Structural basis of a rationally rewired protein-protein interface critical to bacterial signaling

Abstract

Two-component signal transduction systems typically involve a sensor histidine kinase that specifically phosphorylates a single, cognate response regulator. This protein-protein interaction relies on molecular recognition via a small set of residues in each protein. To better understand how these residues determine the specificity of kinase-substrate interactions, we rationally rewired the interaction interface of a Thermotoga maritima two-component system, HK853-RR468, to match that found in a different two-component system, Escherichia coli PhoR-PhoB. The rewired proteins interacted robustly with each other, but no longer interacted with the parent proteins. Analysis of the crystal structures of the wild-type and mutant protein complexes and a systematic mutagenesis study reveal how individual mutations contribute to the rewiring of interaction specificity. Our approach and conclusions have implications for studies of other protein-protein interactions and protein evolution and for the design of novel protein interfaces.

Figure 1. Specificity residues in two-component signaling proteins

(A) Multiple sequence alignment of histidine kinases (DHp domain only) and response regulator (receiver domain only), with specificity residues and highly conserved residues highlighted. Species abbreviations: (Ec) Escherichia coli; (Sa) Staphylococcus aureus; (Tm) Thermotoga maritima. Interacting partners are arranged in the same order in both HK and RR alignments. Sequences are numbered according to the Tm proteins, with the last digit of each number positioned above the relevant amino acid residue. (B) HK853 and (C) PhoR phosphotransfer specificity. Each histidine kinase construct was autophosphorylated with [³²P-γ]ATP and then incubated with the response regulator indicated at room temperature. Samples were taken at the time points indicated and phosphotransfer assessed by SDS-PAGE and phosphorimaging. Arrowheads indicate the position of autophosphorylated kinase or phosphorylated response regulator. Also see Figure S1.

Figure 2. Rational rewiring of phosphotransfer specificity

Phosphotransfer assays for wild-type and mutant two-component proteins. In each panel, the histidine kinase indicated was autophosphorylated with [³²P-γ]ATP and then incubated with the response regulator indicated at 4 °C. Samples were taken at the time points indicated and phosphotransfer assessed by SDS-PAGE and phosphorimaging. (A) Wild-type HK853 and (B) HK853* (which harbors the substitutions A268V, A271G, T275M, V294T, D297E) were tested for phosphotransfer to RR468, RR468*, PhoB, and PhoB*. RR468* contains the substitutions V13P, L14I, I17M, and N21V. PhoB* contains the substitutions P13V, I14L, M17I, and V21N. (C) HK853**, which harbors the substitutions A268V, A271G, and T275M was tested for phosphotransfer to RR468 and RR468*. Also see Figures S2–S3.

Figure 3. Crystal structures of the wild-type complex HK-RR, the rewired and functional HK*-RR* complex, and the impaired HK-RR* complex

Cartoon representation of (A) HK-RR complex with HK853 bound to ADP and RR468 D53 bound to SO₄, (B) HK*-RR* complex with HK853* bound to ADP and RR468* D53 bound to BeF₃⁻ and (C) HK-RR* complex with HK853 bound to ADP and RR468* D53 bound to BeF₃⁻. Left; cartoon representations of the overall structure of the three complexes formed by a homodimeric HK (blue colored with one subunit transparent) bound to two molecules of RR (yellow-green colored with one molecule transparent). In each complex, the ATP-lid in the HK and the β3-α3 linker in the RR are colored in black; the phosphorylatable residues H260 and D53 as well as bound ligands ADP, sulfate (SO₄) and beryllium trifluoride (BeF₃) are shown as sticks. Middle; the angle formed between the interacting helices HK α1 (246–279) and RR α1 (12–26) for each complex is shown. Right; HK-RR interface shown by the DHp domain (with one subunit transparent) bound to one RR with the critical specificity residues (13, 14, 17 and 21 in red for RR468 and 268, 271, 275, 294 and 297 in orange for HK853) highlighted in space-filling spheres. Also see Figures S4–S6.

Figure 4. Comparison of the active center in the HK-RR and HK*-RR* complexes

Close-up view of the active center with a superposition of HK-RR (in cyan) and HK*-RR* (in orange) in cartoon representation. The phosphorylatable residues H260 and D53, residue M55, and the bound ligands (sulfate (SO₄) in the HK-RR complex and beryllium trifluoride (BeF₃) in the HK*-RR* complex) are shown as sticks. Distances are shown by dashed lines; in black color for Cα of H260 in HK-RR with the sulfur atom of SO₄ (8.3 Å) and for Cα of H260 in HK*-RR* with the Be atom of BeF₃ (7.9 Å); in red color for εN of H260 in HK-RR with the sulfur atom of SO₄ (4.8 Å) and with the Be atom of BeF₃⁻ (3.6 Å). Also see Figures S4–S6.

Figure 5. Differential interactions in free and complex structures

(A) DHp domains with a superposition of HKf (in green) and HKf* (in blue) to show new interactions resulting from the mutations introduced into HKf*: A268V, A271G, T275M, V294T and D297E. (B) DHp domains with a superposition of HKf* and HK853* from the HK*-RR* complex (in orange) to show changes in the interactions for M275. (C) Superposition of RR468 in RRf* (green), HK-RR (cyan), HK*-RR* (orange) and HK-RR* (magenta) structures shows the interaction M17-F107 and the different conformations of F107 and the β5-α5 linker in the RR alone or in complex. All the structures are shown in cartoon representation with the selected residues labeled in black and drawn as sticks. Also see Figures S4–S6.

Figure 6. Phosphotransfer between all possible mutational intermediates separating HK-RR and HK*-RR*

(A) Superposition of HK-RR (in cyan) and HK*-RR* (in orange) complexes highlighting how mutations in DHp α1 (A268V, A271G and T275M) affect interactions with mutations in RR α1 (V13P, L14I, I17M and N21V) and F20. A dashed line represents a polar interaction between N21 and T275 in the HK-RR complex. Also see Figures S4–S6. (B) Phosphotransfer assays for wild-type HK853 and HK853 harboring all possible combinations of one, two, or three PhoR-like specificity substitutions present in HK853** (A268V, A271G and T275M). Each lane represents the incubation of the indicated autophosphorylated kinase with the indicated response regulator for 15 seconds at room temperature. Reactions 1–11 and 12–16 were run on separate SDS-PAGE gels; the resulting phosphorimages were contrasted identically and stitched together. (C) The histidine kinase (HK) and response regulator (RR) bands from the phosphotransfer experiments in panel B were quantified and plotted. For each mutational pairing, the x-axis value indicates the intensity of the autophosphorylated HK band (HK~P) normalized to the intensity of the autophosphorylated kinase band and the y-axis value indicates the intensity of the phosphorylated response regulator band (RR~P). In each case, band intensities were normalized to the intensity of the autophosphorylated kinase incubated without response regulator (lane 1 of each gel in panel B). Green points indicate the pairs HK853-RR468 and HK853*-RR468*. The box in the lower left indicates pairings deemed functional; a low level of both the kinase and regulator bands reflects efficient phosphotransfer and dephosphorylation. The 43 functional pairings are underlined in panel B. (D) One example of a mutational path from the wild-type to the rewired complex in which each intermediate state is functional. (E) An example of a mutational path in which all mutations to the kinase occur in three successive steps.