High-resolution protein complexes from integrating genomic information with molecular simulation

Abstract

Bacteria use two-component signal transduction systems (TCS) extensively to sense and react to external stimuli. In these, a membrane-bound sensor histidine kinase (SK) autophosphorylates in response to an environmental stimulus and transfers the phosphoryl group to a transcription factor/response regulator (RR) that mediates the cellular response. The complex between these two proteins is ruled by transient interactions, which provides a challenge to experimental structure determination techniques. The functional and structural homolog of an SK/RR pair Spo0B/Spo0F, however, has been structurally resolved. Here, we describe a method capable of generating structural models of such transient protein complexes. By using existing structures of the individual proteins, our method combines bioinformatically derived contact residue information with molecular dynamics simulations. We find crystal resolution accuracy with existing crystallographic data when reconstituting the known system Spo0B/Spo0F. Using this approach, we introduce a complex structure of TM0853/TM0468 as an exemplary SK/RR TCS, consistent with all experimentally available data.

Fig. 1.

Flow-chart of our approach. Given the target sequence of an unknown protein complex, direct-coupling analysis investigates the mutational pattern of sequential homologues in databases and suggests pair-wise contacts defining an interaction surface. Similarly, unbound structures for the given target sequences can be either directly extracted from a structural database or generated by structure-prediction methods like homology modeling. This information of the unbound structures and interaction surface contacts is sufficient information for docking simulations in computationally efficient structure-based models, providing both insight into the mechanism of docking and making a prediction of the protein complex. To improve the quality of the prediction, it can be additionally relaxed in physics-based empirical force fields.

Fig. 2.

Crystal structure and prediction of the Spo0B/0F complex. (A) The crystal structure of the complex [PDB ID code 1f51 (11)] of Spo0B (blue, mobile C-terminal region transparent) and Spo0F (white). His-30 and Asp-54, the phosphoryl transferring groups, are highlighted in red. (B) DCA predicts contacts between six amino acid pairs (yellow). Our docking simulations based on the unbound proteins [1ixm (13), 1pey (12)] and these additional contacts predict the bound complex with a backbone-RMSD of roughly 3 Å (floppy C/N termini and the mobile ATPase of Spo0B are disregarded) reaching crystallographic accuracy. Additional inclusion of the Spo0B-His Spo0F-Asp contact (red) improves the accuracy to 2.5 Å.

Fig. 3.

Contact maps and helical interfaces for Spo0B/Spo0F and TM0853/TM0468. (A) The axes denote the consecutively numbered amino acids of the Spo0B/Spo0F system (1–192, 193–384, 385+). The contact maps derived from the complexed crystal structure (red) and the predicted structure (blue) agree well. The DCA-contacts are highlighted by circles (six contacts+His-Asp). (B) The prediction of the TM0853/TM0468 (1–200, 201–400, 401+) system shows a similar contact map (blue, nine contacts+His-Asp). (C) For the SpooB/Spo0F-system, only helix 1 of Spo0B has significant contacts with helix 1 of the Spo0F (coloring scheme as in Fig. 2, the interfacial amino acids are shown additionally as ball-and-stick). (D) For the predicted complex structure of TM0853/TM0468, both helices 1 and 2 of the HK are oriented tighter toward helix 1 of the RR. DCA accordingly predicts contacts between helix 1–helix 1 and helix 2–helix 1.

Fig. 4.

Prediction of the TM0853/TM0468 complex. The cytoplasmic domain of the sensor histidine-kinase protein TM0853 [blue, C-terminal ATP-binding domain transparent, PDB ID code 2c2a (24)] and the response regulator TM0468 [white, homologue model based on the structure of 1mvo (26), 45% sequence identity] transfers a phosphoryl group between the two residues. The crucial interaction between TM0853-His-260 and TM0468-Asp-53 (red) is responsible for this transfer and requires the two residues to be in direct contact. Both residues are perfectly conserved over a large set of homologues and are therefore impossible to detect as a contact by DCA. (A) Docking simulations based purely on the contacts predicted by DCA (yellow) converge to a structure with high similarity to the Spo0F/Spo0B complex, but with a large distance between the His-Asp pair (20 Å between C_α). (B) When additionally including the crucial His-Asp interaction as a contact, TM0468 docks at a slightly different angle, which brings His and Asp in direct contact (12 Å between C_α). (C and D) After an additional relaxation in an empirical force-field (C) and rotamer-correction (D), we introduce this structure as a docked complex (PDB file in SI Text), whereas structure (A) might represent an intermediate step during the RR SK binding event. (C) The graph shows a 25-ns relaxation in an empirical force field. The trajectories are compared by the Cα-RMSD (floppy C and N termini as well as the ATPase region are excluded) to the structure right after structure-based docking (red), the structure after 10 ns (green), and the final structure after 25 ns (blue). The relaxation aims at removing artifacts from the structure-based docking procedure and has only limited effect on the backbone because a 3 Å shift can be considered small given the size of the system. (D) The rotamers D9, D10, K105, and H260 (yellow, H260 highlighted by a black circle) of the predicted TM853/TM0468 complex need slight correction to facilitate the phophortransferase reaction: Right after docking TM0853-H260's rotamer is not in line with the TM0468-D53. This can be easily corrected leaving sufficient space for phosphoryl between the two residues.