Assembly of the transmembrane domain of E. coli PhoQ histidine kinase: implications for signal transduction from molecular simulations

Abstract

The PhoQP two-component system is a signaling complex essential for bacterial virulence and cationic antimicrobial peptide resistance. PhoQ is the histidine kinase chemoreceptor of this tandem machine and assembles in a homodimer conformation spanning the bacterial inner membrane. Currently, a full understanding of the PhoQ signal transduction is hindered by the lack of a complete atomistic structure. In this study, an atomistic model of the key transmembrane (TM) domain is assembled by using molecular simulations, guided by experimental cross-linking data. The formation of a polar pocket involving Asn202 in the lumen of the tetrameric TM bundle is crucial for the assembly and solvation of the domain. Moreover, a concerted displacement of the TM helices at the periplasmic side is found to modulate a rotation at the cytoplasmic end, supporting the transduction of the chemical signal through a combination of scissoring and rotational movement of the TM helices.

Figure 1. Schematic representation of the PhoQ histidine kinase in two-component systems.

(A) The sequence topology of PhoQ is related to its (B) 3D homo-dimer schematic arrangement. The following available structures are used to model the global PhoQ structure: the sensor domain solved from E. coli (PDB code 3BQ8 [9]); the cytoplasmic HAMP NMR structure from Archaeoglobus fulgidus (PDB code 2L7H [30]), and the histidine kinase crystal structure from Thermotoga maritima (PDB code 2C2A [58]). The sensor domain harbors several acidic residues (148—EDDDDAE—154, shown in space-filled representation). They have been proposed to be involved in the sensing of divalent cations and AMP at the membrane surface. The ADP and the phosphorylation site (His277) are highlighted in space-filled representation. During the phosphor-transfer reaction, the phosphate group is translated from His277 to the PhoP aspartic acid (Asp51) (green arrow).

Figure 2. Assembly of the TM domain.

(A) Models of individual TM1 and TM2 helices equilibrated in an all-atom membrane bilayer. Gly32 and Pro208 produce a kink in TM1 and TM2, respectively. (B) Stable TM dimers as obtained from MD in a united-atom membrane bilayer. (C) Tetrameric TM model structure produced using TM dimers, MD simulations and cross-linking spatial restraints. From left to right, the model is presented embedded in the membrane from the periplasmic top view, and with a focus on the H-bond network formed by residues Tyr32 and Asn202, which contributes to stabilize the bundle.

Figure 3. TM structural validation using disulfide cross-linking scanning.

MD-averaged contact maps for (A) TM1 and (B) TM2 interfaces within the assembled TM domain. A direct comparison with cross-linking efficiency of (A) TM1 and (B) TM2 is reported in the inset, and shows a strong correlation between the cross-linking (1-efficiency) (in black) and the MD-averaged Cα distance measured for the TM model structure (in red). The cross-linking efficiency for the whole TM1 and TM2 regions is reported in Figure S1.

Figure 4. Effects of Asn202 mutation on the solvation of the TM domain.

The kernel density estimation of water molecules for MD simulations of the wild-type TM bundle, and three relevant Asn202 mutants: N202A, N202H, and H202R. Residue 202 is localized in the middle of the membrane (at 0 Å). Conservative mutations preserve the hydration of the TM core, while substitution with alanine prevents water to enter the bundle. Distribution is calculated along the axis orthogonal to the membrane bilayer, and the transmembrane portion is schematically indicated by the grey area defined by the MD-averaged distance between bilayer polar heads (namely, phosphorus atoms).

Figure 5. Solvation-dependent dynamic features of the TM domain.

(A) The rotation of the Cα atoms around the TM2 helix main axis is computed based on a principal component analysis, and the angle distribution is characterized by three major modes, that can be fitted using three Gaussians, (B) MD time series of the TM2 residue state corresponding to the angle distribution. The system is initially in a metastable state (black), before switching to a solvated state where the TM1-TM1 interface is tighter (grey). After ∼30 ns, the system, passing through to a metastable state, shifts to a stable state characterized by a larger TM1-TM1 distance (light grey). (C) The rotation per residue related to the switch between the 2 most relevant states in MD (B) is calculated. TM2 Pro208 acts like a hinge, and transforms the large movement of the N-terminus into a mild rotation (∼20 degrees) of the TM2 residue at the cytoplasmic interface.

Figure 6. Free energy landscape for the TM conformational change.

The free energy landscape defined by sampling inter-helical distances between TM1 C-termini and TM2 N-termini is reported. The conformational change observed in the unbiased MD simulations (orange points) occurs along a free energy valley, that connects a main equilibrium state (F₀) and a high-energy conformation, and can be associated with relevant states during the signaling process (F₁, ∼5 kcal/mol higher in free energy).

Figure 7. TM connectivity and implications for signaling mechanism.

The panels (A) and (B) indicate the projection for the F₀ and F₁ states, respectively, on the periplasmic (top) and cytoplasmic (bottom) surface of the membrane of the helical termini of the TM model. The contour lines represent the position of TM1 and TM2 during MD, and the dots represent the position of SD and HAMP available structures at the same section surface. (Central panel) At high mM concentration of Mg²⁺ and Ca²⁺ cations, metal bridges are formed between the SD acidic cluster and the negatively charged membrane. This conformation can be associated to the F₀ state of our model and to a kinase-dominant state (K⁺). When the concentration decreases, the metal ion bridges are disrupted, leading to repulsion between the membrane and SD active site. This triggers a conformational change of the TM domain (F₁ state associated to a phosphatase-dominant conformation, P⁺), and results in a rotation of TM2 at the cytoplasmic interface, that is then transmitted to the linked HAMP domain.