Figure 1.
Homology model of SpaK based on PDB entries 2c2a and 2ftk.
Modeled region: 219–459. The 218-residue long N-terminal membrane spanning region (residues 1–218) was not modeled. A: Model of the oligomeric state: homodimer. Coloring scheme reflects in each modeled monomer a consecutive ordering of amino acids in the N-to-C-terminal direction, whereby N-most residues are colored blue and C-most residues are red. Blue-cyan (residues 219–300): central four-helix bundle formed by interaction of 2 helixes from each monomer; Green-red (residues 301–459): C-terminal ATPase-c domain. The labels H247 and G392 show the location of two residues that were changed using site-directed mutagenesis to construct mutants for the phosphorylation studies (see Materials and Methods). B: Homology model of SpaK with marked domains: P1 (dark pink; 219–254), P2 (pink helix; 255–305), P3 (brown; 306–310), P4 (red; 311–455), and P5 (pink strand; 456–459) that are considered as 5 separate functional units. Characteristic sequence motifs (“boxes”) are colored as follows: H (yellow), N (plum), G1 (pale green), F (blue), and G2 (green). Highlighted motifs correspond to those in Fig. 1 from [41] (see Table 3).
Figure 2.
Homology model of the SpaR N-terminal (residues 1–117) and C-terminal (residues 118–220) domains.
Modeling of the N-terminal domain was based on PDB template 1mvo_A, and the C-terminal domain was based on PDB template 2gwr_A. The conformation between domains was modeled based on 2gwr (response regulator protein MTRA from Mycobacterium tuberculosis). Coloring scheme reflects consecutive ordering of amino acids from the N-terminal region (blue) to the C-terminal region (red). Residues in SpaR that correspond to the functional residues in response regulator 2ftk (Spo0F; see Table 2B) are displayed as sticks.
Table 1.
Candidate domain-fusion templates for structure modeling of a SpaK/SpaR complex.
Figure 3.
Homology model of a SpaK-SpaR complex.
A: Model is based on the A and E chains of SPO0B, a phosphotransferase, complexed with SPO0F, a beryllofluoride (PDB template 2ftk). Blue, red: monomers of SpaK; Green: SpaR. B: Close up view of interacting residues (SpaK: H247; SpaR: D8, D9, D51; shown as stick) believed to mediate transfer of phosphate group from SpaK to SpaR.
Table 2.
Residue-residue correspondences between functional motifs in domain-fusion template 2ftk and SpaK (A) or SpaR (B) homology models.
Table 3.
Examples of pairwise residue-residue correspondences between SpaK, Beryllofluoride Spo0F, and CheA histidine kinase.
Figure 4.
In vitro phosphorylation studies of SpaK and SpaR.
A, B: SDS-PAGE of 6xHis-SpaK and 6xHis-SpaR in isolation or in combination and at various mass ratios, in the presence of ATP. A: Coomassie blue staining. B: Autoradiography; lane a: molecular weight markers. C: Phosphorimage analysis of SpaK incubated with [g-32P]-ATP (lane 1) followed by addition of 4 mM (lane 2), 10 mM (lane3), or 50 mM non-labeled (cold) ATP. D: PEI cellulose thin-layer chromatography of 6xHis-SpaK in isolation, or in combination with 6xHis-SpaR with and without EDTA.
Figure 5.
In vitro phosphorylation studies involving SpaK mutants.
A, B: Polyacrylamide gel electrophoresis of 6xHis-SpaR and 6xHis-SpaK wild type or mutants in isolation or in combination, in the presence of ATP. Lanes 1, 7: molecular weight markers. A: Coomassie blue staining. B: Autoradiography. Mutant1: H247Q, Mutant 2: G392A.