Different Conformations of the Kinase-On and Kinase-Off Signaling States in the Aer HAMP Domain

Abstract

HAMP domains are sensory transduction modules that connect input and output domains in diverse signaling proteins from archaea, bacteria, and lower eukaryotes. Here, we employed in vivo disulfide crosslinking to explore the structure of the HAMP domain in the Escherichia coli aerotaxis receptor Aer. Using an Aer HAMP model based on the structure of Archaeoglobus fulgidus Af1503-HAMP, the closest residue pairs at the interface of the HAMP AS-1 and AS-2 helices were determined and then replaced with cysteines and cross-linked in vivo. Except for a unique discontinuity in AS-2, the data suggest that the Aer HAMP domain forms a parallel four-helix bundle that is similar to the structure of Af1503. The HAMP discontinuity was associated with a segment of AS-2 that was recently shown to interact with the Aer-PAS sensing domain. The four-helix HAMP bundle and its discontinuity were maintained in both the kinase-on and kinase-off states of Aer, although differences in the rates of disulfide formation also indicated the existence of different HAMP conformations in the kinase-on and kinase-off states. In particular, the kinase-on state was accompanied by significantly increased disulfide formation rates at the distal end of the HAMP four-helix bundle. This indicates that HAMP signaling may be associated with a tilting of the AS-1 and AS-2 helices, which may be the signal that is transmitted to the kinase control region of Aer.HAMP domains (which are found in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, phosphatases [4], and some diguanylate cyclases and phosphodiesterases [12]) are signal transduction modules that transduce diverse input signals into output signals in proteins from archaea, bacteria, and lower eukaryotes (12,25). Currently, more than 12,000 proteins are predicted to contain HAMP domains, and many of these proteins regulate two-component signaling pathways (SMART [http://smart.embl-heidelberg.de/]). The abundance of these domains, and their conservation across a wide range of species, argues for a strategic role for HAMP domains in signaling. This universal role and their mechanism of action are the focus of extensive current research.In Escherichia coli, methyl-accepting chemoreceptors like Tsr and Tar each contain two subunits with two transmembrane helices, the second of which links a periplasmic sensing module with cytosolic HAMP and signal output (kinase control) domains. When an attractant molecule binds to the periplasmic sensing domain of these receptors, transmembrane helix 2 (TM2) is displaced ϳ2 Å toward the cytoplasm, shifting the conformation of the HAMP domain to the signal-off conformation (reviewed in reference 13). In contrast with Tsr and Tar, the E. coli aerotaxis receptor Aer lacks a periplasmic domain. Aer is anchored in the membrane, but its sensing and HAMP domains are cytosolic (31) (Fig. 1). The cytoplasmic sensor is an N-terminal PAS (Per-ARNT-Sim) (24) domain with a bound flavin adenine dinucleotide (FAD) cofactor that monitors intracellular redox po...

Mechanistic Insights Revealed by the Crystal Structure of a Histidine Kinase with Signal Transducer and Sensor Domains

“…This model was further supported by a series of experimental structures of the HAMP mutants and detailed bioinformatics analyses [15],[16]. Several other laboratories reached a consensus conclusion using homologous HAMP domains from different TCSs that the dynamic properties of the HAMP domains are essential in mediating signal transduction [17]–[22].…”

Structure and Proposed Mechanism for the pH-Sensing Helicobacter pylori Chemoreceptor TlpB

“…Extensive studies of transmembrane receptors of the chemotaxis and histidine kinase families have led to the generally accepted view that signal transmission across the membrane consists of a piston-like motion of transmembrane helix 2 (tm2) relative to tm1, with amplitude 1–2Å (Chervitz and Falke, 1996; Cheung and Hendrickson, 2009; Falke and Erbse, 2009; Moore and Hendrickson, 2009; Ottemann et al, 1999). Within the cytoplasm, the HAMP domain evidently constitutes a switch region that translates the piston-like motion into a different type of transition within the distal portions of the receptor (see recent summary by Watts et al and references therein (Watts et al, 2011)).…”

The HAMP Signal Relay Domain Adopts Multiple Conformational States through Collective Piston and Tilt Motions

“…[11], Falke et al confirmed the NMR structure of Af1503-HAMP as a structural template for the Tar HAMP domain and proposed, based on activity studies of Tar, a pivot model in which an initial piston motion may be able to tilt the helices from different subunits of HAMP with respect to each other. Helical tilting is also proposed as a model for signal relay based on in vivo cross-linking studies of a HAMP domain in the membrane based Aer sensor monitoring the intracellular redox potential [17]. Interestingly, this study found that the N-terminal helix in one monomer tilts in concert with the C-terminal helix in the other monomer.…”

“…Several models exist to describe the functional motions involved in the signal transduction mechanism of HAMP, including the gearbox model [3], the piston model [14]–[16] and a model describing helical tilting [11], [17]. Hulko et al compared the complementary packing mode of the prototype structure and the knobs-into-holes packing of a typical coiled-coil structure, showing that a concerted helix rotation by would convert the conformation into the canonical packing [3].…”