Conformational Changes in Nitric Oxide Synthases Induced by Chlorzoxazone and Nitroindazoles: Crystallographic and Computational Analyses of Inhibitor Potency

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• 作者：Robin J. Rosenfeld ; Elsa D. Garcin ; Koustubh Panda ; Gunilla Andersson ; Anders Å ; berg ; Alan V. Wallace ; Garrett M. Morris ; Arthur J. Olson ; Dennis J. Stuehr ; John A. Tainer ; and Elizabeth D. Getzo
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：November 26, 2002
• 年：2002
• 卷：41
• 期：47
• 页码：13915 - 13925
• 全文大小：744K
• 年卷期：v.41,no.47(November 26, 2002)
• ISSN：1520-4995

文摘

Nitric oxide is a key signaling molecule in many biological processes, making regulation ofnitric oxide levels highly desirable for human medicine and for advancing our understanding of basicphysiology. Designing inhibitors to specifically target one of the three nitric oxide synthase (NOS) isozymesthat form nitric oxide from the L-Arg substrate poses a significant challenge due to the overwhelminglyconserved active sites. We report here 10 new X-ray crystallographic structures of inducible and endothelialNOS oxygenase domains cocrystallized with chlorzoxazone and four nitroindazoles: 5-nitroindazole,6-nitroindazole, 7-nitroindazole, and 3-bromo-7-nitroindazole. Each of these bicyclic aromatic inhibitorshas only one hydrogen bond donor and therefore cannot form the bidentate hydrogen bonds that theL-Arg substrate makes with Glu371. Instead, all of these inhibitors induce a conformational change inGlu371, creating an active site with altered molecular recognition properties. The cost of this conformationalchange is ~1-2 kcal, based on our measured constants for inhibitor binding to the wild-type and E371Amutant proteins. These inhibitors derive affinity by -stacking above the heme and replacing bothintramolecular (Glu371-Met368) and intermolecular (substrate-Trp366) hydrogen bonds to the -sheetarchitecture underlying the active site. When bound to NOS, high-affinity inhibitors in this class areplanar, whereas weaker inhibitors are nonplanar. Isozyme differences were observed in the pterin cofactorsite, the heme propionate, and inhibitor positions. Computational docking predictions match thecrystallographic results, including the Glu371 conformational change and inhibitor-binding orientations,and support a combined crystallographic and computational approach to isozyme-specific NOS inhibitoranalysis and design.