Structure of the Conserved HAMP Domain in an Intact, Membrane-Bound Chemoreceptor:  A Disulfide Mapping Study

Abstract

The HAMP domain is a conserved motif widely distributed in prokaryotic and lower eukaryotic organisms, where it is often found in transmembrane receptors that regulate two-component signaling pathways. The motif links receptor input and output modules and is essential to receptor structure and signal transduction. Recently, a structure was determined for a HAMP domain isolated from an unusual archeal membrane protein of unknown function [Hulko, M., et al. (2006) Cell 126, 929-940]. This study uses cysteine and disulfide chemistry to test this archeal HAMP model in the full-length, membrane-bound aspartate receptor of bacterial chemotaxis. The chemical reactivities of engineered Cys residues scanned throughout the aspartate receptor HAMP region are highly correlated with the degrees of solvent exposure of corresponding positions in the archeal HAMP structure. Both domains are homodimeric, and the individual subunits of both domains share the same helix-connector-helix organization with the same helical packing faces. Moreover, disulfide mapping reveals that the four helices of the aspartate receptor HAMP domain are arranged in the same parallel, four-helix bundle architecture observed in the archeal HAMP structure. One detectable difference is the packing of the extended connector between helices, which is not conserved. Finally, activity studies of the aspartate receptor indicate that contacts between HAMP helices 1 and 2' at the subunit interface play a critical role in modulating receptor on-off switching. Disulfide bonds linking this interface trap the receptor in its kinase-activating on-state, or its kinase inactivating off-state, depending on their location. Overall, the evidence suggests that the archeal HAMP structure accurately depicts the architecture of the conserved HAMP motif in transmembrane chemoreceptors. Both the on- and off-states of the aspartate receptor HAMP domain closely resemble the archeal HAMP structure, and only a small structural rearrangement occurs upon on-off switching. A model incorporating HAMP into the full receptor structure is proposed.

Differential Role of HAMP-like Linkers in Regulating the Functionality of the Group III Histidine Kinase DhNik1p

“…Our shape-function studies promote that regulation of the histidine kinase activity by the upstream poly-HAMP module could be through an in-line mechanism than the side-on interaction as suggested earlier (9). Several recent studies indicated variations in the HAMP domain structure arising out of different helix rotation, helix translation, and helix-helix crossing angle (12,13,27). Similarly, a different mechanism of signal propagation through HAMP domains had been proposed.…”

“…Structurally, HAMP domains are arranged as symmetric homodimeric parallel coiled coils with each protomer having two ␣-helices, AS1 and AS2, connected by a flexible linker segment (12,13,27,37). The four-helix bundle is held together by a hydrophobic core.…”

“…HAMP domains have been established as signal transduction modules in prokaryotic sensor kinases, where single HAMP domains connect a membrane bound sensing (input) domain to the cytoplasmic output domain. Genetic and structural studies on bacterial transmembrane sensor kinases suggested stimulus induced conformational changes in the HAMP domain structure as the mode of signal transmission (12,13). Nik1 orthologs have been identified from both pathogenic and nonpathogenic fungi.…”

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

“…6) (see Fig. 1A) and has since been confirmed in HAMP domains from other proteins (13)(14)(15). The Af1503 HAMP domain adopts an unusual complementary x-da conformation that is related to the canonical coiled coil form by a 26°axial rotation of all four helices (Fig.…”

Bacterial chemoreceptor dynamics correlate with activity state and are coupled over long distances

“…The HAMP AS2 helices may undergo a scissoropening motion when transitioning from kinase off to kinase on (14,19). Attractant-sensitive disulfide bond formation between CheA and receptor suggests that the N-terminal CD1 helices rotate in the PIR on ligand binding (20).…”