Structural stability and heat-induced conformational change of two complement inhibitors: C4b-binding protein and factor H

doi:10.1110/ps.03516504

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Structural stability and heat-induced conformational change of two complement inhibitors: C4b-binding protein and factor H

Lena Kask¹, Bruno O. Villoutreix², Mårten Steen¹, Bala Ramesh³, Björn Dahlbäck¹ and Anna M. Blom¹

¹ Lund University, The Wallenberg Laboratory, Department of Clinical Chemistry, University Hospital Malmö, S-205 02 Malmö, Sweden
² French National Institute of Health and Medical Research (INSERM) U428, University Paris V, Paris, France
³ Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School, London NW3 PF, UK

(RECEIVED November 17, 2003; FINAL REVISION January 23, 2004; ACCEPTED February 1, 2004)

Abstract

The complement inhibitors C4b-binding protein (C4BP) and factorH (FH) both consist of complement control protein (CCP) domains.Here we examined the secondary structure of both proteins bycircular dichroism and Fourier-transform infrared techniqueat temperatures ranging from 30°C–90°C. We foundthat predominantly ${beta}$ -sheet structure of both proteins was stableup to 70°C, and that a reversible conformational changetoward ${alpha}$ -helix was apparent at temperatures ranging from 70°Cto 90°C. The ability of both proteins to inhibit complementwas not impaired after incubation at 95°C, exposure to extremepH conditions, and storage at room temperature for several months.Similar remarkable stability was previously observed for vacciniavirus control protein (VCP), which is also composed of CCP domains;it therefore seems to be a general property of CCP-containingproteins. A typical CCP domain has a hydrophobic core, whichis wrapped in ${beta}$ -sheets and stabilized by two disulphide bridges.How the CCP domains tolerate harsh conditions is unclear, butit could be due to a combination of high content of prolines,hydrophobic residues, and the presence of two disulphide bridgeswithin each domain. These findings are of interest because CCP-containingcomplement inhibitors have been proposed as clinical agentsto be used to control unwanted complement activation that contributesto many diseases.

Keywords: complement inhibitors; structural stability; C4b-binding protein; factor H

Abbreviations: C4BP, C4b-binding protein • FH, factor H • CCP, complement control protein • CD, circular dichroism • FTIR, Fourier transform-infrared spectroscopy • PT, prothrombin • VCP, vaccinia virus complement control protein

Reprint requests to: Anna Blom, Lund University, Department of Clinical Chemistry, University Hospital Malmö, S-205 02 Malmö, Sweden; e-mail: Anna.Blom{at}klkemi.mas.lu.se; fax: (46) 40-33-70-44.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03516504.

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