The tie that binds the dynamic duo: the connector between AS1 and AS2 in the HAMP domain of the Escherichia coli Tsr chemoreceptor.

A phospholipid bilayer membrane provides a selective permeability barrier that separates a cell’s world into “me” and “not me.” A large fraction of a bacterial genome is devoted to encoding proteins that allow only the desired molecules to breach this barrier and become concentrated in, or excluded from, the intracellular space. The membrane also filters information about the environment using transmembrane receptors to communicate what is important to the cell. Transmembrane signaling is therefore one of the key biological activities to understand. In this issue, Ames et al. (1) present a high-resolution genetic analysis of one critical 14-residue segment of the Escherichia coli Tsr chemoreceptor. Their results suggest that a detailed mechanistic understanding of one type of transmembrane signaling is within reach. Bacterial chemotaxis remains the system of choice for studying a number of fundamental questions about sensory transduction, partly because of the simplicity of the behavior and partly because of the tractability of bacteria as experimental subjects. E. coli cells swim in a three-dimensional random walk that consists of several seconds of swimming in gentle curves, called runs, which are punctuated by brief intervals ( 0.1 s) of rapid, undirected reorientation, called tumbles. Runs correspond to the counterclockwise (CCW) rotation of all four to six left-handed helical flagellar filaments. Tumbles occur when at least one flagellum in the bundle rotates clockwise (CW). Runs toward higher attractant concentrations are extended because tumbles are suppressed, and the random walk is thus biased in a favorable direction (for a review of chemotaxis, see reference 17). Bacterial chemoreceptors can be thought of as transmembrane allosteric enzymes. In Tsr, the regulatory site at which serine binds is near the apex of the extracellular (periplasmic) domain, and the catalytic subunit is the CheA kinase bound to the membrane-distal tip of the cytoplasmic domain (Fig. 1). These two regions are separated by a phospholipid bilayer and about 350 Å, a large distance on the molecular scale. Tsr is a homodimer, and serine binds at the dimer interface. The critical conformational change associated with serine binding is probably a downward, 1to 2-Å piston-like displacement of the second transmembrane helix (TM2) into the cytoplasm. This event translates into a thousandfold decrease in the receptorstimulated activity of CheA, which phosphorylates the response regulator CheY. The binding of phospho-CheY to flagellar motors promotes CW rotation, and decreased CheA activity inhibits tumbling and lengthens up-gradient runs (for a review of chemoreceptor function, see reference 8). To migrate in an attractant gradient, a cell must continually adjust its sensitivity. Serine binding increases the rate of methylation of certain glutamate residues in the adaptation domain, whose negative charge is thereby neutralized. As a result, the stimulation of CheA is restored, the serine-induced displacement of TM2 is reversed (12), and the affinity of the regulatory site for serine (13) decreases, so the cells can now respond to further increases in serine concentration. How can serine binding, manifested as a modest displacement of TM2, lead to drastic changes in the output activity of the receptor? A large part of the answer lies within the HAMP domain (hereafter simply HAMP), which connects TM2 to the rest of the cytoplasmic domain. HAMP contains two amphipathic helices of about 18 residues (6) joined by a flexible connector of 14 residues (Fig. 2A). HAMPs are widely distributed linkers of functional domains of homodimeric proteins in many organisms (4, 9), and they are often found immediately after a membrane-spanning helix. In Tsr, HAMP serves as a two-way conduit for information passed between the extracellular and intracellular domains. A nuclear magnetic resonance structure of the Af1503 HAMP from the thermophilic archaeon Archeoglobus fulgidis (11) reveals a parallel four-helix bundle in which the connector nestles up against the bundle. The helices show knob-on-knob (x-da) packing rather than the more usual knob-in-hole (a-d-a) packing. Tsr HAMP has been modeled based on the Af1503 structure (Fig. 2B). Interactions of the connector with the bundle may stabilize knob-on-knob packing and serve as a regulator of receptor activity. Ames et al. (1) thus focused their attention on the connector. They began with the premise that Tsr behaves as a two-state device, existing either in an “on” (CheA-stimulating) or “off” (CheA-inhibiting) form (2, 5). The equilibrium between these two states, both within an individual receptor and within the receptor patch (10, 14), determines the overall receptor activity and, thus, the intracellular level of phospho-CheY, to which the motors are exquisitely sensitive (7). If the connector stabilizes one or more conformations of HAMP, rather subtle changes could have profound effects on receptor function. Accordingly, each of the 14 residues in the connector (positions 234 through 247) was randomized by degenerate codon mutagenesis, and 179 of the 266 possible single-residue substitutions were identified. All of the mutant receptors were then tested for their ability to mediate serine taxis. Only four connector residues appear to be specifically important for TSR connector function (Fig. 2C). All four residues are similar or identical between Tsr and the closely related Tar (aspartate) receptor (Fig. 2A). One, Gly-245, is part * Mailing address: Department of Biology, Texas A&M University, 3258 TAMU, College Station, TX 77843. Phone: (979) 845-5158. Fax: (979) 845-2891. E-mail: [email protected]. Published ahead of print on 15 August 2008.