Structural diversity in twin-arginine signal peptide-binding proteins

*Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom;
^‡Center for Molecular and Biomolecular Informatics, Nijmegen Center for Molecular Life Sciences, and
**Protein Biophysics Group, Institute for Molecules and Materials, Radboud University, 6525 ED, Nijmegen, The Netherlands; and
^¶Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom

Edited by William M. Clemons, California Institute of Technology, Pasadena, CA, and accepted by the Editorial Board August 15, 2007 (received for review April 30, 2007)

Abstract

The twin-arginine transport (Tat) system is dedicated to the translocation of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat system by signal peptides containing a twin-arginine motif. In Escherichia coli, many Tat substrates bind redox-active cofactors in the cytoplasm before transport. Coordination of cofactor insertion with protein export involves a “Tat proofreading” process in which chaperones bind twin-arginine signal peptides, thus preventing premature export. The initial Tat signal-binding proteins described belonged to the TorD family, which are required for assembly of N- and S-oxide reductases. Here, we report that E. coli NapD is a Tat signal peptide-binding chaperone involved in biosynthesis of the Tat-dependent nitrate reductase NapA. NapD binds tightly and specifically to the NapA twin-arginine signal peptide and suppresses signal peptide translocation activity such that transport via the Tat pathway is retarded. High-resolution, heteronuclear, multidimensional NMR spectroscopy reveals the 3D solution structure of NapD. The chaperone adopts a ferredoxin-type fold, which is completely distinct from the TorD family. Thus, NapD represents a new family of twin-arginine signal-peptide-binding proteins.

Footnotes

^††To whom correspondence should be addressed. E-mail: f.sargent{at}dundee.ac.uk
Author contributions: J.M. and C.A.E.M.S. contributed equally to this work; J.M., C.A.E.M.S., G.W.V., and F.S. designed research; J.M., C.A.E.M.S., G.B., V.L., T.P., and F.S. performed research; D.J.R., T.P., G.W.V., and F.S. analyzed data; and J.M., C.A.E.M.S., D.J.R., T.P., G.W.V., and F.S. wrote the paper.
↵ ^†Present address: Environment Naturel, Architectural et Construit–Institut des Sciences et Technologies de l'Environhement/Laboratoire de Biotechnologie Environnementale, cole Polytechnique Fédérale Lausanne, Bâtiment Chimie-B Ecublens, CH-1015 Lausanne, Switzerland.
↵ ^§Present address: Spronk NMR Consultancy, Pylimo Gatve 6, LT-01117 Vilnius, Lithuania.
↵ ^‖Present address: College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. W.M.C. is a guest editor invited by the Editorial Board.
Data deposition: The resonance assignments have been deposited in the Biological Magnetic Resonance Data Bank, www.bmrb.wisc.edu (BMRB accession no. 15381), and the atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2jsx).
This article contains supporting information online at www.pnas.org/cgi/content/full/0703967104/DC1.
Abbreviations:

HSQC,

heteronuclear single quantum correlation;

ITC,

isothermal titration calorimetry;

Tat,

twin-arginine translocation.