Ionic interactions in the formation of the thrombin-hirudin complex.
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ABSTRACT:
SUBMITTER: Betz A
PROVIDER: S-EPMC1150126 | biostudies-other | 1991 May
REPOSITORIES: biostudies-other
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Project description:Thrombin is the pivotal serine protease enzyme in the blood cascade system and thus a target of drug design for control of its activity. The most efficient nonphysiologic inhibitor of thrombin is hirudin, a naturally occurring small protein. Hirudin and its synthetic mimics employ a range of hydrogen bonding, salt bridging, and hydrophobic interactions with thrombin to achieve tight binding with K(i) values in the nano- to femtomolar range. The one-dimensional (1)H nuclear magnetic resonance spectrum recorded at 600 MHz reveals a resonance 15.33 ppm downfield from silanes in complexes between human α-thrombin and r-hirudin in pH 5.6-8.8 buffers and between 5 and 35 °C. There is also a resonance between 15.17 and 15.54 ppm seen in complexes of human α-thrombin with hirunorm IV, hirunorm V, an Nα(Me)Arg peptide, RGD-hirudin, and Nα-2-naphthylsulfonyl-glycyl-DL-4-amidinophenylalanyl-piperidide acetate salt (NAPAP), while there is no such low-field resonance observed in a complex of porcine trypsin and NAPAP. The chemical shifts suggest that these resonances represent H-bonded environments. H-Donor-acceptor distances in the corresponding H-bonds are estimated to be <2.7 Å. Addition of Phe-Pro-Arg-chloromethylketone (PPACK) to a complex of human α-thrombin with r-hirudin results in an additional signal at 18.03 ppm, which is 0.10 ppm upfield from the observed signal [Kovach, I. M., et al. (2009) Biochemistry 48, 7296-7304] for thrombin covalently modified with PPACK. In contrast, the peak at 15.33 ppm remains unchanged. The fractionation factors for the thrombin-hirudin complexes are near 1.0 within 20% error. The most likely site of the short H-bond in complexes of thrombin with the hirudin family of inhibitors is in the hydrophobic patch of the C-terminus of hirudin where Glu(57') and Glu(58') are embedded and interact with Arg(75) and Arg(77) and their solvate water (on thrombin). Glu(57') and Glu(58') present in the hirudin family of inhibitors make up a key binding epitope of fibrinogen, thrombin's prime substrate, which lends substantial interest to the short hydrogen bond as a binding element at the fibrinogen recognition site.
| S-EPMC3750058 | biostudies-literature
Project description:The importance of interactions with residues 15-21 in the core domain of hirudin for the formation of the complex with thrombin has been investigated by site-directed mutagenesis. Contacts made by Leu-15 were found to be particularly important; replacement of this residue by alanine led to a decrease in the binding energy (delta delta Gbo) of 15 kJ.mol-1. Comparison with effects obtained in previous mutagenesis studies indicate that interactions with the side chain of Leu-15 contribute more to the stability of the complex than those of any other hirudin residues. Interactions with the side chains of Glu-17, Asn-20 and Val-21 also contributed significantly to binding energy; the delta delta Gbo value for these mutations was between 3 and 6 kJ.mol-1. Examination of the crystal structure of the thrombin-hirudin complex suggested the possibility that ionic interactions that would increase binding energy could be engineered by mutating Ser-19, Asn-20 and Gln-49 to acidic residues. The stability of the thrombin-hirudin complex was not, however, increased by these substitutions. The results obtained are discussed in terms of the crystal structure of the thrombin-hirudin complex.
| S-EPMC1137969 | biostudies-other
Project description: Not available
| S-EPMC2530905 | biostudies-literature
Project description:The metabolic fate of the anticoagulant protein, hirudin, and its complex with thrombin are presently unknown. Therefore we have labelled hirudin and human thrombin-hirudin complex with the residualizing label dilactitol-125I-tyramine (*I-DLT) in order to identify their tissue sites of catabolism in the rat. The rapid plasma clearance of hirudin after intravenous injection was unaffected by *I-DLT labelling, and by 2 h 6% or less of the injected dose remained in the blood. The majority (80.3 +/- 4.0%, n = 2) of *I-DLT-hirudin radioactivity recovered in tissues was found in kidney, and kidney was also at least 150 times more active in taking up hirudin, on a weight basis, than any other tissue examined (liver, spleen, skin, muscle, intestine, fat, lung). *I-DLT-hirudin which bound to thrombin was isolated by chromatography on concanavalin A-Sepharose; hirudin itself does not bind to concanavalin A. Radioactivity from thrombin-*I-DLT-hirudin was precipitable by anti-thrombin antibody and *I-DLT-thrombin-hirudin was precipitable by anti-hirudin antibody. By 1 h after injection of labelled thrombin-hirudin complexes, the recoveries of radioactivity from hirudin and thrombin in liver were comparable (38.6 +/- 3.0 and 36.4 +/- 4.1%, n = 3), whereas more radioactivity was recovered in kidney from hirudin than from thrombin (27.6 +/- 8.7 compared with 13.6 +/- 4.5%) and less was recovered in lung (0.4 +/- 0.2 compared with 17.7 +/- 2.9%). We conclude that hirudin is catabolized predominantly in kidney, whereas the thrombin-hirudin complex is catabolized by both liver and kidney.
| S-EPMC1137762 | biostudies-other
Project description:BackgroundHirudin is an anti-coagulation protein produced by the salivary glands of the medicinal leech Hirudomedicinalis. It is a powerful and specific thrombin inhibitor. The novel recombinant hirudin, RGD-hirudin, which contains an RGD motif, competitively inhibits the binding of fibrinogen to GPIIb/IIIa on platelets, thus inhibiting platelet aggregation while maintaining its anticoagulant activity.ResultsRecombinant RGD-hirudin and six mutant variants (Y3A, S50A, Q53A, D55A, E57A and I59A), designed based on molecular simulations, were expressed in Pichia pastoris. The proteins were refolded and purified to homogeneity as monomers by gel filtration and anion exchange chromatography. The anti-thrombin activity of the six mutants and RGD-hirudin was tested. Further, we evaluated the binding of the mutant variants and RGD-hirudin to thrombin using BIAcore surface plasmon resonance analysis (SPR). Kinetics and affinity constants showed that the KD values of all six mutant proteins were higher than that of RGD-hirudin.ConclusionsThese findings contribute to a novel understanding of the interaction between RGD-hirudin and thrombin.
| S-EPMC4304120 | biostudies-literature
Project description:A novel class of synthetic, multisite-directed thrombin inhibitors, known as hirunorms, has been described recently. These compounds were designed to mimic the binding mode of hirudin, and they have been proven to be very strong and selective thrombin inhibitors. Here we report the crystal structure of the complex formed by human alpha-thrombin and hirunorm V, a 26-residue polypeptide containing non-natural amino acids, determined at 2.1 A resolution and refined to an R-factor of 0.176. The structure reveals that the inhibitor binding mode is distinctive of a true hirudin mimetic, and it highlights the molecular basis of the high inhibitory potency (Ki is in the picomolar range) and the strong selectivity of hirunorm V. Hirunorm V interacts through the N-terminal tetrapeptide with the thrombin active site in a nonsubstrate mode; at the same time, this inhibitor specifically binds through the C-terminal segment to the fibrinogen recognition exosite. The backbone of the N-terminal tetrapeptide Chg1"-Val2"-2-Nal3"-Thr4" (Chg, cyclohexyl-glycine; 2-Nal, beta-(2-naphthyl)-alanine) forms a short beta-strand parallel to thrombin main-chain residues Ser214-Gly219. The Chg1" side chain fills the S2 subsite, Val2" is located at the entrance of S1, whereas 2-Nal3" side chain occupies the aryl-binding site. Such backbone orientation is very close to that observed for the N-terminal residues of hirudin, and it is similar to that of the synthetic retro-binding peptide BMS-183507, but it is opposite to the proposed binding mode of fibrinogen and of small synthetic substrates. Hirunorm V C-terminal segment binds to the fibrinogen recognition exosite, similarly to what observed for hirudin C-termninal tail and related compounds. The linker polypeptide segment connecting hirunorm V N-and C-terminal regions is not observable in the electron density maps. The crystallographic analysis proves the correctness of the design and it provides a compelling proof on the interaction mechanism for this novel class of high potency multisite-directed synthetic thrombin inhibitors.
| S-EPMC2143932 | biostudies-other
Project description:7-Azatryptophan (AW), a noncoded isostere of tryptophan (W), possesses interesting spectral properties. In particular, the presence of a nitrogen atom at position 7 in the indolyl nucleus of AW results in a red shift of the absorption maximum and fluorescence emission by 10 and 46 nm, respectively, compared to W. In the present work, we report the chemical synthesis and the conformational and functional characterization of an analog (denoted as Y3AW) of the N-terminal domain 1-47 of hirudin, a highly potent thrombin inhibitor, in which Tyr 3 has been replaced by AW. The results obtained were compared with those of the corresponding Y3W analog. We found that the replacement W --> AW reduces affinity for thrombin by 10-fold, likely because of the lower hydrophobicity of AW compared with that of W. Measurements of the resonance energy transfer effect, which was observed between Tyr13 and the amino acid at position 3 upon disulfide-coupled folding, demonstrate that AW behaves as a better energy acceptor than W for studying protein renaturation. The interaction of Y3AW with thrombin was studied by exciting the sample at 320 nm and recording the change in fluorescence of Y3AW on binding to the enzyme. Our results indicate that the fluorescence of AW of hirudin 1-47 in the Y3AW-thrombin complex is strongly quenched, possibly because of the presence of two structural water molecules at the hirudin-thrombin interface that can promote the nonradiative decay of AW in the excited state. The data herein reported demonstrate that the incorporation of AW can be of broad applicability in the study of protein folding and protein-protein interaction.
| S-EPMC2279979 | biostudies-literature
Project description:Preterm birth (PTB) related health problems take over one million lives each year, and currently, no clinical analysis is available to determine if a fetus is at risk for PTB. Here, we describe the preparation of a key PTB risk biomarker, thrombin-antithrombin (TAT), and characterize it using dot blots, MS, and microchip electrophoresis (µCE). The pH for fluorescently labeling TAT was also optimized using spectrofluorometry and spectrophotometry. The LOD of TAT was measured in µCE. Lastly, TAT was combined with six other PTB risk biomarkers and separated in µCE. The ability to make and characterize TAT is an important step toward the development of an integrated microfluidic diagnostic for PTB risk.
| S-EPMC6829041 | biostudies-literature
Project description:Transcriptome sequencing data (6.5 Gb) of the salivary glands of the haematophagous leech Hirudo nipponia was obtained by using the BGIseq-500 platform. After identification and analysis, one transcript (Unigene5370) was annotated to hirudin HV3 from Hirudo medicinalis with an e-value of 1e-29 and was named hirudin-HN. This transcript was a new thrombin inhibitor gene belonging to the proteinase inhibitor I14 (hirudin) family. Hirudin-HN, with a 270-bp cDNA, encodes an 89-aa protein containing a 20-aa signal peptide. The mature hirudin-HN protein contains the typical structural characteristics of hirudin, e.g., three conserved disulfide bonds and the PKP and DFxxIP motifs. Proteins (Hir and M-Hir) were obtained via prokaryotic expression, and the mature hirudin-HN protein was shown to have anticoagulant activity and thrombin affinity by using the chromogenic substrate S2238 and surface plasmon resonance (SPR) interaction analysis, respectively. The N-terminal structure of the mature hirudin-HN protein was shown to be important for anticoagulant activity by comparing the activity and thrombin affinity of Hir and M-Hir. The abundances of Hirudin-HN mRNA and protein were higher in the salivary glands of starving animals than in those of feeding or fed leeches. These results provided a foundation for further study on the structure-function relationship of hirudin-HN with thrombin.
| S-EPMC6776071 | biostudies-literature
Project description:We develop a procedure to characterize the association of protein structures into homodimers using coevolutionary couplings extracted from Direct Coupling Analysis (DCA) in combination with Structure Based Models (SBM). Identification of dimerization contacts using DCA is more challenging than intradomain contacts since direct couplings are mixed with monomeric contacts. Therefore a systematic way to extract dimerization signals has been elusive. We provide evidence that the prediction of homodimeric complexes is possible with high accuracy for all the cases we studied which have rich sequence information. For the most accurate conformations of the structurally diverse dimeric complexes studied the mean and interfacial RMSDs are 1.95Å and 1.44Å, respectively. This methodology is also able to identify distinct dimerization conformations as for the case of the family of response regulators, which dimerize upon activation. The identification of dimeric complexes can provide interesting molecular insights in the construction of large oligomeric complexes and be useful in the study of aggregation related diseases like Alzheimer's or Parkinson's.
| S-EPMC4559900 | biostudies-literature