1ql9
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FACTOR XA SPECIFIC INHIBITOR IN COMPLEX WITH RAT TRYPSIN MUTANT X99RT
OverviewOverview
In order to investigate issues of selectivity and specificity in, protein-ligand interactions, we have undertaken the reconstruction of the, binding pocket of human factor Xa in the structurally related rat trypsin, by site-directed mutagenesis. Three sequential regions (the "99"-, the, "175"- and the "190"- loops) were selected as representing the major, structural differences between the ligand binding sites of the two, enzymes. Wild-type rat trypsin and variants X99rT and X(99/175/190)rT were, expressed in yeast, and analysed for their interaction with factor Xa and, trypsin inhibitors. For most of the inhibitors studied, progressive loop, replacement at the trypsin surface resulted in inhibitory profiles akin to, factor Xa. Crystals of the variants were obtained in the presence of, benzamidine (3), and could be soaked with the highly specific factor Xa, inhibitor (1). Binding of the latter to X99rT results in a series of, structural adaptations to the ligand, including the establishment of an, "aromatic box" characteristic of factor Xa. In X(99/175/190)rT, introduction of the 175-loop results in a surprising re-orientation of the, "intermediate helix", otherwise common to trypsin and factor Xa. The, re-orientation is accompanied by an isomerisation of the Cys168-Cys182, disulphide bond, and burial of the critical Phe174 side-chain. In the, presence of (1), a major re-organisation of the binding site takes place, to yield a geometry identical to that of factor Xa. In all, binding of (1), to trypsin and its variants results in significant structural, rearrangements, inducing a binding surface strongly reminiscent of factor, Xa, against which the inhibitor was optimised. The structural data reveal, a plasticity of the intermediate helix, which has been implicated in the, functional cofactor dependency of many trypsin-like serine proteinases., This approach of grafting loops onto scaffolds of known related structures, may serve to bridge the gap between structural genomics and drug design.
About this StructureAbout this Structure
1QL9 is a Single protein structure of sequence from Rattus norvegicus with , and as ligands. Active as Trypsin, with EC number 3.4.21.4 Known structural/functional Sites: , , and . Full crystallographic information is available from OCA.
ReferenceReference
Reconstructing the binding site of factor Xa in trypsin reveals ligand-induced structural plasticity., Reyda S, Sohn C, Klebe G, Rall K, Ullmann D, Jakubke HD, Stubbs MT, J Mol Biol. 2003 Jan 31;325(5):963-77. PMID:12527302
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