Electrostatic Interactions Guide the Active Site Face of a Structure-Specific Ribonuclease to Its RNA Substrate

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• 作者：Matthew J. Plantinga ; Alexei V. Korennykh ; Joseph A. Piccirilli ; Carl C. Correll
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：August 26, 2008
• 年：2008
• 卷：47
• 期：34
• 页码：8912-8918
• 全文大小：296K
• 年卷期：v.47,no.34(August 26, 2008)
• ISSN：1520-4995

文摘

Restrictocin, a member of the α-sarcin family of site-specific endoribonucleases, uses electrostatic interactions to bind to the ribosome and to RNA oligonucleotides, including the minimal specific substrate, the sarcin/ricin loop (SRL) of 23S−28S rRNA. Restrictocin binds to the SRL by forming a ground-state E:S complex that is stabilized predominantly by Coulomb interactions and depends on neither the sequence nor structure of the RNA, suggesting a nonspecific complex. The 22 cationic residues of restrictocin are dispersed throughout this protein surface, complicating a priori identification of a Coulomb interacting surface. Structural studies have identified an enzyme−substrate interface, which is expected to overlap with the electrostatic E:S interface. Here, we identified restrictocin residues that contribute to binding in the E:S complex by determining the salt dependence [∂ log(k₂/K_1/2)/∂ log[KCl]] of cleavage of the minimal SRL substrate for eight point mutants within the protein designed to disrupt contacts in the crystallographically defined interface. Relative to the wild-type salt dependence of −4.1, a subset of the mutants clustering near the active site shows significant changes in salt dependence, with differences of magnitude being ≥0.4. This same subset was identified using calculated salt dependencies for each mutant derived from solutions to the nonlinear Poisson−Boltzmann equation. Our findings support a mechanism in which specific residues on the active site face of restrictocin (primarily K110, K111, and K113) contribute to formation of the E:S complex, thereby positioning the SRL substrate for site-specific cleavage. The same restrictocin residues are expected to facilitate targeting of the SRL on the surface of the ribosome.