Noncollagenous region of the streptococcal collagen‐like protein is a trimerization domain that supports refolding of adjacent homologous and heterologous collagenous domains

• Published in: Protein science 2010-04, Vol.19 (4), p.775-785
• Main Authors: Yu, Zhuoxin, Mirochnitchenko, Oleg, Xu, Chunying, Yoshizumi, Ayumi, Brodsky, Barbara, Inouye, Masayori
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Calorimetry, Differential Scanning; Carrier Proteins - chemistry; Carrier Proteins - metabolism; Clostridium perfringens; collagen; Collagen - chemistry; Dimerization; folding; Models, Molecular; Molecular Sequence Data; Protein Conformation; Protein Folding; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; streptococcus; Streptococcus pyogenes; Streptococcus pyogenes - metabolism; Structure-Activity Relationship; trimerization; triple‐helix
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.356

Proper folding of the (Gly‐Xaa‐Yaa)n sequence of animal collagens requires adjacent N‐ or C‐terminal noncollagenous trimerization domains which often contain coiled‐coil or beta sheet structure. Collagen‐like proteins have been found recently in a number of bacteria, but little is known about their folding mechanism. The Scl2 collagen‐like protein from Streptococcus pyogenes has an N‐terminal globular domain, designated Vsp, adjacent to its triple‐helix domain. The Vsp domain is required for proper refolding of the Scl2 protein in vitro. Here, recombinant Vsp domain alone is shown to form trimers with a significant α‐helix content and to have a thermal stability of Tm = 45°C. Examination of a new construct shows that the Vsp domain facilitates efficient in vitro refolding only when it is located N‐terminal to the triple‐helix domain but not when C‐terminal to the triple‐helix domain. Fusion of the Vsp domain N‐terminal to a heterologous (Gly‐Xaa‐Yaa)n sequence from Clostridium perfringens led to correct folding and refolding of this triple‐helix, which was unable to fold into a triple‐helical, soluble protein on its own. These results suggest that placement of a functional trimerization module adjacent to a heterologous Gly‐Xaa‐Yaa repeating sequence can lead to proper folding in some cases but also shows specificity in the relative location of the trimerization and triple‐helix domains. This information about their modular nature can be used in the production of novel types of bacterial collagen for biomaterial applications.