A zipped-helix cap potentiates HAMP domain control of chemoreceptor signaling

Abstract

Environmental awareness is an essential attribute for all organisms. The chemotaxis system of provides a powerful experimental model for the investigation of stimulus detection and signaling mechanisms at the molecular level. These bacteria sense chemical gradients with transmembrane proteins [methyl-accepting chemotaxis proteins (MCPs)] that have an extracellular ligand-binding domain and intracellular histidine kinases, adenylate cyclases, methyl-accepting proteins, and phosphatases (HAMP) and signaling domains that govern locomotor behavior. HAMP domains are versatile input-output elements that operate in a variety of bacterial signaling proteins, including the sensor kinases of two-component regulatory systems. The MCP HAMP domain receives stimulus information and in turn modulates output signaling activity. This study describes mutants of the serine chemoreceptor, Tsr, that identify a heptad-repeat structural motif (LLF) at the membrane-proximal end of the receptor signaling domain that is critical for HAMP output control. The homodimeric Tsr signaling domain is an extended, antiparallel, four-helix bundle that controls the activity of an associated kinase. The N terminus of each subunit adjoins the HAMP domain; the LLF residues lie at the C terminus of the methylation-helix bundle. We found, by using in vivo Förster resonance energy transfer kinase assays, that most amino acid replacements at any of the LLF residues abrogate chemotactic responses to serine and lock Tsr output in a kinase-active state, impervious to HAMP-mediated down-regulation. We present evidence that the LLF residues may function like a leucine zipper to promote stable association of the C-terminal signaling helices, thereby creating a metastable helix-packing platform for the N-terminal signaling helices that facilitates conformational control by the HAMP domains in MCP-family chemoreceptors.

Hydrogen exchange of chemoreceptors in functional complexes suggests protein stabilization mediates long-range allosteric coupling

“…This suggests that MH2/MH2' forms the dimer interface, since MH1 shows no evidence of stable packing (log(PF) ≤ 2.4). Such a model is consistent with a recent mutagenesis study suggesting stable MH2/MH2' packing at the membrane proximal end of MH2 in the Ser receptor (25), but is difficult, however, to reconcile with the existence of disulfide crosslinked MH1 /MH1' dimers within this region that retain activity (5,26).…”

Conformational shifts in a chemoreceptor helical hairpin control kinase signaling inEscherichia coli

“…Dashed horizontal lines indicate K 1/2 values for wild-type Tsr in the UU2567 (R−B−; black line) and UU2700 (R+B+; gray line) hosts. In our laboratory, SDs for K 1/2 measurements are typically less than ±25%; those for Hill coefficients and kinase activities are typically less than ±50% (16, 26, 46, 71, 78–81). The two wild-type plasmids (pPA114 and pRR53) produced slightly different K 1/2 values in UU2700; so the dashed gray line is at the average of those two values.…”

Structure and function of the juxtamembrane GAF domain of potassium biosensor KdpD

“…The more common and better studied HAMP domain histidine kinases also have a four-helix bundle organization involved in input-output transition during signaling. 7,[39][40][41][42][43][44][45] Various structure based mechanistic models have been developed for HAMP containing effector modules invoke either helix rotation, helix tilting or bending, helix kinks or lever-like motions of the helix to accomplish conformational changes necessary to move from inactive to active states and vice versa. 42,[44][45][46][47][48][49] Crux to these models is the presence of a central four-helix bundle and the reorientation of these helices relative to each other around a 50 which introduces a His6-TeV protease site tag at the N-terminal of the translated fusion protein (Tables S1 and S2).…”

“…The more common and better studied HAMP domain histidine kinases also have a four‐helix bundle organization involved in input–output transition during signaling 7,39–45 . Various structure based mechanistic models have been developed for HAMP containing effector modules invoke either helix rotation, helix tilting or bending, helix kinks or lever‐like motions of the helix to accomplish conformational changes necessary to move from inactive to active states and vice versa 42,44–49 .…”