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On the origins of enzyme inhibitor selectivity and promiscuity: a case study of protein kinase binding to staurosporine.

ABSTRACT: Relationships between ligand binding and the shapes of the binding sites in families of homologous enzymes are investigated by comparing matrices of distances between key binding site atoms. Multiple linear regression is used to help identify key distances that influence ligand binding affinity. In order to illustrate the utility of this generic approach, we study protein kinase binding sites for ATP and the promiscuous competitive inhibitor, staurosporine. We show that the size of the gatekeeper residue and the closure between the first glycine of the GXGXXG motif and the aspartate of the DFG loop act together to promote tight binding. Our web-based tool, 'mapping analogous hetero-atoms onto residue interactions' (MAHORI), indicates that the greater the number of hydrogen bonds made by the kinase around the methylamine group of staurosporine, the tighter the binding. The conservation of surrounding atoms identified using our novel grid-based method clearly demonstrates that the most structurally conserved part of the binding site for staurosporine is the main chain of the hinge region. The critical role of interactions that are not dependent on side-chain identities is consistent with the promiscuous nature of this inhibitor.

SUBMITTER: Tanramluk D

PROVIDER: S-EPMC2737611 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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On the origins of enzyme inhibitor selectivity and promiscuity: a case study of protein kinase binding to staurosporine.

Tanramluk Duangrudee D Schreyer Adrian A Pitt William R WR Blundell Tom L TL

Chemical biology & drug design 20090701 1

Relationships between ligand binding and the shapes of the binding sites in families of homologous enzymes are investigated by comparing matrices of distances between key binding site atoms. Multiple linear regression is used to help identify key distances that influence ligand binding affinity. In order to illustrate the utility of this generic approach, we study protein kinase binding sites for ATP and the promiscuous competitive inhibitor, staurosporine. We show that the size of the gatekeepe ...[more]

PMID: 19519740

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Project description:Staurosporine, a potent protein kinase C (PKC) inhibitor, was studied for its effects on the binding of phorbol 12,13-dibutyrate (PDBu) to human polymorphonuclear leucocytes (PMNs). Treatment of PMNs with staurosporine concentrations in the range 50 nM-2 microM at 37 degrees C (but not at 4 degrees C) and with 1 nM [3H]PDBu at both temperatures enhanced specific PDBu binding to PMNs by approx. 10-600% relative to control values. The potentiation was rapid (detectable within 1 min) and reached a plateau after 10 min of cell treatment. Scatchard analysis of the binding showed that staurosporine increased the total number of PDBu-binding sites (Bmax) from (178 +/- 9) x 10(3) (control) to (324 +/- 15) x 10(3) sites per PMN and lowered the apparent dissociation constant (Kd) from 9.6 +/- 0.8 (control) to 3.3 +/- 0.3 nM. In Ca(2+)-depleted cells, staurosporine induced similar changes in Kd values, whereas the Bmax. increased by 60%. Treatment of PMNs with 500 nM staurosporine enhanced PDBu binding in the particulate fraction by 120 +/- 7% and decreased PDBu binding in the soluble fraction by 69 +/- 4%, whereas PKC histone-phosphorylating activity of both fractions was almost completely inhibited. Incubation of staurosporine-pretreated particulate fractions with soluble fractions enriched the particulate fraction in PDBu-binding sites at the expense of the soluble fraction, without altering the binding affinity of PDBu for either fraction. Stimulation of PMNs with chemotactic N-formyl peptides induced a transient increase in PDBu binding. This effect was potentiated by approx. 52% by staurosporine. These results show that, in addition to its interference with PKC protein-phosphorylating activity, staurosporine enhances both PDBu-binding capacity and affinity to PMNs, through a mechanism involving Ca(2+)-dependent and -independent processes. Alterations of PDBu binding to soluble and particulate fractions suggest that the enhanced binding capacity in intact PMNs may be due to translocation of PDBu receptors, presumably PKC units. This phenomenon may affect PKC-dependent cellular responses mediated by physiological stimulation.

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On the origins of enzyme inhibitor selectivity and promiscuity: a case study of protein kinase binding to staurosporine.

Publications

On the origins of enzyme inhibitor selectivity and promiscuity: a case study of protein kinase binding to staurosporine.

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