Opening and closing of the periplasmic gate in lactose permease

doi:10.1073/pnas.0800825105

Opening and closing of the periplasmic gate in lactose permease

Departments of *Physiology and
^‡Microbiology, Immunology, and Molecular Genetics,
^§Molecular Biology Institute, University of California, Los Angeles, CA 90095-1662; and
^†Department of Physiology and Biophysics, University of California, Irvine, CA 92697-4560

Contributed by H. Ronald Kaback, January 28, 2008 (received for review January 18, 2007)

Abstract

X-ray crystal structures of lactose permease (LacY) reveal pseudosymmetrically arranged N- and C-terminal six-transmembrane helix bundles surrounding a deep internal cavity open on the cytoplasmic side and completely closed on the periplasmic side. The residues essential for sugar recognition and H⁺ translocation are located at the apex of the cavity and are inaccessible from the outside. On the periplasmic side, helices I/II and VII from the N- and C- six helix bundles, respectively, participate in sealing the cavity from the outside. Three paired double-Cys mutants—Ile-40 → Cys/Asn-245 → Cys, Thr-45 → Cys/Asn-245 → Cys, and Ile-32 → Cys/Asn-245 → Cys—located in the interface between helices I/II and VII on the periplasmic side of LacY were constructed. After cross-linking with homobifunctional reagents less than ≈15 Å in length, all three mutants lose the ability to catalyze lactose transport. Strikingly, however, full or partial activity is observed when cross-linking is mediated by flexible reagents greater than ≈15 Å in length. The results provide direct support for the argument that transport via LacY involves opening and closing of a large periplasmic cavity.

Footnotes

^¶To whom correspondence should be addressed. E-mail: rkaback{at}mednet.ucla.edu
Author contributions: Y.Z., L.G., and H.R.K. designed research; Y.Z. and J.A.F. performed research; Y.Z., L.G., and H.R.K. analyzed data; and Y.Z., L.G., J.A.F., and H.R.K. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0800825105/DC1.