1sy1
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1.0 A Crystal Structure of T121V Mutant of Nitrophorin 4 Complexed with Nitric Oxide
OverviewOverview
Nitrophorins 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.
About this StructureAbout this Structure
1SY1 is a Single protein structure of sequence from Rhodnius prolixus with PO4, HEM and NO as ligands. Full crystallographic information is available from OCA.
ReferenceReference
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
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