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J. Biol. Chem., Vol. 283, Issue 29, 20069-20076, July 18, 2008

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Influences of the N700S Thrombospondin-1 Polymorphism on Protein Structure and Stability^*

From the Departments of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706

Thrombospondins (THBSs) are multimodular, secreted proteins characterized by a signature domain comprising a unique set of 13 calcium-binding repeats flanked by epidermal growth factor (EGF)-like and lectin-like modules. A polymorphism that changes a conserved Asn to Ser at residue 700 in the most N-terminal calcium-binding repeat of THBS-1 (repeat 1C) is found in 8–10% of European populations and has been linked to increased risk of premature coronary artery disease. The Ser substitution leads to altered stability in the EGF-like and wire modules of the THBS-1 signature domain as assessed by differential scanning calorimetry carried out in 2 mM or 200 µM calcium. Studies of the melting profiles of the THBS-2 signature domain proteins with Asn or Ser at position 702 (homologous to 700 in THBS-1) revealed that the impact of the Ser allele is similar in both THBS-1 and THBS-2. Structure determination of the Ser⁷⁰² THBS-2 variant in 2 mM calcium showed that repeat 1C contains two bound calcium ions as in the crystal of the Asn⁷⁰² protein, including the ion that is coordinated by Asn⁷⁰², and is associated with changes in conformation of repeat 1C and the adjacent EGF-like modules. The Ser substitution leads to the decreased ability of soluble THBS-2 signature domain protein to bind 4B6.13, a conformation-sensitive monoclonal antibody that recognizes an epitope in repeat 1C. These results indicate that although THBS harboring the Ser allele binds a full complement of calcium ions, repeat 1C is altered, leading to destabilization of surrounding structures.

Received for publication, January 9, 2008 , and in revised form, April 25, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant HL54462 (to D. F. M.). This work was also supported by a Shaw Foundation for Medical Research grant (to J. L. K.). Differential scanning calorimetry data were obtained at the University of Wisconsin-Madison Biophysics Instrumentation Facility, which was established with support from the University of Wisconsin-Madison, National Science Foundation Grant BIR-9512577, and National Institutes of Health Grant S10 RR13790. 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 supplemental Figs. S1 and S2.

The atomic coordinates and structure factors (code 2RPH) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¹ Supported by National Institutes of Health Training Grant HL07899.

² To whom correspondence should be addressed: 4285 Medical Sciences Center, 1300 University Ave., University of Wisconsin, Madison, WI 53706. Tel.: 608-262-1576; Fax: 608-263-4969; E-mail: dfmosher{at}wisc.edu.

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				C. B. Carlson, K. A. Gunderson, and D. F. Mosher Mutations Targeting Intermodular Interfaces or Calcium Binding Destabilize the Thrombospondin-2 Signature Domain J. Biol. Chem., October 3, 2008; 283(40): 27089 - 27099. [Abstract] [Full Text] [PDF]