Spectroscopic characterization of Mn2+ and Cd2+ coordination to phosphorothioates in the conserved A9 metal site of the hammerhead ribozyme

Hunsicker-Wang, Laura M.; Vogt, Matthew J.; Hoogstraten, Charles G.; Cosper, Nathaniel J.; Davenport, Audrey M.; Hendon, Christopher H.; Scott, Robert A.; Britt, R. David; DeRose, Victoria J.

doi:10.1016/j.jinorgbio.2022.111754

Title: Spectroscopic characterization of Mn²⁺ and Cd²⁺ coordination to phosphorothioates in the conserved A9 metal site of the hammerhead ribozyme

Journal Article · 11 February 2022 · Journal of Inorganic Biochemistry

DOI: https://doi.org/10.1016/j.jinorgbio.2022.111754 · OSTI ID:1981670

Hunsicker-Wang, Laura M. ^[1]; Vogt, Matthew J. ^[2]; Hoogstraten, Charles G. ^[3]; Cosper, Nathaniel J. ^[4]; Davenport, Audrey M. ^[5]; Hendon, Christopher H. ^[5]; Scott, Robert A. ^[6]; Britt, R. David ^[7]; DeRose, Victoria J. ^[5]

Trinity University, San Antonio, TX (United States)
National Institute of Allergy and Infectious Diseases, Bethesda, MD (United States)
Michigan State University, East Lansing, MI (United States)
Brady-Built, Inc., Auburn, MA (United States)
University of Oregon, Eugene, OR (United States)
University of Georgia, Athens, GA (United States)
University of California-Davis, CA (United States)

Phosphorothioate modifications have widespread use in the field of nucleic acids. As substitution of sulfur for oxygen can alter metal coordination preferences, the phosphorothioate metal-rescue experiment is a powerful method for identifying metal coordination sites that influence specific properties in a large RNAs. The A9/G10.1 metal binding site of the hammerhead ribozyme (HHRz) has previously been shown to be functionally important through phosphorothioate rescue experiments. While an A9-S_Rp substitution is inhibitory in Mg²⁺, thiophilic Cd²⁺ rescues HHRz activity. Mn²⁺ is also often used in phosphorothioate metal-rescue studies but does not support activity for the A9-S_Rp HHRz. Here, we use EPR, electron spin-echo envelope modulation (ESEEM), and X-ray absorption spectroscopic methods to directly probe the structural consequences of Mn²⁺ and Cd²⁺ coordination to R_p and S_p phosphorothioate modifications at the A9/G10.1 site in the truncated hammerhead ribozyme (tHHRz). The results demonstrate that while Cd²⁺ does indeed bind to S in the thio-substituted ligand, Mn²⁺ coordinates to the non–sulfur oxo group of this phosphorothioate, regardless of isomer. Computational models demonstrate the energetic preference of Mn—O over Mn—S coordination in metal-dimethylthiophosphate models. In the case of the tHHRz, the resulting Mn²⁺ coordination preference of oxygen in either R_p or S_p A9 phosphorothioates differentially tunes catalytic activity, with Mn—O coordination in the A9-S_Rp phosphorothioate enzyme being inhibitory.

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Research Organization:: SLAC National Accelerator Laboratory, Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)

Sponsoring Organization:: National Science Foundation (NSF); USDOE

Grant/Contract Number:: AC02-76SF00515

OSTI ID:: 1981670

Journal Information:: Journal of Inorganic Biochemistry, Journal Name: Journal of Inorganic Biochemistry Journal Issue: C Vol. 230; ISSN 0162-0134

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
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Title: Spectroscopic characterization of Mn2+ and Cd2+ coordination to phosphorothioates in the conserved A9 metal site of the hammerhead ribozyme

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Title: Spectroscopic characterization of Mn²⁺ and Cd²⁺ coordination to phosphorothioates in the conserved A9 metal site of the hammerhead ribozyme