Transmembrane signaling by the aspartate receptor: Engineered disulfides reveal static regions of the subunit interface

Autor: Joseph J. Falke, Christina M. Lin, Stephen A. Chervitz
Rok vydání: 1995
Předmět:
Zdroj: Biochemistry. 34:9722-9733
ISSN: 1520-4995
0006-2960
Popis: The aspartate receptor of Escherichia coli, Salmonella typhimurium, and related bacteria serves as an initial chemosensor in a pathway which regulates taxis toward chemoattractants and away from repellents [reviewed by Stock and Surrette (1995), Parkinson (1993), Hazelbauer (1992), and Bourret et al. (1991)]. The periplasmic domain of this transmembrane receptor binds aspartate and transmits a conformational signal through the bilayer to its cytoplasmic domain, which together with the histidine kinase CheA and the coupling protein CheW exists as a kinetically stable ternary complex (Schuster et al., 1993; Gegner et al., 1992; Ninfa et al., 1991; Borkovich & Simon, 1989). The function of this receptor–CheW–CheA ternary complex is to phosphorylate the response regulator protein CheY in a reaction which is inhibited by ligand binding to the receptor. Ultimately, the steady-state level of phospho-CheY controls the switching of the flagellar motor between its two directions of rotation, thereby enabling chemotaxis. Homologous histidine kinase signaling pathways regulated by specific environmental or physiological stimuli are present in all prokaryotic organisms examined to date and appear to be widespread in eukaryotes as well (Alex & Simon, 1994; Ota & Varshavsky, 1993; Chang et al., 1993). The present work investigates the mechanism of transmembrane signaling by the aspartate receptor. The focus is the structural interface joining the two subunits of this homodimeric protein, where the following questions are addressed. Does the signal require a change in the association state of the dimer or a rearrangement of the packing interface between subunits within the dimer? Does the signal trigger movement of specific transmembrane helices outside the subunit interface? The answers to such questions have broader relevance, since the aspartate receptor is representative of a large class of membrane-spanning receptor proteins exhibiting direct primary and transmembrane structure homologies, found in both the prokaryotic and eukaryotic versions of histidine kinase pathways [reviewed by Ecker (1995), Stock and Surrette (1995), Alex and Simon (1994), Parkinson (1993), and Hazelbauer (1992)]. Mechanistic studies of the aspartate receptor are facilitated by the availability of extensive structural information regarding its periplasmic ligand binding and transmembrane domains, which together generate the transmembrane signal. Schematic outlines of both domains are presented in Figure 1. FIGURE 1 Schematic structure of the aspartate receptor, including the locations of the engineered cysteines. (A) Schematic representation of the periplasmic helices, looking down their long axes toward the cytoplasm (Milburn et al., 1991). The periplasmic domain ... The crystallographic structure of the periplasmic ligand binding domain (Milburn et al., 1991; Yeh et al., 1993) reveals a dimer of two identical subunits, each a four-helix bundle with nearly parallel long axes (Figure 1). Two symmetric aspartate binding sites lie at the interface between the two subunits, near the extreme periplasmic end distal to the membrane. Contacts between the two subunits are primarily between their N-terminal helices α1 and α1′ (the prime distinguishes helices from different subunits), which form a coiled-coil about the C2 rotational symmetry axis at the center of the dimer (Scott et al., 1993). The transmembrane domain of the receptor consists of four membrane-spanning helices, whose packing has been mapped by engineered disulfide studies (Scott & Stoddard, 1994; Pakula & Simon, 1992b; Lynch & Koshland, 1991; Falke et al., 1988). At the center of the transmembrane domain lie the two N-terminal helices, one from each subunit of the dimer, which begin in the cytoplasm then span the bilayer where they are labeled TM1 and TM1′. These helices emerge in the periplasm where they are believed to be continuous with the α1 and α1′ helices, respectively, lying at the subunit interface in the periplasmic domain. The remaining two transmembrane helices, TM2 and TM2′, are proposed to be extensions of the periplasmic helices α4 and α4′. respectively, and are observed to lie farther from the center of the helix cluster spanning the bilayer (Figure 1). Two different classes of models have been proposed for the conformational change triggered by aspartate: one class proposes that the two subunits move relative to one another, thereby altering the subunit interface, while the other class suggests that the signal is carried by more localized structural changes within each subunit (Lee et al., 1995; Kim, 1994; Danielson et al., 1994; Scott & Stoddard, 1994; Parkinson, 1993: Pakula & Simon, 1992a; Stoddard et al., 1992; Lynch & Koshland, 1992; Milligan & Koshland, 1991; Mowbray & Koshland, 1987). In order to probe the role of the subunit interface in transmembrane signaling, the present study engineers free cysteines and disulfide bonds into the interface, thereby generating targetted perturbations and covalent links between α1 and α1′ in the perplasmic domain, or between TM1 and TM1′ in the transmembrane domain. These alterations provide a powerful tool with which to resolve different models for the transmembrane signal. For example, models which propose significant structural rearrangements of the subunit interface (a change in the number of subunits, or movement of the subunits relative to one another) predict that the stability conferred to the interface by an engineered disulfide bond will disrupt signal transmission. In contrast, models which propose that the interface is static, such that its structure is unaltered by ligand binding, predict that an intersubunit disulfide may have little or no effect on the transmembrane signal. An in vitro method is employed to quantitate the effects of engineered cysteines and disulfides on signal transmission across the bilayer. This direct activity assay measures ligand-sensitive, transmembrane regulation of the kinase CheA in the reconstituted receptor–CheW–CheA ternary complex (Borkovich et al., 1989). The results indicate that (i) the periplasmic and transmembrane regions of the subunit interface are effectively static during the transmembrane signal, while (ii) some engineered perturbations of the interface significantly distort the receptor structure. For comparison, the effects of engineered cysteines and disulfides on in vitro methylation of the receptor are also examined. The methylation and phosphorylation results are generally in agreement, although exceptions to this rule indicate that the two assays may monitor different features of receptor structure.
Databáze: OpenAIRE
Externí odkaz: