Probing Conservation of HAMP Linker Structure and Signal Transduction Mechanism through Analysis of Hybrid Sensor Kinases

Abstract

The HAMP linker, a predicted structural element observed in many sensor kinases and methyl-accepting chemotaxis proteins, transmits signals between sensory input modules and output modules. HAMP linkers are located immediately inside the cytoplasmic membrane and are predicted to form two short amphipathic ␣-helices (AS-1 and AS-2) joined by an unstructured connector. HAMP linkers are found in the Escherichia coli nitrate-and nitrite-responsive sensor kinases NarX and NarQ (which respond to ligand by increasing kinase activity) and the sensor kinase CpxA (which responds to ligand by decreasing kinase activity). We constructed a series of hybrids with fusion points throughout the HAMP linker, in which the sensory modules of NarX or NarQ are fused to the transmitter modules of NarX, NarQ, or CpxA. A hybrid of the NarX sensor module and the CpxA HAMP linker and transmitter module (NarX-CpxA-1) responded to nitrate by decreasing kinase activity, whereas a hybrid in which the HAMP linker of NarX was replaced by that of CpxA (NarX-CpxANarX-1) responded to nitrate by increasing kinase activity. However, sequence variations between HAMP linkers do not allow free exchange of HAMP linkers or their components. Certain deletions in the NarX HAMP linker resulted in characteristic abnormal responses to ligand; similar deletions in the NarQ and NarX-CpxA-1 HAMP linkers resulted in responses to ligand generally similar to those seen in NarX. We conclude that the structure and action of the HAMP linker are conserved and that the HAMP linker transmits a signal to the output domain that ligand is bound.Two-component regulatory systems are used by all domains of life to sense and respond to environmental changes (48). Many of the sensor components of two-component regulatory systems and most methyl-accepting chemotaxis proteins (MCPs) share a common molecular architecture. These proteins are dimers in the cytoplasmic membrane and have a modular structure. The sensory module of each monomer consists of a short, amino-terminal cytoplasmic tail, a transmembrane ␣-helix (TM-1), a periplasmic domain which may interact with a specific ligand, a second transmembrane ␣-helix (TM-2), and, in many but not all sensor proteins, a HAMP linker. The cytoplasmic output module interacts with a response regulator (14). Signal transduction requires that information about ligand binding in the exterior sensory input module of the protein cross the cytoplasmic membrane and regulate the activity of the output module in the interior of the cell. The output modules of MCPs interact with an associated soluble histidine kinase, CheA, whereas those of the two-component sensors are histidine kinases in equilibrium between kinase and phosphatase activities.Many of these sensors appear to use a common structural element for signal transduction between external inputs and cytoplasmic outputs. The HAMP (histidine kinase, adenylyl cyclase, MCP, and phosphatase) linker or P-type linker is predicted to be a structure of approximately 50 aminoacyl residues connecting ...

The HAMP Linker in Histidine Kinase Dimeric Receptors Is Critical for Symmetric Transmembrane Signal Transduction

“…Our previous study (12) has suggested that specific interactions between the two predicted helices in the HAMP linker are critical for the linker function, as the two helices have to be derived from the same protein for construction of a functional Tar-EnvZ hybrid receptor. A similar conclusion was drawn from a recent report characterizing a set of NarX-NarQ, NarX-CpxA, and NarQ-CpxA hybrid receptors (13). Our present data further support the previous proposal that the HAMP linker adapts a four-helix bundle structure in a dimer receptor as shown in Fig.…”

“…In Tez1A1 and Tez1PQ, the sensor module of Tar is fused to the transmitter module of EnvZ using the EnvZ linker with one Ala insertion at the N-terminal end and the EnvZ linker with a Pro to Gln mutation at position 185 (the position number is based on EnvZ sequence), respectively (12). A number of functional hybrid histidine kinases have also been constructed by fusing the sensor module of one histidine kinase with the transmitter module of another histidine kinase at the HAMP linker region (NarQ-NarX, NarX-NarQ, and NarX-CpxA fusions) or by replacing the HAMP linker of one histidine kinase (NarX) with the HAMP linker of another histidine kinase (CpxA) (13). In addition, functional hybrid chemoreceptors have been constructed in which Tar-Tsr (14), Tap-Tar (15), and Trg-Tsr (16) fusions are made using the Tsr, Tar, and Tsr linkers, respectively, and a Aer-Tsr fusion is made using the Aer linker (17).…”

LapD is a bis-(3′,5′)-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0–1

“…These domains typically relay signals across the cytoplasmic membrane, altering their conformation in response to activation of an extracellular input domain and propagating that conformational change to a cytoplasmic output domain (20). Structure/function studies of HAMP-domain proteins have shown that mutations in this domain can have profound effects, constitutively activating or inactivating signaling output (23). To investigate a role for this domain in regulating LapD function, we mutated E203, one of the few amino acids that are well conserved among all HAMP domains (24).…”

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

“…One of these regions is the HAMP domain, first found in histidine kinases, adenylyl cyclases (ACs), 2 methyl-accepting chemotaxis proteins, and phosphatases (3) but also present in many other proteins (4). A co-evolution between individual families of HAMP domains and output domains is evident (4) and several examples in which HAMP domains were swapped without loss of function suggest a common mechanism for signal propagation (5)(6)(7)(8).…”