic equations. Label-free surface plasmon resonance was used to assess the role of sodium ion in LMWL binding to thrombin at a fixed ionic strength. RESULTS AND CONCLUSIONS:Exosite 2-induced conformational change in thrombin's active site is strongly dependent on the structure of the ligand, which has consequences with respect to regulation of thrombin. The ionic and non-ionic contributions to binding affinity and the thermodynamic signature were highly ligand specific. Interestingly, LMWLs display preference for the sodium-bound form of thrombin, which supports the existence of an energetic coupling between exosite 2 and sodium-binding site of thrombin.
| S-EPMC5943173 | biostudies-literature
Project description:The serine proteinase alpha-thrombin plays a pivotal role in the regulation of blood fluidity, and therefore constitutes a primary target in the treatment of various haemostatic disorders. Haemadin is a slow tight- binding thrombin inhibitor from the land-living leech Haemadipsa sylvestris. Here we present the 3.1 A crystal structure of the human alpha-thrombin- haemadin complex. The N-terminal segment of haemadin binds to the active site of thrombin, forming a parallel beta-strand with residues Ser214-Gly216 of the proteinase. This mode of binding is similar to that observed in another leech-derived inhibitor, hirudin. In contrast to hirudin, however, the markedly acidic C-terminal peptide of haemadin does not bind the fibrinogen-recognition exosite, but interacts with the heparin-binding exosite of thrombin. Thus, haemadin binds to thrombin according to a novel mechanism, despite an overall structural similarity with hirudin. Haemadin inhibits both free and thrombomodulin-bound alpha-thrombin, but not intermediate activation forms such as meizothrombin. This specific anticoagulant ability of haemadin makes it an ideal candidate for an antithrombotic agent, as well as a starting point for the design of novel antithrombotics.
| S-EPMC305786 | biostudies-literature
Project description:Activation of platelets by the serine protease thrombin is a critical event in haemostasis. This process involves the binding of thrombin to glycoprotein Ibα (GpIbα) and cleavage of protease-activated receptors (PARs). The N-terminal extracellular domain of GpIbα contains an acidic peptide stretch that has been identified as the main thrombin binding site, and both anion binding exosites of thrombin have been implicated in GpIbα binding, but it remains unclear how they are involved. This issue is of critical importance for the mechanism of platelet activation by thrombin. If both exosites bind to GpIbα, thrombin could potentially act as a platelet adhesion molecule or receptor dimerisation trigger. Alternatively, if only a single site is involved, GpIbα may serve as a cofactor for PAR-1 activation by thrombin. To determine the involvement of thrombin's two exosites in GpIbα binding, we employed the complementary methods of mutational analysis, binding studies, X-ray crystallography and NMR spectroscopy. Our results indicate that the peptide corresponding to the C-terminal portion of GpIbα and the entire extracellular domain bind exclusively to thrombin's exosite II. The interaction of thrombin with GpIbα thus serves to recruit thrombin activity to the platelet surface while leaving exosite I free for PAR-1 recognition.
| S-EPMC3919161 | biostudies-literature
Project description:Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed.
| S-EPMC5224481 | biostudies-literature
Project description:Thrombin, derived from zymogen prothrombin (ProT), is a serine protease involved in procoagulation, anticoagulation, and platelet activation. Thrombin's actions are regulated through anion-binding exosites I and II (ABE I and ABE II) that undergo maturation during activation. Mature ABEs can utilize exosite-based communication to fulfill thrombin functions. However, the conformational basis behind such long-range communication and the resultant ligand binding affinities are not well understood. Protease activated receptors (PARs), involved in platelet activation and aggregation, are known to target thrombin ABE I. Unexpectedly, PAR3 (44-56) can already bind to pro-ABE I of ProT. Nuclear magnetic resonance (NMR) ligand-enzyme titrations were used to characterize how individual PAR1 (49-62) residues interact with pro-ABE I and mature ABE I. 1D proton line broadening studies demonstrated that binding affinities for native PAR1P (49-62, P54) and for the weak binding variant PAR1G (49-62, P54G) increased as ProT was converted to mature thrombin. 1H,15N-HSQC titrations revealed that PAR1G residues K51, E53, F55, D58, and E60 exhibited less affinity to pro-ABE I than comparable residues in PAR3G (44-56, P51G). Individual PAR1G residues then displayed tighter binding upon exosite maturation. Long-range communication between thrombin exosites was examined by saturating ABE II with phosphorylated GpIb? (269-282, 3Yp) and monitoring the binding of PAR1 and PAR3 peptides to ABE I. Individual PAR residues exhibited increased affinities in this dual-ligand environment supporting the presence of interexosite allostery. Exosite maturation and beneficial long-range allostery are proposed to help stabilize an ABE I conformation that can effectively bind PAR ligands.
| S-EPMC6716794 | biostudies-literature
Project description:Polyphosphate (a linear polymer of inorganic phosphate) is secreted from platelet dense granules, and we recently showed that it accelerates factor V activation by thrombin.To examine the interaction of polyphosphate with thrombin.Thrombin, but not prothrombin, altered the electrophoretic migration of polyphosphate in gel mobility assays. Thrombin binding to polyphosphate was influenced by ionic strength, and was evident even in plasma. Two positively charged exosites on thrombin mediate its interactions with other proteins and accessory molecules: exosite I (mainly with thrombin substrates), and exosite II (mainly with certain anionic polymers). Free thrombin, thrombin in complex with hirudin's C-terminal dodecapeptide and gamma-thrombin all bound polyphosphate similarly, excluding exosite I involvement. Mutations within exosite II, but not within exosite I, the Na(+)-binding site or hydrophobic pocket, weakened thrombin binding to polyphosphate as revealed by NaCl dependence. Surface plasmon resonance demonstrated tight interaction of polyphosphate with thrombin (K(d) approximately 5 nm) but reduced interaction with a thrombin exosite II mutant. Certain glycosaminoglycans, including heparin, only partially competed with polyphosphate for binding to thrombin, and polyphosphate did not reduce heparin-catalyzed inactivation of thrombin by antithrombin.Polyphosphate interacts with thrombin's exosite II at a site that partially overlaps with, but is not identical to, the heparin-binding site. Polyphosphate interactions with thrombin may be physiologically relevant, as the polyphosphate concentrations achievable following platelet activation are far above the approximately 5 nM K(d) for the polyphosphate-thrombin interaction.
| S-EPMC2856763 | biostudies-literature