Signal transduction in histidine kinases: insights from new structures

Abstract

Histidine kinases (HKs) are major players in bacterial signaling. There has been an explosion of new HK crystal structures in the last 5 years. We globally analyze the structures of HKs to yield insights into the mechanisms by which signals are transmitted to and across protein structures in this family. We interpret known enzymological data in the context of new structural data to show how asymmetry across the dimer interface is a key feature of signal transduction in HKs, and discuss how different HK domains undergo asymmetric to symmetric transitions during signal transduction and catalysis. A thermodynamic framework for signaling that encompasses these various properties is presented, and the consequences of weak thermodynamic coupling are discussed. The synthesis of observations from enzymology, structural biology, protein engineering, and thermodynamics paves the way for a deeper molecular understanding of HK signal transduction.

Figure 1. Two-component system architecture

(A) Schematic representation of a canonical TCS. Many of these domains are repeated in other protein classes. The DHp is the catalytic core of Histidine Kinases. (B) Variable domain structures from three sensor and signal transducing domains colored to highlight key features. The yellow helix (top) shows the dimeric interface. The white and black helices (bottom) show input and output helices. (C) Structures of conserved domains, DHp and catalytic, colored to highlight various features. DHp dimeric structure (top) has the N-terminal helix colored in orange and the C-terminal helix in blue. Critical regions for function are labeled and color-coded in the CA structure (bottom).

Figure 2. Catalytic core of Histidine Kinases

A) Surface representation of the catalytic domain (green) and the response regulator (blue) are shown on their respective docking sites on the DHp bundle (left). Structural alignment of DHp bundles from various HKs (right) show that the core of the bundle is highly symmetric and conserved while the ends of the bundle vary. B) A sequence logo for the helices of DHp domains (based on Pfam 00512) with conserved positions. An acidic residue always follows the catalytic histidine. There is a loop of variable length between the two helices. C) A schematic showing how the handedness of the loop between the two DHp helices determines auto-phosphorylation geometry in the DHp bundle.

Figure 3. Asymmetry in CA-DHp distances

A) The distances between the catalytic His (on the DHp) and an ATP-binding Asn (on the CA) on each side of the dimer are correlated for all known HK structures. Structures representing the autophosphorylation Michaelis complex show significant asymmetry B) The DHp-CA distance is correlated with the angle between the Gripper helix and the DHp helix. The Gripper helix shows a very specific angular preference in in the autokinase Michaelis structures.

Figure 4. Nucleotide dependent placement of the Gripper helix

Structural representations of the DHp and CA domains are shown in ADP- (left) and ATP-(right) bound states show different associations with the Gripper. On the inactive side, Gripper residues interact with the DHp stem to form a 3 helix bundle, with the Phe (cyan) wedged into the dimeric interface. This conformation is also seen on both monomers in the ADP-bound state. On the active side, Gripper is released to facilitate autokinase activity. This conformation is only seen in one monomer of the ATP-bound state. DHp helices are colored as before. Gripper residues are conserved across several HKs.

Figure 5. Asymmetry in the DHp bundle

A) 23 structures of DHp domains were structurally aligned such that the z-axis coincided with the dimer axis (see supplement). X,Y coordinates extracted at 4 different slices across the bundle show that the core of the bundle is highly invariant whereas the top of the bundle varies significantly. Inter-monomer Cα-Cα distance distributions are also shown. B) Structures that represent ADP-bound, Apo and inactive states show a symmetric diamond like geometry (blue) in the XY coordinates at slice 4, whereas ATP-bound Michaelis complex structures show a distorted kite-like geometry (red). C) A third correlation plot of distances from the central helical axis to chain A vs. chain B shows that slice 2 is highly symmetric whereas slice 4 is asymmetric.

Figure 6. HAMP Domains

A) Sequence alignment of six HAMP domains of known structure show a conserved Gly and a semi-conserved Glu. B) Three different mechanisms proposed for HAMP signaling are described in cartoon form. C) An aligned ensemble of 26 HAMP domains was used to extract XY coordinates and distance distributions at 2 locations as in Fig. 5. The distribution for helix 1 (orange) is skewed outwards at the bottom of the bundle whereas helix 2 is skewed inwards (blue). D) The negative slope of the correlation between intermonomer Cα-Cα distances show that displacements in helix 1 and 2 are anti-correlated. E) The two overlaid structures, from Af1503 (2LFR,dark) and Aer2 (1I44, light) show a diagonal displacement of helices.

Figure 7. Symmetry-asymmetry transitions through polar linkers

A) Polar clusters located a few residues above the catalytic histidine are present in several HKs. Alternate packing arrangements of the polar clusters result in asymmetric bending of the linker as seen in structures of the temperature-sensitive DesK (B, left) and Af1503-EnvZ chimera (C, left). The asymmetry profiles (B,C right) plot the Cα RMSD of computed over a 7-residue moving window for chains A and B superimposed on chains B and A respectively, and the Cα-Cα distance for each residue across the dimer. There is striking asymmetry in the linker in many HK structures.

Figure 8. The TM and periplasmic sensory elements

A) Two structural states of the transmembrane bundle of PhoQ inferred using Bayesian modeling of disulfide crosslinking data. The states differ in their helical packing by a diagonal or reciprocal displacement of the helices. B) A simplified cartoon of three proposed models for conformational changes in the sensor domain. C) The magnitude of the displacement in the z and xy coordinates of the p-helices of Tar (left) and DctB (right) upon ligand binding. A small z-shift (pistoning) and a larger xy-shift (scissoring) are seen in both cases as the helix enters the membrane.

Figure 9. A thermodynamic framework for signal transduction

A) shows a single domain that has two states, an active state (X) or an inactive state (0). The population of the active state is directly related to the energy difference between these two states i.e. the equilibrium constant. B) shows two thermodynamically coupled domains. The fraction of the active state depends on two equilibrium constants and a coupling energy. C) extends this formalism to three linked domains. In the limit of rigid, all-or-nothing coupling between the domains, the system reduces to a 2 state system but in the limit of weak coupling, multiple intermediates contribute to the active population.