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J. Biol. Chem., Vol. 281, Issue 9, 5908-5915, March 3, 2006

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Topology of the Porin MspA in the Outer Membrane of Mycobacterium smegmatis^*

Maysa Mahfoud, Suja Sukumaran, Peter Hülsmann¹, Katrin Grieger, and Michae Niederweis²

From the Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, D-91058 Erlangen, Germany and the Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294

MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the outer membrane (OM). It is the prototype of a new family of octameric porins with a single central channel of 9.6 nm in length and consists of two hydrophobic -barrels of 3.7 nm in length and a more hydrophilic, globular rim domain. The length of the hydrophobic domain of MspA does not match the thicknesses of mycobacterial OMs of 5-12 nm as derived from electron micrographs. Further, the membrane topology of MspA is unknown as it is for any other mycobacterial OM protein. We used MspA as a molecular ruler to define the boundaries of the OM of M. smegmatis by surface labeling of single cysteine mutants. Seventeen mutants covered the surface of the rim domain and were biotinylated with a membrane-impermeable reagent. The label efficiencies in vitro were remarkably similar to the predicted accessibilities of the cysteines. By contrast, six of these mutants were protected from biotinylation in M. smegmatis cells. Tryptophan 21 defines a horizontal plane that dissects the surface-exposed versus the membrane-protected residues of MspA. The 8 phenylalanines at position 99 form a ring at the periplasmic end of the hydrophobic -barrel domain. These results indicated that (i) the membrane boundaries of MspA are defined by aromatic girdles as in porins of Gram-negative bacteria and (ii) loops and a 3.4-nmlong part of the hydrophilic rim domain are embedded into the OM of M. smegmatis. This is the first report suggesting that elements other than hydrophobic -helices or -sheets are integrated into a lipid membrane.

Received for publication, October 27, 2005 , and in revised form, December 12, 2005.

^* This work was funded by grants from the Deutsche Forschungsgemeinschaft (Grant Ni 412), the Volkswagen-Stiftung (Grant I/77 729), and the Fonds der Chemischen Industrie (to M. N.). 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 five supplemental figures and a supplemental table.

¹ Present Address: Institut für Klinische und Molekulare Virologie, Am Schlossgarten 4, 91058 Erlangen, Germany.

² To whom correspondence should be addressed. Tel.: 205-996-2711; Fax: 205-934-9256; E-mail: Mnieder{at}Uab.Edu.

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