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Hirunorms are true hirudin mimetics. The crystal structure of human alpha-thrombin-hirunorm V complex.

De Simone G, Lombardi A, Galdiero S, Nastri F, Della Morte R, Staiano N, Pedone C, Bolognesi M, Pavone V
Protein Sci 7 243-53 (1998)
Cited: 14 times
EuropePMC logo PMID: 9521099

Abstract

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.

Reviews - 5gds mentioned but not cited (2)

  1. Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands. Troisi R, Balasco N, Autiero I, Vitagliano L, Sica F. Int J Mol Sci 22 10803 (2021)
  2. Non-Canonical Amino Acids in Analyses of Protease Structure and Function. Goettig P, Koch NG, Budisa N. Int J Mol Sci 24 14035 (2023)


Reviews citing this publication (2)

  1. Proton Bridging in Catalysis by and Inhibition of Serine Proteases of the Blood Cascade System. Kovach IM. Life (Basel) 11 396 (2021)
  2. Amino acid modifications for conformationally constraining naturally occurring and engineered peptide backbones: Insights from the Protein Data Bank. Di Costanzo L, Dutta S, Burley SK. Biopolymers 109 e23230 (2018)

Articles citing this publication (10)

  1. Crystal structure of the human alpha-thrombin-haemadin complex: an exosite II-binding inhibitor. Richardson JL, Kröger B, Hoeffken W, Sadler JE, Pereira P, Huber R, Bode W, Fuentes-Prior P. EMBO J 19 5650-5660 (2000)
  2. From natural to synthetic multisite thrombin inhibitors. Lombardi A, De Simone G, Galdiero S, Staiano N, Nastri F, Pavone V. Biopolymers 51 19-39 (1999)
  3. Design and evaluation of novel bivalent thrombin inhibitors based on amidinophenylalanines. Steinmetzer T, Renatus M, Künzel S, Eichinger A, Bode W, Wikström P, Hauptmann J, Stürzebecher J. Eur J Biochem 265 598-605 (1999)
  4. Structure based antibody-like peptidomimetics. Murali R, Greene MI. Pharmaceuticals (Basel) 5 209-235 (2012)
  5. The crystal structure of alpha-thrombin-hirunorm IV complex reveals a novel specificity site recognition mode. Lombardi A, De Simone G, Nastri F, Galdiero S, Della Morte R, Staiano N, Pedone C, Bolognesi M, Pavone V. Protein Sci 8 91-95 (1999)
  6. Protease inhibitors. Part 2. Weakly basic thrombin inhibitors incorporating sulfonyl-aminoguanidine moieties as S1 anchoring groups: synthesis and structure-activity correlations. Clare BW, Scozzafava A, Briganti F, Iorga B, Supuran CT. J Enzyme Inhib 15 235-264 (2000)
  7. Branched porphyrins as functional scaffolds for multisite bioconjugation. Engelen M, Lombardi A, Vitale R, Lista L, Maglio O, Pavone V, Nastri F. Biotechnol Appl Biochem 62 383-392 (2015)
  8. Novel Inhibitors and Activity-Based Probes Targeting Trypsin-Like Serine Proteases. Ferguson TEG, Reihill JA, Martin SL, Walker B. Front Chem 10 782608 (2022)
  9. Proton bridging in the interactions of thrombin with hirudin and its mimics. Kovach IM, Kakalis L, Jordan F, Zhang D. Biochemistry 52 2472-2481 (2013)
  10. Historical Article Vincenzo Pavone: Friend, mentor and inspiring scientist. Nastri F, Maglio O, Lombardi A. Biopolymers 109 e23234 (2018)


Related citations provided by authors (2)

  1. Rational Design of True Hirudin Mimetics: Synthesis and Characterization of Multisite-Directed Alpha-Thrombin Inhibitors. Lombardi A, Nastri F, Della Morte R, Rossi A, De Rosa A, Staiano N, Pedone C, Pavone V J. Med. Chem. 39 2008- (1996)
  2. Refined Structure of the Hirudin-Thrombin Complex. Rydel TJ, Tulinsky A, Bode W, Huber R J. Mol. Biol. 221 583- (1991)

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