1c1s

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Revision as of 09:58, 20 November 2007

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spinqualitylabelsall models 1c1s, resolution 1.63Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

RECRUITING ZINC TO MEDIATE POTENT, SPECIFIC INHIBITION OF SERINE PROTEASES

Overview

As regulators of ubiquitous biological processes, serine proteases can, cause disease states when inappropriately expressed or regulated, and are, thus rational targets for inhibition by drugs. Recently we described a new, inhibition mechanism applicable for the development of potent, selective, small molecule serine protease inhibitors that recruit physiological Zn2+, to mediate high affinity (sub-nanomolar) binding. To demonstrate some of, the structural principles by which the selectivity of Zn2+-mediated serine, protease inhibitors can be developed toward or against a particular, target, here we determine and describe the structures of, thrombin-BABIM-Zn2+, -keto-BABIM-Zn2+, and -hemi-BABIM-Zn2+ (where BABIM, is bis(5-amidino-2-benzimidazolyl)methane, keto-BABIM is, bis(5-amidino-2-benzimidazolyl)methane ketone, and hemi-BABIM is, (5-amidino-2-benzimidazolyl)(2-benzimidazolyl)methane), and compare them, with the corresponding trypsin-inhibitor-Zn2+ complexes. Inhibitor binding, is mediated by a Zn ion tetrahedrally coordinated by two benzimidazole, nitrogen atoms of the inhibitor, by N(epsilon2)His57, and by, O(gamma)Ser195. The structures of Zn2+-free trypsin-BABIM and -hemi-BABIM, were also determined at selected pH values for comparison with the, corresponding Zn2+-mediated complexes. To assess some of the physiological, parameters important for harnessing Zn2+ as a co-inhibitor, crystal, structures at multiple pH and [Zn2+] values were determined for, trypsin-keto-BABIM. The Kdvalue of Zn2+ for the binary trypsin-keto-BABIM, complex was estimated to be <12 nM at pH 7.06 by crystallographic, determination of the occupancy of bound Zn2+ in trypsin-keto-BABIM, crystals soaked at this pH in synthetic mother liquor containing inhibitor, and 100 nM Zn2+. In synthetic mother liquor saturated in Zn2+, trypsin-bound keto-BABIM is unhydrated at pH 9.00 and 9.93, and has an sp2, hybridized ketone carbon bridging the 5-amidinobenzimidazoles, whereas at, pH 7.00 and 8.00 it undergoes hydration and a change in geometry upon, addition of water to the bridging carbonyl group. To show how Zn2+ could, be recruited as a co-inhibitor of other enzymes, a method was developed, for locating in protein crystals Zn2+ binding sites where design of, Zn2+-mediated ligands can be attempted. Thus, by soaking trypsin crystals, in high concentrations of Zn2+ in the absence of a molecular inhibitor, the site where Zn2+ mediates binding of BABIM and analogs was identified, as well as another Zn2+ binding site.

About this Structure

1C1S is a Single protein structure of sequence from Bos taurus with CA, NA, PO4 and BAB as ligands. Active as Trypsin, with EC number 3.4.21.4 Full crystallographic information is available from OCA.

Reference

Recruiting Zn2+ to mediate potent, specific inhibition of serine proteases., Katz BA, Luong C, J Mol Biol. 1999 Sep 24;292(3):669-84. PMID:10497030

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