A general method for determining helix packing in membrane proteinsin situ: Helices I and II are close to helix VII in the lactose permease ofEscherichia coli

Abstract

It was previously shown that coexpression of the lactose permease of Escherichia coli in two contiguous fragments leads to functional complementation. We demonstrate here that site-directed thiol crosslinking of coexpressed permease fragments can be used to determine helix proximity in situ without the necessity of purifying the permease. After coexpression of the six N-terminal (N 6 ) and six C-terminal (C 6 ) The lactose (lac) permease of Escherichia coli is a paradigm for secondary transport proteins that transduce free energy stored in an electrochemical ion gradient into a solute concentration gradient. This integral membrane protein that catalyzes the coupled stoichiometric translocation of ␤-galactosides and H ϩ has been solubilized, purified to homogeneity, reconstituted, and shown to be solely responsible for ␤-galactoside transport (1) as a monomer (see ref.2). Based on circular dichroism and hydropathy analysis, a secondary-structure model was proposed (3) in which the permease contains 12 ␣-helical rods that traverse the membrane in zig-zag fashion connected by loops with the N and C termini on the cytoplasmic face. Evidence favoring many aspects of the model has been obtained from a variety of approaches (reviewed in ref. 4, in addition see refs. 5-7), and analysis of an extensive series of lac permeasealkaline phosphatase (lacY-phoA) fusions has provided unequivocal support for the topological predictions of the 12-helix model (8).Site-directed and Cys-scanning mutagenesis have allowed delineation of amino acid residues in the permease that are important for active transport and͞or substrate binding (reviewed in refs. 9 and 10). However, static and dynamic information at high resolution are required to understand the role of these residues in the transport mechanism. Because hydrophobic membrane proteins are notoriously difficult to crystallize, a high-resolution structure of lac permease is not available, and development of alternative methods for obtaining structural information is essential. In this respect, a functional permease molecule has been engineered in which all of the native Cys residues have been replaced (C-less permease), and single Cys residues have been placed at every position in the molecule. In addition to providing evidence that remarkably few amino acid residues are irreplaceable with regards to insertion, stability, or activity, the Cys-replacement mutants represent unique molecules that are being used to study permease structure-function relationships (reviewed in ref. 11).A helix packing model of the C-terminal half of lac permease has been formulated (12) based on site-directed excimer fluorescence, which demonstrates that helix VIII (Glu-269) is close to helix X (His-322), helix IX (Arg-302) is close to helix X (Glu-325), and that helix X is in ␣-helical conformation (also see ref. 13). Furthermore, the presence of two pairs of charged residues that interact functionally-Asp-237 (helix VII) with Lys-358 (helix XI) (14-18) and Asp-240 (helix VII) with Lys-319 (he...

Opening and closing of the periplasmic gate in lactose permease

“…Homobifunctional thiol-reactive reagents (Fig. 2) combined with site-directed factor Xa proteolysis (21,22) were used to study cross-linking on the periplasmic side of LacY between the N-and C-terminal six-helix bundles (Fig. 1).…”

Increased and controlled expression of the Rickettsia prowazekii ATP/ADP translocase and analysis of cysteine-less mutant translocase

“…Various thiol reagents are available and can be used to study protein topology by EPR, NMR, diffraction and a variety of other methods. In combination with cysteinescanning mutagenesis, these methods have been used to elucidate the importance of specific residues for transport activity in the E. coli lactose permease (Wu & Kaback, 1996, and references therein). We intend to use these methods on the cys-less translocase produced by the pETl la/BLR(DE3)pLysS expression system in future mutagenesis studies to identify the specific amino acids in the ATP/ADP translocase protein which are important for transport activity and to determine the structure-function relationships of this unusual transport system.…”

Function, Structure, and Evolution of the Major Facilitator Superfamily: The LacY Manifesto

“…Because LacY and many other MFS transporters were apparently so resistant to traditional means of structural analysis, alternative approaches were developed to approximate the overall 3D fold, as well as local interactions. LacY has been the test bed for the development of most of these methodologies with transport proteins, and they include mapping transmembrane segments by phoA fusions [57], protein insertion into loops and Advances in Biology 5 deletion analysis [58][59][60], accessibility/reactivity of natural or uniquely engineered Cys residues to membrane-permeant and impermeant reagents [61], determination of secondary structure by circular dichroism (CD) [62], laser Raman [63], or Fourier transform infrared spectroscopy (FTIR) [64], identifying long-range contacts by suppressor analysis [65,66], thiol cross-linking [67,68], excimer fluorescence [69], engineered Mn(II) binding sites [70], site-directed electron paramagnetic resonance [71,72], or discontinuous mAb epitope mapping [73,74].…”