Stability and Conformation of a Chemoreceptor HAMP Domain Chimera Correlates with Signaling Properties

Abstract

HAMP domains are dimeric, four-helix bundles that transduce conformational signals in bacterial receptors. Genetic studies of the Escherichia coli serine receptor (Tsr) provide an opportunity to understand HAMP conformational behavior in terms of functional output. To increase its stability, the Tsr HAMP domain was spliced into a poly-HAMP unit from the Pseudomonas aeruginosa Aer2 receptor. Within the chimera, the Tsr HAMP undergoes a thermal melting transition at a temperature much lower than that of the Aer2 HAMP domains. Pulse-dipolar electron spin resonance spectroscopy and site-specific spin-labeling confirm that the Tsr HAMP maintains a four-helix bundle. Pulse-dipolar electron spin resonance spectroscopy was also used to study three well-characterized HAMP mutational phenotypes: those that cause flagella rotation that is counterclockwise (CCW) A and kinase-off; CCW B and also kinase-off; and, clockwise (CW) and kinase-on. Conformational properties of the three HAMP variants support a biphasic model of dynamic bundle stability, but also indicate distinct conformational changes within the helix bundle. Functional kinase-on (CW) and kinase-off (CCW A) states differ by concerted changes in the positions of spin-label sites at the base of the bundle. Opposite shifts in the subunit separation distances of neighboring residues at the C-termini of the α1 and α2 helices are consistent with a helix scissors motion or a gearbox rotational model of HAMP activation. In the drastic kinase-off lesion of CCW B, the α1 helices unfold and the α2 helices form a tight two-helix coiled-coil. The substitution of a critical residue in the Tsr N-terminal linker or control cable reduces conformational heterogeneity at the N-terminus of α1 but does not affect structure at the C-terminus of α2. Overall, the data suggest that transitions from on- to off-states involve decreased motional amplitudes of the Tsr HAMP coupled with helix rotations and movements toward a two-helix packing mode.

Figure 1

Schematic representations of the recombinant Tsr-Aer2 HAMP domains. (A) Domain organization of the chimeric Tsr-Aer2H1–3 protein that is composed of the Tsr HAMP (Tsr H) and the PaAer2 poly-HAMP domains (H1, H2, and H3). Residue numbers for each domain are: Aer2H1, 8–56; Aer2H2–H3, 62–172; Aer2H1–2 linker, 57–62; Tsr HAMP, 216–264. The chimera maintains the helical registers of all four HAMP domains, based on the alignment of Tsr HAMP to Aer2 H2. (B) Schematic diagram of the domain architecture of the recombinant Tsr-Aer2 HAMP domains. The Tsr HAMP domain is fused between the PaAer2H1–2 domains with the Aer2H1–2 linkers at both ends. The α-helices are depicted as cylinders. (C) Ribbon diagram showing the α-helical structure of the chimeric Tsr-Aer2H1–3 protein.

Figure 2

Helical content and thermostability of the chimeric Tsr-Aer2H1–3 protein compared to that of Aer2H1–3. (A) The CD wavelength scans show the overall α-helical structure of Tsr-Aer2H1–3 and Aer2H1–3 alone. (B) The CD melting curves show the two-step unfolding character of the chimeric protein. An additional transition in the chimera at 26°C is not present in the Aer2H–13 domain, which has a melting temperature of 53°C.

Figure 3

Structural properties of the Tsr HAMP domain as revealed by PDS. (A) Intersubunit spin-probe distances of nitroxide-labeled proteins measured by PDS. Chosen sites are marked on the α1 (light blue) and α2 (dark blue) helices of the Tsr HAMP domain. Pairwise distance distributions [P(r)] were generated for each pair of spins on adjacent subunits. Based on sequence alignments (12, 28), the heptad positions of the spin-label sites are defined as follows—α1: 220, f; 224, c; 230, b; 232, d; and α2: 250, b; 254, f; 258, c; 260, e; 261, f. (B) Homology model of the Tsr HAMP domain. The atomic coordinates for the model were obtained by threading the Tsr HAMP sequence onto the Af1503 A291F HAMP using SWISS-MODEL. (C) Ribbon diagram. The Tsr HAMP domain is depicted as a dimeric four-helix bundle with the α1 helices in light blue, the α2 helices in dark blue, and the CTR in gold. The spin-label positions are shown on only one subunit; α1 on the left and α2 on the right. (D) Helical wheel representation of the heptad repeat positions of a parallel four-helix bundle with a-d, knobs-into-holes, packing.

Figure 4

Effects of single residue substitutions on the secondary structure and thermal stability of the Tsr HAMP domain. (A) The CD spectra of the recombinant HAMP domains containing the WT Tsr HAMP (blue) compared to the E248L (red), M222P (green), and A233P (black) variants. The recombinant proteins with the WT Tsr, E248L, and A233P variants maintain their overall α-helical structure. The M222P variant shows the most reduced helical content. (B) CD thermal melts of the variant Tsr-Aer2H1–3 proteins. The E248L and A233P mutations do not affect the two-step unfolding of the recombinant protein, but have altered transition temperatures. The E248L variant has melting transitions at 33 and 54°C, and the A233P variant has transitions at 27 and 48°C. In contrast, the M222P variant unfolds in a single step at 39°C. (Inset) Upper-right panel shows a schematic representation of the dynamic bundle model of HAMP domain activity (adapted from the literature (9, 28, 32)). The measured relative stabilities against thermal unfolding for the WT and variant HAMP domains are mapped onto a hypothetical bell-shaped curve relating helical bundle stabilities to physiological output.

Figure 5

Effects of the nonfunctional kinase-off [CCW(B)] substitution on the structural properties of the Tsr-Aer2H1–3 chimeric protein. (A) PDS distance distributions for the Tsr WT (blue) and M222P – CCW(B) (green). Schematic diagrams for the Tsr HAMP are marked with the spin-label sites. (B) Ribbon diagrams showing the position of M222P on α1 in the hydrophobic core of the Tsr.

Figure 6

Effects of the functional kinase-off [CCW(A)] and kinase-on (CW) substitutions on the structural properties of the Tsr-Aer2H1–3 chimeric protein. (A) PDS distance distributions. Schematic diagrams for the Tsr HAMP are marked with the spin-label sites. Distance distributions [P(r)] measured by PDS are shown for the Tsr WT (blue) and variants E248L – CCW(A) (red), and A233P – CW (black). (B) Schematic diagram summarizing the possible conformational changes relating CCW(A) and CW as inferred by the four spin-label positions at the base of α1 and α2. For α1, increase in the d-d separation and decrease in the b-b separation suggest a counterclockwise rotation in switching from the CCW(A)-kinase-off state to the CW kinase-on state. For α2, decrease in the e-e separation indicates a corresponding clockwise rotation. The c-c position of α2 shows little change, probably due to breadth of the distribution. (C) Ribbon diagrams showing the positions of E248 at the very N-terminus of α2 in a predicted f position and A233 at an e position on the N-terminus of α1.