Dynamics and activation in response regulators: the β4-α4 loop

Abstract

Two-component signal transduction systems of microbes are a primary means to respond to signals emanating from environmental and metabolic fluctuations as well as to signals coordinating the cell cycle with macromolecular syntheses, among a large variety of other essential roles. Signals are recognized by a sensor domain of a histidine kinase which serves to convert signal binding to an active transmissible phosphoryl group through a signal-induced ATP-dependent autophosphorylation reaction directed to histidine residue. The sensor kinase is specifically mated to a response regulator, to which it transfers the phosphoryl group that activates the response regulator's function, most commonly gene repression or activation but also interaction with other regulatory proteins. Two-component systems have been genetically amplified to control a wide variety of cellular processes; for example, both Escherichia coli and Pseudomonas aeruginosa have 60 plus confirmed and putative two-component systems. Bacillus subtilis has 30 plus and Nostoc punctiformis over 100. As genetic amplification does not result in changes in the basic structural folds of the catalytic domains of the sensor kinase or response regulators, each sensor kinase must recognize its partner through subtle changes in residues at the interaction surface between the two proteins. Additionally, the response regulator must prepare itself for efficient activation by the phosphorylation event. In this short review, we discuss the contributions of the critical β4-α4 recognition loop in response regulators to their function. In particular, we focus on this region's microsecond-millisecond timescale dynamics propensities and discuss how these motions play a major role in response regulator recognition and activation.

Keywords: NMR; millisecond dynamics; phosphorylation; response regulator; β4-α4 recognition loop.

Figure 1

Schematic representations of the two-component system (top) and the phosphorelay (bottom).

Figure 2. Co-crystal structures of the complexes

(A) Spo0F-Spo0B, (B) CheY-CheZ, and (C) Ydp1-Sln1. Spo0F, CheY, Sln1=response regulators; Spo0B=phosphotransferase; CheZ=phosphatase; Ydp1=domain of kinase. Interaction surfaces are highlighted in ‘wheat’ color.

Figure 3. Hydrophobic surfaces for homology models of the N-terminal regulatory domains of the response regulators

(A) OmpR, (B) PhoB, and (C) ArcA. The α1-helix/α1-α5 interface region is highlighted in a dashed box. The color-coded hydrophobic scale used is also shown (see text).

Figure 4. Dynamics of NtrC

(A) Superimposition of the NMR structures of the N-terminal domain of unphosphorylated, inactive NtrC (blue, PDB 1DC7) and active NtrC (orange, PDB 1DC8) states are superimposed. (B) NMR ¹⁵N backbone R_ex values (millisecond-microsecond motions) superimposed on unphosphorylated, inactive NtrC (PDB 1DC7). These data were adapted with permission from Ref (21). The NMR exchange term R_ex is shown on a continuous color scale from red to blue. The color scale indicates the extent of conformational exchange between states that sense different chemical environments.

Figure 5. Conformational families of Spo0F

(A) Unphosphorylated-inactive Spo0F occupies two distinct conformational families (blue and orange, PDB code 1FSP) in the critical β4-α4 recognition loop. (B) One of these families structurally overlaps with the phosphorylated-active conformation of Spo0F (pink, PDB code 1PUX). (C) Magnified view of the β4-α4 recognition loop in its unphosphorylated-inactive form (blue and orange) and in its phosphorylated-active form (pink).

Figure 6

The NMR structure of the β4-α4 recognition loop from H101A Spo0F (yellow, PDB code 2JVI), overlaid with the unphos-horylated-inactive (blue and orange) and activated structures of Spo0F (pink).

Figure 7. Molecular dynamics simulation data

(A) Wild-type Spo0F and (B) L66A Spo0F (PDB code 2JVK). In (A), the two structures shown (orange and yellow) represent the structures from the wild-type Spo0F molecular dynamics ensemble which have the greatest root mean square deviations (RMSDs) with respect to one another. In (B), the two structures shown (red and green) represent the structures from the L66A Spo0F molecular dynamics ensemble which have the greatest RMSDs with respect to one another. The large difference in the β4-α4 recognition loop region (indicated by black arrows) for wild-type Spo0F (A) suggests significant motional propensity in this region. In the case of L66A Spo0F (B), this motional propensity is restricted.

Figure 8. Coupling of residues which co-vary across the sensor kinase-response regulator interaction surface

The residues comprise six color-coded clusters. These are superimposed on the sensor kinase HK853 (T. maritime, PDB code 2C2A) on the left and the response regulator Spo0F (B. subtilis) on the right. The aspartate and histidine residues which are involved in the phosphotransfer are highlighted.