The refined 1.9‐Å X‐ray crystal structure of d‐Phe‐Pro‐Arg chloromethylketone‐inhibited human α‐thrombin: Structure analysis, overall structure, electrostatic properties, detailed active‐site geometry, and structure‐function relationships

Abstract

Thrombin is a multifunctional serine proteinase that plays a key role in coagulation while exhibiting several other key cellular bioregulatory functions. The X-ray crystal structure of human alpha-thrombin was determined in its complex with the specific thrombin inhibitor D-Phe-Pro-Arg chloromethylketone (PPACK) using Patterson search methods and a search model derived from trypsinlike proteinases of known spatial structure (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S.R., & Hofsteenge, J., 1989, EMBO J. 8, 3467-3475). The crystallographic refinement of the PPACK-thrombin model has now been completed at an R value of 0.156 (8 to 1.92 A); in particular, the amino- and the carboxy-termini of the thrombin A-chain are now defined and all side-chain atoms localized; only proline 37 was found to be in a cis-peptidyl conformation. The thrombin B-chain exhibits the characteristic polypeptide fold of trypsinlike serine proteinases; 195 residues occupy topologically equivalent positions with residues in bovine trypsin and 190 with those in bovine chymotrypsin with a root-mean-square (r.m.s.) deviation of 0.8 A for their alpha-carbon atoms. Most of the inserted residues constitute novel surface loops. A chymotrypsinogen numbering is suggested for thrombin based on the topological equivalences. The thrombin A-chain is arranged in a boomeranglike shape against the B-chain globule opposite to the active site; it resembles somewhat the propeptide of chymotrypsin(ogen) and is similarly not involved in substrate and inhibitor binding. Thrombin possesses an exceptionally large proportion of charged residues. The negatively and positively charged residues are not distributed uniformly over the whole molecule, but are clustered to form a sandwichlike electrostatic potential; in particular, two extended patches of mainly positively charged residues occur close to the carboxy-terminal B-chain helix (forming the presumed heparin-binding site) and on the surface of loop segment 70-80 (the fibrin[ogen] secondary binding exosite), respectively; the negatively charged residues are more clustered in the ringlike region between both poles, particularly around the active site. Several of the charged residues are involved in salt bridges; most are on the surface, but 10 charged protein groups form completely buried salt bridges and clusters. These electrostatic interactions play a particularly important role in the intrachain stabilization of the A-chain, in the coherence between the A- and the B-chain, and in the surface structure of the fibrin(ogen) secondary binding exosite (loop segment 67-80).(ABSTRACT TRUNCATED AT 400 WORDS)

Role of the A chain in thrombin function

“…When the structure of thrombin was first solved [2], it was noted that the A chain is rich in charged residues engaged in intra-and inter-molecular interactions. Specific interactions that stabilize the fold are the Asp1a-Lys9 and Arg14d-Glu13 ion pairs and the Arg4-Glu8-Asp14-Glu14c ion quartet (Fig.…”

“…This could also explain the similarity of phenotypes observed between deletions of Lys9 or Lys10 [8] with those of prothrombins Denver (E8K and E14cK) [5] that affect the ion quartet directly. Interactions between the A chain (stick model, residues labeled in black) and B chain (wheat surface, residues labeled in white) of thrombin based on the structure 1PPB [2]. The A chain is stabilized by the Asp1a-Lys9 and Arg14d-Glu13 ion pairs and the ion quartet Arg4-Glu8-Asp14-Glu14c (H bonds are in green).…”

“…Because catalytic function and all known regulatory interactions involve the B chain, the shorter A chain has received little attention in functional and mutagenesis studies and is considered an appendage from the activation process of prothrombin. The original structure of thrombin solved by Bode [2] has cemented this view in a widely used orientation where the B chain is displayed prominently with its catalytic site in the middle and eclipsing the A chain in its back. The role of the A chain of thrombin has not been studied in terms of Ala-scanning mutagenesis [3], so most of our knowledge is currently based on a previous study on bovine thrombin where the A and B chains were separated after reduction of all disulfide bonds including Cys1-Cys122, denaturation, removal of the A chain and refolding of the B chain [4].…”

Structural identification of the pathway of long-range communication in an allosteric enzyme

“…The Na ϩ -bound form E:Na ϩ is the high-activity conformation corresponding to the procoagulant fast form (18,43), and its structural signatures are well established (21,29). The Na ϩ -free form is partitioned into two conformations, E and E*, of which only E can interact with Na ϩ and corresponds to the low-activity, anticoagulant slow form (18,43).…”

“…Structural details on how Na ϩ binding influences allosterically the active site have emerged recently (12,21), but mutagenesis and spectroscopic studies vouch for more extensive, global effects of Na ϩ binding on the conformation of the enzyme (12,19,23). In addition to the allosteric effect of Na ϩ , thrombin activity and specificity is influenced allosterically by binding of ligands to exosite I (24-28), a domain located 25-Å away from the active site and on the opposite pole of the molecule relative to the Na ϩ site (16,29). The exact mechanism of this long-range communication remains controversial (30,31).…”