Published online 10 January 2006
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The role of positively charged amino acids and electrostatic interactions in the complex of U1A protein and U1 hairpin II RNA
1Department of Biochemistry and Molecular Biology, University of Southern California Los Angeles, CA 90089-9176, USA 2Department of Surgery, Keck School of Medicine, University of Southern California Los Angeles, CA 90089-9176, USA 3Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California Los Angeles, CA 90089-9176, USA 4MRC Laboratory of Molecular Biology Hills Road, Cambridge CB2 2QH, UK 5Karolinska Institutet, Department of Biosciences at Novum SE-141 57 Huddinge, Sweden
*To whom correspondence should be addressed. Tel: +1 323 865 0655; Fax: +1 323 865 0158; Email: ilaird{at}usc.edu
Received October 14, 2005. Revised December 22, 2005. Accepted December 22, 2005.
Previous kinetic investigations of the N-terminal RNA recognition motif (RRM) domain of spliceosomal protein U1A, interacting with its RNA target U1 hairpin II, provided experimental evidence for a ‘lure and lock’ model of binding in which electrostatic interactions first guide the RNA to the protein, and close range interactions then lock the two molecules together. To further investigate the ‘lure’ step, here we examined the electrostatic roles of two sets of positively charged amino acids in U1A that do not make hydrogen bonds to the RNA: Lys20, Lys22 and Lys23 close to the RNA-binding site, and Arg7, Lys60 and Arg70, located on ‘top’ of the RRM domain, away from the RNA. Surface plasmon resonance-based kinetic studies, supplemented with salt dependence experiments and molecular dynamics simulation, indicate that Lys20 predominantly plays a role in association, while nearby residues Lys22 and Lys23 appear to be at least as important for complex stability. In contrast, kinetic analyses of residues away from the RNA indicate that they have a minimal effect on association and stability. Thus, well-positioned positively charged residues can be important for both initial complex formation and complex maintenance, illustrating the multiple roles of electrostatic interactions in protein–RNA complexes.
Present addresses: Michael E. Linde, Graduate Program in Immunology, Johns Hopkins School of Medicine, Richard Starr Ross Research Building, Suite 1071S, Baltimore, MD 21205, USA
Phinikoula S. Katsamba, Center for Biomolecular Interaction Analysis, School of Medicine, University of Utah, Salt Lake City, UT 84132, USA
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