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J. Biol. Chem., Vol. 279, Issue 47, 49131-49137, November 19, 2004

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Structural and Dynamic Independence of Isopeptide-linked RanGAP1 and SUMO-1^*

Matthew S. Macauley, Wesley J. Errington, Mark Okon, Manuela Schärpf, Cameron D. Mackereth, Brenda A. Schulman, and Lawrence P. McIntosh, A Canadian Institutes of Health Research Scientist¶

From the Department of Biochemistry and Molecular Biology, the Department of Chemistry, and the Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada and the Departments of Structural Biology and Genetics/Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105

Although sumoylation regulates a diverse and growing number of recognized biological processes, the molecular mechanisms by which the covalent attachment of the ubiquitin-like protein SUMO can alter the properties of a target protein remain to be established. To address this question, we have used NMR spectroscopy to characterize the complex of mature SUMO-1 with the C-terminal domain of human RanGAP1. Based on amide chemical shift and ¹⁵N relaxation measurements, we show that the C terminus of SUMO-1 and the loop containing the consensus sumoylation site in RanGAP1 are both conformationally flexible. Furthermore, the overall structure and backbone dynamics of each protein remain unchanged upon the covalent linkage of Lys⁵²⁴ in RanGAP1 to the C-terminal Gly⁹⁷ of SUMO-1. Therefore, SUMO-1 and RanGAP1 behave as "beads-on-a-string," connected by a flexible isopeptide tether. Accordingly, the sumoylation-dependent interaction of RanGAP1 with the nucleoporin RanBP2 may arise through the bipartite recognition of both RanGAP1 and SUMO-1 rather than through a new binding surface induced in either individual protein upon their covalent linkage. We hypothesize that this conformational flexibility may be a general feature contributing to the recognition of ubiquitin-like modified proteins by their downstream effector machineries.

Received for publication, July 30, 2004 , and in revised form, August 26, 2004.

The assigned chemical shift list has been deposited in the BioMagRes-Bank under accession numbers 6304, 6305, and 6306.

^* This research was supported by grants from the National Cancer Institute of Canada with funds from the Canadian Cancer Society (to L. P. M.) as well as National Institutes of Health Grant R01GM69530 (B. A. S). Instrument support was provided by the Government of Canada's Network of Centres of Excellence Program supported by the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council (NSERC) through the Protein Engineering Network of Centres of Excellence (PENCE, Inc.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains additional data on RanGAP1_c and SUMO-1_gg in the form of supplemental Figs. S1 and S2 and Table S1.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry, 2146 Health Sciences Mall, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. Tel.: 604-822-3341; Fax: 604-822-5227; E-mail: mcintosh{at}otter.biochem.ubc.ca.

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