Binding Of Benzamidine Research Articles

Cross-Polarization of Insensitive Nuclei from Water Protons for Detection of Protein-Ligand Binding.

Hyperpolarization derived from water protons enhances the NMR signal of 15N nuclei in a small molecule, enabling the sensitive detection of a protein-ligand interaction. The water hyperpolarized by dissolution dynamic nuclear polarization (D-DNP) acts as a universal signal enhancement agent. The 15N signal of benzamidine was increased by 1480-fold through continuous polarization transfer by J-coupling-mediated cross-polarization (J-CP) via the exchangeable protons. The signal enhancement factor favorably compares to factors of 110- or 17-fold using non-CP-based polarization transfer mechanisms. The hyperpolarization enabled detection of the binding of benzamidine to the target protein trypsin with a single-scan measurement of 15N R2 relaxation. J-CP provides an efficient polarization mechanism for 15N or other low-frequency nuclei near an exchangeable proton. The hyperpolarization transfer sustained within the relaxation time limit of water protons additionally can be applied for the study of macromolecular structure and biological processes.

Water regulates the residence time of Benzamidine in Trypsin

The process of ligand-protein unbinding is crucial in biophysics. Water is an essential part of any biological system and yet, many aspects of its role remain elusive. Here, we simulate with state-of-the-art enhanced sampling techniques the binding of Benzamidine to Trypsin which is a much studied and paradigmatic ligand-protein system. We use machine learning methods to determine efficient collective coordinates for the complex non-local network of water. These coordinates are used to perform On-the-fly Probability Enhanced Sampling simulations, which we adapt to calculate also the ligand residence time. Our results, both static and dynamic, are in good agreement with experiments. We find that the presence of a water molecule located at the bottom of the binding pocket allows via a network of hydrogen bonds the ligand to be released into the solution. On a finer scale, even when unbinding is allowed, another water molecule further modulates the exit time.

Changes in Protonation States of In-Pathway Residues can Alter Ligand Binding Pathways Obtained From Spontaneous Binding Molecular Dynamics Simulations.

Protein-ligand binding processes often involve changes in protonation states that can be key to recognize and orient the ligand in the binding site. The pathways through which (bio)molecules interplay to attain productively bound complexes are intricate and involve a series of interconnected intermediate and transition states. Molecular dynamics (MD) simulations and enhanced sampling techniques are commonly used to characterize the spontaneous binding of a ligand to its receptor. However, the effect of protonation state changes of in-pathway residues in spontaneous binding MD simulations remained mostly unexplored. Here, we used molecular dynamics simulations to reconstruct the trypsin-benzamidine binding pathway considering different protonation states of His57. This residue is part of the trypsin catalytic triad and is located more than 10 Å away from Asp189, which is responsible for benzamidine binding in the trypsin S1 pocket. Our MD simulations showed that the binding pathways that benzamidine follow to target the S1 binding site are critically dependent on the His57 protonation state. Binding of benzamidine frequently occurs when His57 is protonated in the delta nitrogen while the binding process is significantly less frequent when His57 is positively charged. Constant-pH MD simulations retrieved the equilibrium populations of His57 protonation states at trypsin active pH offering a clearer picture of benzamidine recognition and binding. These results indicate that properly accounting for protonation states of distal residues can be important in spontaneous binding MD simulations.

Exploring the Ligand Binding/Unbinding Pathway by Selectively Enhanced Sampling of Ligand in a Protein-Ligand Complex.

Understanding the protein-ligand binding is of fundamental biological interest and is essential for structure-based drug design. The difficulty in capturing the dynamic process, however, poses a great challenge for current experimental and theoretical simulation techniques. A selective integrated-tempering-sampling molecular dynamics (SITSMD) method offering an option for selectively enhanced sampling of the ligand in a protein-ligand complex was utilized to quantitatively illuminate the binding of benzamidine to the wild-type trypsin protease and its two mutants (S214E and S214K). The SITSMD simulations could produce consistent results as the extensive conventional MD simulation and gave additional insights into the binding pathway for the test protein-ligand complex system using significantly saved computational resource and time, indicating the potential of such a method in investigating protein-ligand binding. Additionally, the simulations identified the different roles of trypsin-benzamidine van der Waals (vdW) and electrostatic interactions in the binding: the former interaction works as the driving force for dragging the benzamidine close to the native binding pocket, and the latter interaction mainly contributes to stabilizing the benzamidine inside the pocket. The S214E mutation introduces more favorable electrostatic interactions, and as a result, both vdW and electrostatic interactions drive the benzamidine binding, lowering the binding and unbinding free energy barrier. In contrast, the S214K mutation prohibits the binding of the benzamidine to the native ligand binding pocket by introducing disliked charge-charge interactions. In summary, these findings suggest that the change in specific residues could modify the protein druggability, including the binding kinetics and thermodynamics.

Deciphering the canonical blockade of activated Hageman factor (FXIIa) by benzamidine in the coagulation cascade: A thorough dynamical perspective.

The experimental inhibitory potency of benzamidine (BEN) paved way for further design and development of inhibitors that target β-FXIIa. Structural dynamics of the loops and catalytic residues that encompass the binding pocket of β-FXIIa and all serine proteases are crucial to their overall activity. Employing molecular dynamics and post-MD analysis, this study sorts to unravel the structural and molecular events that accompany the inhibitory activity of BEN on human β-FXIIa upon selective non-covalent binding. Analysis of conformational dynamics of crucial loops revealed prominent alterations of the original conformational posture of FXIIa, evidenced by increased flexibility, decreased compactness, and an increased exposure to solvent upon binding of BEN, which could have in turn interfered with the essential roles of these loops in enhancing their procoagulation interactions with biological substrates and cofactors, altogether resulting in the consequential inactivation of FXIIa. A sustained interaction of the catalytic triad residues and key residues of the autolysis loop impeded their roles in catalysis which equally enhanced the inhibitory potency of BEN toward β-FXIIa evidenced by a favorable binding. Findings provide essential structural and molecular insights that could facilitate the structure-based design of novel antithrombotic compounds with enhanced inhibitory activities and low therapeutic risk.

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.

Na+-mimicking ligands stabilize the inactive state of leukotriene B4 receptor BLT1.

Most G-protein-coupled receptors (GPCRs) are stabilized in common in the inactive state by the formation of the sodium ion-centered water cluster with the conserved Asp2.50 inside the seven-transmembrane domain. We determined the crystal structure of the leukotriene B4 (LTB4) receptor BLT1 bound with BIIL260, a chemical bearing a benzamidine moiety. Surprisingly, the amidine group occupies the sodium ion and water locations, interacts with D662.50, and mimics the entire sodium ion-centered water cluster. Thus, BLT1 is fixed in the inactive state, and the transmembrane helices cannot change their conformations to form the active state. Moreover, the benzamidine molecule alone serves as a negative allosteric modulator for BLT1. As the residues involved in the benzamidine binding are widely conserved among GPCRs, the unprecedented inverse-agonist mechanism by the benzamidine moiety could be adapted to other GPCRs. Consequently, the present structure will enable the rational development of inverse agonists specific for each GPCR.

Comparative molecular modeling analysis of­5‐amidinoindole and benzamidine binding to thrombin and trypsin: specific H‐bond formation contributes to high 5‐amidinoindole potency and selectivity for thrombin and factor Xa

Comparative molecular modeling analysis of­5‐amidinoindole and benzamidine binding to thrombin and trypsin: specific H‐bond formation contributes to high 5‐amidinoindole potency and selectivity for thrombin and factor Xa

Comparison of the active-site conformations of bovine alpha-thrombin and meizothrombin(desF1) by electron spin resonance.

The active site of the prothrombin activation intermediate meizothrombin(desF1) was probed using several fluorosulfonylphenyl spin labels specific for the active serine hydroxyl of serine proteases. The mobilities of the thrombin species inhibited with the nitroxide spin labels m-IV [4-(2,2,6,6-tetramethyl-piperidine-1-oxyl) -m-(fluorosulfonyl)benzamide] and m-V [3-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl) -m-(fluorosulfonyl)benzamide], which are sensitive to differences between alpha- and gamma-thrombin, were quite similar to that of alpha-thrombin. That is, no major conformational differences between meizothrombin(desF1) and alpha-thrombin were observed in this region of the extended active site. On the other hand, p-IV [4-(2,2,6,6-tetramethyl piperidine-1-oxyl)-p-(fluorosulfonyl)benzamide], p-V [3-(2,2,5,5-tetramethylpyrrolidine-1-oxyl) -p-(fluorosulfonyl)benzamide], and m-VII [N-[m- (fluorosulfonyl)phenyl]-4-N-(2,2,6,6-tetramethyl- piperidine-1-oxyl)urea], which probe an apolar binding region of bovine thrombin, exhibited large differences in mobility between alpha-thrombin and meizothrombin(desF1). The conformational consequences of indole binding to spin-labeled thrombin species demonstrated that both species also possess an indole-binding site. However, the nitroxide mobility changes upon indole binding to the spin-labeled protein derivative were somewhat different between the two thrombin species under study. In addition, the effects of the benzamidine binding were quite similar for each labeled protein. Thus is appears that, while both species posses a fully functional active site, the region in meizothrombin(desF1) probed by spin labels p-IV, p-V, and m-VII, which corresponds to the apolar binding region, differs in conformation from alpha-thrombin.

Inhibition of bovine β-trypsin, human α-thrombin and porcine pancreatic β-kallikrein-B by 4′,6-diamidino-2-phenylindole, 6-amidinoindole and benzamidine: a comparative thermodynamic and X-ray structural study

The inhibitory effect of 4′,6-diamidino-2-phenylindole (DAPI) and 6-amidinoindole on the catalytic properties of bovine β-trypsin (trypsin), human α-thrombin (thrombin) and porcine pancreatic β-kallikrein-B (kallikrein) was investigated (between pH 3.0 and 7.0, I = 0.1 M; T = 30.0 ± 0.5°C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Next, the X-ray crystal structure of the trypsin: DAPI complex was solved at 1.9 Å resolution ( R = 0.161). Over the whole pH range explored, values of the association inhibition constant ( K i) for DAPI and 6-amidinoindole binding to trypsin, thrombin and kallikrein are higher than those found for benzamidine association, suggesting a binding mode of DAPI to the enzyme primary specificity pocket-based on the indole moiety of the inhibitor. On lowering the pH from 5.5 to 3.0, the decrease in affinity for DAPI, 6-amidinoindole and benzamidine binding to trypsin, thrombin and kallikrein reflects the acidic p K shift of the Asp189 invariant residue, present at the bottom of the primary specificity subsite of the serine proteinases considered, from 4.5, in the free enzyme, to 3.7, in the proteinase:inhibitor complexes. Inspection of the refined crystal structure of the trypsin: DAPI complex, however, does not allow a unique interpretation of the inhibitor binding mode. The present data were analysed in parallel with those reported for related serine (pro)enzyme/inhibitor systems.

Analysis of the aromatic 1H-NMR spectrum of the kringle 5 domain from human plasminogen. Evidence for a conserved kringle fold.

A kringle 5 domain fragment from human plasminogen has been investigated by 1H-NMR spectroscopy at 300 MHz and 620 MHz. The study focuses on the kringle 5 aromatic spectrum as aromatic side chains appear to mediate the binding of benzamidine. Spin-echo experiments and acid/base-titration studies in conjunction with two-dimensional double-quantum and chemical-shift-correlated spectroscopies were used to identify individual spin systems. Sequence-specific assignments of aromatic resonances are derived from direct comparison of the kringle 5 spectrum with spectra of the homologous kringle 1 and kringle 4 domains of plasminogen. As previously observed for kringles 1 and 4, the pattern we detect for Tyr9 in kringle 5 reflects a slow conformational exchange between two states in equilibrium, one in which the Tyr9 ring is freely mobile and one in which its flip dynamics are constrained. Proton Overhauser experiments in 1H2O and in 2H2O have been used to probe aromatic ring interactions and to identify residues which are part of the hydrophobic core centered at the Leu46 side chain. Overall, the data indicate a strong structural homology among the three plasminogen kringles.

Zymogen activation: effect of peptides sequentially related to the bovine beta-trypsin N-terminus on Kazal inhibitor and benzamidine binding to bovine trypsinogen.

The activating effect of peptides sequentially related to the Ile 16-Val17-Gly18 N-terminus of bovine beta-trypsin (namely Ile-Val-Gly, Ile-Val, Ile-Leu, Ile-Ala, Val-Val, Leu-Val, and Val-Leu) on the thermodynamic parameters for the binding of the porcine pancreatic secretory trypsin inhibitor (Kazal inhibitor) and benzamidine to bovine trypsinogen was investigated at pH 5.5 (Bis tris-HCl buffer, I = 0.1 M) and T = 21 +/- 0.5 degrees C. Thermodynamic parameters for Kazal inhibitor and benzamidine association to the binary peptide/zymogen adducts are more favorable than those observed for ligand binding to the proenzyme alone, although never as much as those reported for the formation of bovine beta-trypsin/Kazal inhibitor and bovine beta-trypsin/benzamidine adducts. Analogously, the affinity of activating peptides for the binary proenzyme/Kazal inhibitor and binary proenzyme/benzamidine complexes is higher than that observed for peptide binding to free bovine trypsinogen. Differences in affinity for ligand binding to free bovine trypsinogen, to its binary adducts and to bovine beta-trypsin suggest the presence of different activation levels of the proenzyme, none of which structurally coincide with that achieved in bovine beta-trypsin. The existence of different discrete states suggests that the zymogen-to-active enzyme transition should not be considered as a two-state process but as a multistep event.

Refined 2 Å X-ray crystal structure of porcine pancreatic kallikrein A, a specific trypsin-like serine proteinase: Crystallization, structure determination, crystallographic refinement, structure and its comparison with bovine trypsin

Porcine pancreas kallikrein A has been crystallized in the presence of the small inhibitor benzamidine, yielding tetragonal crystals of space group P4 12 12 containing two molecules per asymmetric unit. X-ray data up to 2·05 Å resolution have been collected using normal rotation anode as well as synchrotron radiation. The crystal structure of benzamidine-kallikrein has been determined using multiple isomorphous replacement techniques, and has subsequently been refined to a crystallographic R-value of 0·220 by applying a diagonal matrix least-squares energy constraint refinement procedure. Both crystallographically independent kallikrein molecules 1 and 2 are related by a non-integral screw axis and form open, heterologous “dimer” structures. The root-mean-square deviation of both molecules is 0·37 Å for all main-chain atoms. This value is above the estimated mean positional error of about 0·2 Å and reflects some significant conformational differences, especially at surface loops. The binding site of molecule 1 in the asymmetric unit is in contact with residues of molecule 2, whereas the binding site of the latter is free and accessible to the solvent. In both molecules the characteristic “kallikrein loop”, where the peptide chain of kallikrein A is cleaved, is only partially traceable. The carbohydrate attached to Asn95 in this loop, although detectable chemically, is not defined. A comparison of the refined structures of porcine kallikrein and bovine trypsin indicates spatial homology for these enzymes. The root-mean-square difference is 0·68 Å if we compare only main-chain atoms of internal segments. Remarkably large deviations are found in some external loops most of which surround the binding site and form a more compact rampart around it in kallikrein than in trypsin. This feature might explain the strongly reduced activity and accessibility of kallikrein towards large protein substrates and inhibitors (e.g. as shown by the model-building experiments on inhibitor complexes reported by Chen & Bode. 1983). The conformation of the active site residues is very similar in both enzymes. Tyr99 of kallikrein, which is a leucyl residue in trypsin, protrudes into the binding site and interferes with the binding of peptide substrates (Chen & Bode. 1983). The kallikrein specificity pocket is significantly enlarged compared with trypsin due to a longer peptide segment, 217 to 220, and to the unique outwards orientation of the carbonyl group of cis-Pro219. Further, the side-chain of Ser226 in porcine kallikrein, which is a glycyl residue in trypsin, partially covers Asp 189 at the bottom of the pocket. These features considerably affect the binding geometry and strength of binding of benzamidine.

Acrosin inhibition. Comparisons of membrane-associated and -solubilized enzyme.

Acrosin is an extrinsic membrane proteinase from spermatozoa which functions in the fertilization process. Liposomes were utilized as a model system to determined possible effects of membrane association on acrosin's enzymatic activity. By comparison with solubilized enzyme, liposome-bound acrosin had a substantial reduction in the apparent affinity for "progressive" inhibitors such as leupeptin, lima bean trypsin inhibitor, soy bean trypsin inhibitor, and for a proteinase inhibitor from sperm extracts. In contrast, the liposome-bound and -solubilized enzymes were essentially identical with respect to the binding of benzamidine and p-aminobenzamidine which are competitive acrosin inhibitors. These results suggest membrane association can influence some but not all of acrosin's enzymatic properties.

Kinetic evidence for a two-state, hybrid model for the trypsin activation by modifiers.

Kinetic investigation of the activation of alpha- and beta-trypsins by tosyl-L-arginine methyl ester, in the presence of benzamidine at concentrations higher than 1 mM, indicates that each could exist as a two-state hybrid allosteric system, based on at least two enzyme forms, E and E* (or E and F). Form E* shows much higher affinity for ligands, thus giving rise to ternary complexes of the types SE*S, ME*S, ME*M, SE*M, where the right position identifies binding at the active site, whereas the left position represents binding to the allosteric site. Using a large number of experimental points, measured at different combinations of substrate and modifier concentrations, it was possible to evaluate quantitatively the parameters needed for the description of the model with considerable precision. At high concentrations, benzamidine competes with tosyl-L-arginine methyl ester for an allosteric site, blocking substrate activation of beta-trypsin, but allowing activation of alpha-trypsin. These results imply the participation of an induced fit step during ternary complex formation that gives rise to complexes of the form E*S2 or E*SI, where E* stands for the conformationally changed E* in the complex. The overall picture is that of a two-state model combined with induced-fit. Negative cooperativity (nH = 0.80) was found for the binding of benzamidine (over 1 mM) to trypsin in the presence of substrate. The proposed model is allowing the design of experiments that should lead to an understanding of the mechanism of trypsin activation by modifiers.

Binding of benzamidine and protons to trypsin as measured by difference spectra

The interaction of trypsin and benzamidine causes changes in their ultraviolet absorption spectra; the spectral changes are measured by difference spectroscopy, and it appears that both components contribute to the difference spectrum. The dependence of the height of a difference peak at 248 mμ upon the concentrations of benzamidine and hydrogen ion fits a model in which the two ligands compete for a single class of non-interacting sites on trypsin. The system is characterized by the dissociation constants K l = 2 × 10 −5 m and K h = 2.4 × 10 −5 m, for benzamidine and protons, respectively. A similar difference spectrum is obtained with diisopropyl phosphoryltrypsin, whereas none is obtained with p-toluene sulfonyl- l-lysine chloro ketonetrypsin or with trypsinogen.