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J. Biol. Chem., Vol. 280, Issue 45, 37732-37741, November 11, 2005

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Structural Insights into the Secretin PulD and Its Trypsin-resistant Core^*

Mohamed Chami1, Ingrid Guilvout1, Marco Gregorini, Hervé W. Rémigy, Shirley A. Müller, Marielle Valerio¶, Andreas Engel, Anthony P. Pugsley2, and Nicolas Bayan

From the M. E. Müller Institute, Biozentrum, University of Basel, Klingelbergstrasse 70, CH 4056 Basel, Switzerland, Molecular Genetics Unit, CNRS URA2175, Institut Pasteur, 25 rue du Dr. Roux 75724 Paris cedex 15, France, and ^¶Laboratoire de Modélisation et Ingénierie des Protéines, Université de Paris XI-Orsay, 15, rue G. Clémenceau, 91405 Orsay cedex France

Limited proteolysis, secondary structure and biochemical analyses, mass spectrometry, and mass measurements by scanning transmission electron microscopy were combined with cryo-electron microscopy to generate a three-dimensional model of the homomultimeric complex formed by the outer membrane secretin PulD, an essential channel-forming component of the type II secretion system from Klebsiella oxytoca. The complex is a dodecameric structure composed of two rings that sandwich a closed disc. The two rings form chambers on either side of a central plug that is part of the middle disc. The PulD polypeptide comprises two major, structurally quite distinct domains; an N domain, which forms the walls of one of the chambers, and a trypsin-resistant C domain, which contributes to the outer chamber, the central disc, and the plug. The C domain contains a lower proportion of potentially transmembrane -structure than classical outer membrane proteins, suggesting that only a small part of it is embedded within the outer membrane. Indeed, the C domain probably extends well beyond the confines of the outer membrane bilayer, forming a centrally plugged channel that penetrates both the peptidoglycan on the periplasmic side and the lipopolysaccharide and capsule layers on the cell surface. The inner chamber is proposed to constitute a docking site for the secreted exoprotein pullulanase, whereas the outer chamber could allow displacement of the plug to open the channel and permit the exoprotein to escape.

Received for publication, April 25, 2005 , and in revised form, August 17, 2005.

^* This work was supported in part by European Commission Research and Training Grant HPRN-CT-2000-00075, Swiss National Foundation Grant NF 31-59 415.99 (to A. E.), the Maurice E. Müller Foundation of Switzerland, and the Université de Paris Sud. 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 material.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 33-145688494; Fax: 33-145688960; E-mail: max{at}pasteur.fr.

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