Determination of transmembrane protein structure by disulfide cross-linking: the Escherichia coli Tar receptor.

Abstract

We have devised a generally applicable strategy for analysis of protein structure and have applied it to examine the structure of the transmembrane portion of the Tar receptor of Escherichia coli. The basis of our approach is the use of disulfide cross-linking to identify residues that are within close proximity. To generate and test large numbers of cysteine pairs, we used an unusual method of mutagenesis by which cysteine substitutions can be created randomly at a number of targeted codons. Cysteine-substituted proteins encoded by mutagenized genes may be screened directly for disulfide formation within oligomers or, alternatively, different pools of genes may be randomly recombined to generate gene populations with substitutions in multiple regions. Thus, it is possible to detect a variety of disulfide cross-links between and within individual protein molecules. Interactions between the four membrane-spanning stretches of the Tar dimer were probed by measuring the tendency of 48 cysteine substitutions throughout this region to form disulffide cross-links with one another. We have interpreted these data to suggest a helical-bundle structure for the transmembrane region. The four helices of this bundle are not structurally equivalent: the two TM1 helices interact closely, whereas the TM2 helices are more peripherally located.The Tar receptor of Escherichia coli is a transmembrane signal transducer protein involved in the detection of chemotactic effectors. It is a member of a family of related receptors, the methyl-accepting chemotaxis proteins, which share considerable amino acid sequence similarity (1). Tar appears to act as a dimer (2). It mediates repellent responses to nickel and cobalt ions and attractant responses to aspartate and maltose (3). Each 553-amino acid monomer of Tar is comprised of relatively large periplasmic and intracellular domains as well as two membrane-spanning domains that are thought to be a-helical (4). (The transmembrane topology of Tar is illustrated in Fig. 1.) Ligands bind to the periplasmic region of Tar (5-8) leading the receptor to modulate the activity of the soluble autophosphorylating kinase CheA (9, 10). This initial event leads, through a series of additional intracellular interactions, to altered bacterial swimming behavior.The nature of the protein conformational changes that mediate communication across lipid bilayers is unknown. The Tar receptor resembles the mammalian epidermal growth factor and insulin receptors in that each of these proteins consists of large soluble domains linked by a small number of transmembrane stretches. In contrast to the epidermal growth factor receptor, the oligomeric state of the Tar receptor does not change as a consequence of ligand binding (2); thus any structural changes involved in transmembrane signal transmission occur within the preformed receptor dimer. We presume that ligand binding results in a conformational change in the periplasmic domain and that this change is then propagated by the four transmembrane domains to t...

Cysteine-directed cross-linking localizes regions of the human erythrocyte anion-exchange protein (AE1) relative to the dimeric interface

“…This interpretation would be consistent with the observed restricted dynamics. The lack of cross-linked product formed by reaction of R656C with the homobifunctional maleimides may also be a consequence of the geometric requirement for maleimide cross-linking, and EC4 may be as flexible as the other loops [34].…”

“Frozen” dynamic dimer model for transmembrane signaling in bacterial chemotaxis receptors

“…4B). The 2 long helices from one monomer and those from the other form a quasi-4-helix bundle in the middle of the dimer, suggesting that the 4 transmembrane helices also form a 4-helix bundle (Milburn et al, 1991;Pakula & Simon, 1992). A 3-dimensional model of the receptor without the cytoplasmic domain is shown in Figure 5A.…”

Evidence that the Adaptation Region of the Aspartate Receptor Is a Dynamic Four-Helix Bundle: Cysteine and Disulfide Scanning Studies

“…The structures of the periplasmic and transmembrane regions are wellcharacterized, with each dominated by four-helix bundles (23)(24)(25). In the periplasmic domain, helices α1-4 of each subunit form a four-helix bundle, and the two symmetric bundles associate at the dimer interface dominated by an α1-α1′ coiled-coil interaction.…”