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Biochemistry. 2008 Aug 26;47(34):8912-8. doi: 10.1021/bi800592g. Epub 2008 Aug 2.

Electrostatic interactions guide the active site face of a structure-specific ribonuclease to its RNA substrate.

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  • 1Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA.


Restrictocin, a member of the alpha-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 [partial differential log(k 2/ K 1/2)/ partial differential 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 >or=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.

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