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J. Biol. Chem., Vol. 279, Issue 18, 19302-19314, April 30, 2004

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Structure-Function Analysis of PrsA Reveals Roles for the Parvulin-like and Flanking N- and C-terminal Domains in Protein Folding and Secretion in Bacillus subtilis^*

Marika Vitikainen, Ilkka Lappalainen¶, Raili Seppala, Haike Antelmann||, Harry Boer**, Suvi Taira, Harri Savilahti, Michael Hecker||, Mauno Vihinen¶¶¶, Matti Sarvas, and Vesa P. Kontinen||||

From the Vaccine Development Laboratory, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland, the Faculty of Biosciences, Department of Biological and Environmental Sciences, Division of Biochemistry, P. O. Box 56, FIN-00014 University of Helsinki, Finland, the ^¶Institute of Medical Technology, FIN-33014 University of Tampere, Finland, the ^||Institute für Mikrobiologie und Molecular Biologie, Ernst-Moritz-Arndt-Universität, D-17487 Greifswald, Germany, ^**VTT Biotechnology, P. O. Box 1500, FIN-02044 VTT, Espoo, Finland, the Program in Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, Viikki Biocenter, P. O. Box 56, FIN-00014, University of Helsinki, Finland, and the ^¶¶Research Unit, Tampere University Hospital, FIN-30520 Tampere, Finland

The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall. This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase). In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues. PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain. We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N- and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth. Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro. Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains.

Received for publication, January 26, 2004

^* This work was supported in part by the Academy of Finland (to M. V., I. L., R. S., S. T., H. S., M. V., M. S., and V. P. K.), the Finnish National Technology Agency (TEKES) (to S. T. and H. S), the Medical Research Fund of Tampere University Hospital (to I. L. and M. V.), Genencor International (Palo Alto, CA) (to H. A.), Quality of Life and Management of Living Resource Grants QLK3-CT-1999-00413 from the European Union (to M. V., R. S., H. A., M. H., M. S., and V. P. K.), Research Program 2000-2005, Project No. 64330VTT Industrial Biotechnology from the Academy of Finland, Finnish Centre of Excellence, and grants from the Deutsche Forschungsgemeinschaft (DFG), the Bundesministerium für Bildung, Wissenschaft, Forschung, und Technologie (BMFT), and the Fonds der Chemischen Industrie (to H. A. and M. H.). 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.

Present address: Dept. of Biosciences, Division of General Microbiology, University of Helsinki, P. O. Box 56, FIN 00014, University of Helsinki, Finland.

^|||| To whom correspondence should be addressed. Tel.: 358-9-47448562; Fax: 358-9-47448347; E-mail: Vesa.Kontinen{at}ktl.fi.

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