Transmembrane Signaling in Chimeras of the Escherichia coli Aspartate and Serine Chemotaxis Receptors and Bacterial Class III Adenylyl Cyclases

Abstract

The Escherichia coli chemoreceptors for serine (Tsr) and aspartate (Tar) and several bacterial class III adenylyl cyclases (ACs) share a common molecular architecture; that is, a membrane anchor that is linked via a cytoplasmic HAMP domain to a C-terminal signal output unit. Functionality of both proteins requires homodimerization. The chemotaxis receptors are well characterized, whereas the typical hexahelical membrane anchor (6TM) of class III ACs, suggested to operate as a channel or transporter, has no known function beyond a membrane anchor. We joined the intramolecular networks of Tsr or Tar and two bacterial ACs, Rv3645 from Mycobacterium tuberculosis and CyaG from Arthrospira platensis, across their signal transmission sites, connecting the chemotaxis receptors via different HAMP domains to the catalytic AC domains. AC activity in the chimeras was inhibited by micromolar concentrations of L-serine or L-aspartate in vitro and in vivo. Single point mutations known to abolish ligand binding in Tar (R69E or T154I) or Tsr (R69E or T156K) abrogated AC regulation. Co-expression of mutant pairs, which functionally complement each other, restored regulation in vitro and in vivo. Taken together, these studies demonstrate chemotaxis receptor-mediated regulation of chimeric bacterial ACs and connect chemical sensing and AC regulation.The canonical chemotaxis receptors Tsr for serine and Tar for aspartate of Escherichia coli have been studied extensively as model signaling systems that regulate bacterial swimming behavior (1-5). Ligand binding to the periplasmic domain of the receptor initiates a biochemically defined cascade of events that finally results in reversal of the flagellar beat (4, 6). The chemotaxis receptors are tripartite proteins with a periplasmic receptor anchored in the cytoplasmic membrane by two transmembrane spans followed by a HAMP domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins of chemotaxis, and protein phosphatases (7)) which adjoins the second transmembrane helix and serves as a signal converter followed by an output domain as C terminus (3). Homodimerization of Tsr or Tar chemoreceptors is required for function, and the ligands bind in the periplasmic interface between the subunits (8 -11). The ubiquitous HAMP domains are signal converters of 55-60 amino acids with a homodimeric fourhelical parallel coiled-coil conformation (12). In the chemotaxis receptors the HAMP domain operates as a C-terminal histidine kinase control unit that has no known intrinsic enzymatic activity. As this molecular architecture is predominant in the 30 known sensor kinases of E. coli, a variety of chimeric sensor proteins has been generated successfully in the past that consist of modules derived from different E. coli sensors (see examples in Refs. 13-15).A similar tripartite domain architecture as in chemotaxis receptors exists in several class III AC 2 isoforms in eubacteria. Class III AC isoforms outnumber all others, are prevalent in bacteria, and include all eukaryotic ACs (16 -17). ...

HAMP Domain-mediated Signal Transduction Probed with a Mycobacterial Adenylyl Cyclase as a Reporter

“…These chimeras were regulated in vitro as well as in vivo by their respective ligands L-serine and L-aspartate (17). The biochemical properties of these chemotaxis receptors such as ligand specificity and sensitivity, responses to previously characterized targeted receptor mutations, and requirement for dimerization were identical to those determined in chemotaxis experiments (17). In addition, functionality of the chimeric constructs was demonstrated in vivo (17).…”

“…The chimeras Tsr 1-215 -Tsr HAMP 216 -268 -Rv3645 CHD 331-549 (17) and Tsr 1-215 -Af1503 HAMP 278 -331 -Rv3645 CHD 331-549 (16) were used as templates. Point mutations were introduced by site-directed mutagenesis using restriction sites located nearby or by fusion PCR using corresponding sense and antisense primers (from MWG Biotech and Biomers).…”

“…Bacteria were washed with 50 mM Tris/HCl, 1 mM EDTA, pH 8, and stored at Ϫ80°C. Membrane fractions were prepared as previously described (17) and assayed for AC activity. The integrity of all recombinant proteins was examined by Western blotting using a purified mouse monoclonal RGS-His 4 antibody (Qiagen).…”

“…Formerly, functional chimeras were generated in which the sensor parts of the serine (Tsr) or the aspartate chemotaxis receptors (Tar) were connected via different HAMP domains (from Tsr, Tar, and Rv3645) to the CHD of Rv3645 (17). These chimeras were regulated in vitro as well as in vivo by their respective ligands L-serine and L-aspartate (17).…”

Engineering adenylate cyclases regulated by near-infrared window light

“…2A). The involvement of α-helices in mediating intramolecular signal transduction is not unique to bacteriophytochromes, and other researchers have used such helices for heterologous domain replacements (43)(44)(45)(46)(47)(48)(49).…”