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 alpha-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-CpxA-NarX-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.

FIG. 1.

HAMP linkers in this study. The single-letter amino acid sequences of the three HAMP linkers used in this study are shown. Sequences are aligned about the Pro at the N-terminal end of AS-1 and the Glu at the N-terminal end of AS-2; these residues are underlined. The predicted junction between TM-2 and cytoplasmic amino acid sequences is at the left, and the aminoacyl residue number at the C-terminal end of the HAMP linker is shown at the right for reference. The hydrophobic heptad repeat characteristic of HAMP linkers is shown, with hydrophobic positions capitalized and hydrophobic residues shown in boxes. The extents of the amphipathic sequences 1 and 2 (AS-1 and AS-2) are shown by bold lines, and the connector is shown by a light dotted line. It is not clear whether sequences upstream of the conserved Pro in AS-1 form a continuous α-helix with AS-1; these are indicated by a bold dashed line. Positions in AS-1 and AS-2 are numbered for reference, and deletions analyzed in this study are illustrated by black rectangles.

FIG. 2.

Structures of narX and narQ plasmids. See Materials and Methods for details of construction. The narX (gray) and narQ (white) genes are depicted as rounded rectangles to show the coding regions for the corresponding modules and the HAMP linker. Nucleotide positions of restriction endonuclease sites are indicated. Only selected sites are depicted for each plasmid. The narX transcription initiation point (V. Stewart and P. J. Bledsoe, unpublished data) is indicated by the arrow.

FIG. 3.

Hybrid sensor kinases with NarX sensor modules and CpxA output modules. The single-letter amino acid sequences of the HAMP linkers of hybrid sensor kinases utilizing the NarX sensor module and the CpxA output module are shown. Sequences from NarX are in plain type; sequences from CpxA are on a gray background; aminoacyl residues not native to either NarX or CpxA used for building the hybrids are boxed. The (arbitrarily assigned) endpoints of AS-1 and AS-2 are denoted by dashed lines, and the coordinates of positions within AS-1 and AS-2 are indicated as in Fig. 1. β-Galactosidase specific activity of the ΔcpxA Φ(cpxP-lacZ) strain VJS7220 expressing the indicated hybrid from a plasmid in the presence (+ NO₃⁻) or absence (− NO₃⁻) of 40 mM NaNO₃ is indicated. Approximately the same β-galactosidase activity was seen for strain VJS7220 carrying an empty plasmid vector as with a plasmid expressing narX (data not shown). For details of culture growth during assays, see Materials and Methods.

FIG. 4.

Hybrid sensor kinases containing elements of NarX, NarQ, and CpxA. The single-letter amino acid sequences of the HAMP linkers of hybrid sensor kinases utilizing the NarX or NarQ sensor module and the NarQ or NarX output module are shown. Sequences from NarX are in plain type, sequences from NarQ are on a gray background, and sequences from CpxA are in white on a black background. The (arbitrarily assigned) endpoints of AS-1 and AS-2 are denoted by dashed lines, and the coordinates of positions within AS-1 and AS-2 are indicated. β-Galactosidase specific activity of the ΔnarX narQ Φ(narG-lacZ) strain VJS5054 expressing the indicated hybrid from a plasmid in the presence (+ NO₃⁻) or absence (− NO₃⁻) of 40 mM NaNO₃ is indicated. The specific β-galactosidase activity of VJS5054 carrying an empty plasmid vector was <10 Miller units (data not shown). For details of culture growth during assays, see Materials and Methods.

FIG. 5.

The HAMP linker in NarX, NarQ, CpxA, and hybrid sensor kinases. Sensor kinases which responded to nitrate in this study are depicted in cartoon form. The scale is in aminoacyl residues, and amino (N) and carboxy (C) termini are indicated. Modules or elements from NarX, NarQ, and CpxA are represented by gray, white, and black rounded rectangles, respectively. The absence of the central module in CpxA and the NarX-Cpx-1 and -2 hybrids is denoted by the dashed line. The primary structures of NarX-CpxA-1 and -2 differ by one amino acid, as do those of NarX-CpxA-NarX-1 and -2; the phenotypes of the two variants of each hybrid differ quantitatively but not qualitatively. The activity listed under “ligand bound” indicates the dominant activity of the sensor kinase in the presence of the ligand, nitrate. The conclusion is that the ligand-bound activity of these sensor kinases is determined not by the HAMP linker but by the transmitter module and/or the central module.