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1.0 A Crystal Structure of T121V Mutant of Nitrophorin 4 Complexed with Nitric Oxide
Structural highlights
FunctionNP4_RHOPR Heme-based protein that deliver nitric oxide gas (NO) to the victim while feeding, resulting in vasodilation and inhibition of platelet aggregation. Also bind tightly to histamine, which is released by the host to induce wound healing (By similarity). Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedNitrophorins are ferric heme proteins that transport nitric oxide (NO) from blood-sucking insects to victims. NO binding is tighter at lower pH values, as found in the insect salivary gland, and weaker at the pH of the victim's tissue, facilitating NO release and subsequent vasodilation. Previous structural analyses of nitrophorin 4 (NP4) from Rhodnius prolixus revealed a substantial NO-induced conformational change involving the A-B and G-H loops, which rearrange to desolvate the distal pocket and pack nonpolar residues against the heme-ligated NO. Previous kinetic analyses revealed a slow, biphasic, and pH-dependent NO release, which was proposed to be associated with loop movements. In this study, we created NP4 mutants D30A and D30N (A-B loop), D129A/L130A (G-H loop), and T121V (distal pocket). Eight crystal structures were determined, including complexes with NO, NH(3), and imidazole, to resolutions as high as 1.0 A. The NO-induced conformational change is largely abolished in the loop mutants, but retained in T121V. Kinetic analyses using stopped-flow spectroscopy revealed the pH dependence for NO release is eliminated for D129A/L130A, considerably reduced for D30A and D30N, but retained for T121V. NO association rates were increased 2-5-fold for T121V, but were unchanged in the loop mutants. Taken together, our findings demonstrate that the pH dependency for NO release is linked to loop dynamics and that solvent reorganization is apparently rate-limiting for formation of the initial iron-nitrosyl bond. Interestingly, the multiphasic kinetic behavior of rNPs was not affected by mutations, and its cause remains unclear. Role of binding site loops in controlling nitric oxide release: structure and kinetics of mutant forms of nitrophorin 4.,Maes EM, Weichsel A, Andersen JF, Shepley D, Montfort WR Biochemistry. 2004 Jun 1;43(21):6679-90. PMID:15157102[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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