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J. Biol. Chem., Vol. 281, Issue 8, 5224-5232, February 24, 2006

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Crystal Structure of the Cytomegalovirus DNA Polymerase Subunit UL44 in Complex with the C Terminus from the Catalytic Subunit

DIFFERENCES IN STRUCTURE AND FUNCTION RELATIVE TO UNLIGANDED UL44^*

Brent A. Appleton¹, Justin Brooks^¶2, Arianna Loregian³, David J. Filman, Donald M. Coen, and James M. Hogle⁴

From the Department of Biological Chemistry and Molecular Pharmacology, the Committee on Virology, and the ^¶Summer Honors Undergraduate Research Program, Harvard Medical School, Boston, Massachusetts 02115

The human cytomegalovirus DNA polymerase is composed of a catalytic subunit, UL54, and an accessory protein, UL44, which has a structural fold similar to that of other processivity factors, including herpes simplex virus UL42 and homotrimeric sliding clamps such as proliferating cell nuclear antigen. Several specific residues in the C-terminal region of UL54 and in the "connector loop" of UL44 are required for the association of these proteins. Here, we describe the crystal structure of residues 1-290 of UL44 in complex with a peptide from the extreme C terminus of UL54, which explains this interaction at a molecular level. The UL54 peptide binds to structural elements similar to those used by UL42 and the sliding clamps to associate with their respective binding partners. However, the details of the interaction differ from those of other processivity factor-peptide complexes. Crucial residues include a three-residue hydrophobic "plug" from the UL54 peptide and Ile¹³⁵ of UL44, which forms a critical intramolecular hydrophobic anchor for interactions between the connector loop and the peptide. As was the case for the unliganded UL44 structure, the UL44-peptide complex forms a head-to-head dimer that could potentially form a C-shaped clamp on DNA. However, the peptide-bound structure displays subtle differences in the relative orientation of the two subdomains of the protein, resulting in a more open clamp, which we predicted would affect its association with DNA. Indeed, filter binding assays revealed that peptide-bound UL44 binds DNA with higher affinity. Thus, interaction with the catalytic subunit appears to affect both the structure and function of UL44.

Received for publication, June 24, 2005 , and in revised form, December 2, 2005.

The atomic coordinates and structure factors (code 1YYP) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by National Institutes of Health Grants AI19838 (toD. M. C.) and AI32480 (to J. M. H.). Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source was supported by United States Department of Energy, Office of Energy Research, under Contract W-31-109-ENG-38. 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.

¹ Present address: Dept. of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080.

² Supported in part by Research Experiences for Undergraduates Grant DBI-0243489 from the National Science Foundation. Present address: Washington University Medical School, St. Louis, MO 63110.

³ Present address: Dept. of Histology, Microbiology, and Medical Biotechnologies, Section of Microbiology, University of Padua, 35121 Padua, Italy.

⁴ To whom correspondence should be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, Bldg. C-2, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-3918; Fax: 617-432-4360; E-mail: jhogle{at}hms.harvard.edu.

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