Mechanism of Activation for Transcription Factor PhoB Suggested by Different Modes of Dimerization in the Inactive and Active States

Abstract

Response regulators (RRs), which undergo phosphorylation/dephosphorylation at aspartate residues, are highly prevalent in bacterial signal transduction. RRs typically contain an N-terminal receiver domain that regulates the activities of a C-terminal DNA binding domain in a phosphorylation-dependent manner. We present crystallography and solution NMR data for the receiver domain of Escherichia coli PhoB which show distinct 2-fold symmetric dimers in the inactive and active states. These structures, together with the previously determined structure of the C-terminal domain of PhoB bound to DNA, define the conformation of the active transcription factor and provide a model for the mechanism of activation in the OmpR/PhoB subfamily, the largest group of RRs. In the active state, the receiver domains dimerize with 2-fold rotational symmetry using their alpha4-beta5-alpha5 faces, while the effector domains bind to DNA direct repeats with tandem symmetry, implying a loss of intramolecular interactions.

Phosphorylation-independent activation of the atypical response regulator NblR

“…2a). Consistent with phosphorylation-induced dimerization in RRs of the OmpR/PhoB family (Bachhawat et al, 2005), incubation of RR468 with acetyl phosphate induces a band shift. In contrast, 6His-NblR-RD and 6His-NblR did not change in mobility, suggesting that these proteins were not phosphorylated.…”

“…The quality of the final model was assessed using the PROCHECK suite of programs (Morris et al, 1992). The receiver domain structures of phosphorylated Spo0A from Bacillus stearothermophilus (RCSB code 1QMP) (Lewis et al, 1999), beryllium fluoride-activated PhoB from E. coli (RCSB code 1ZES) (Bachhawat et al, 2005), FrzS from Myxococcus xanthus (RCSB code 2GKG) (Fraser et al, 2007), beryllium fluoride-activated CheY from E. coli (RCSB code 1FQW) , HP1043 from Helicobacter pylori (RCSB code 2PLN) as well as the NblR model were structurally aligned using the LSQKAB program as implemented in the CCP4 suite (Collaborative Computational Project, Number 4, 1994).…”

A Link between Dimerization and Autophosphorylation of the Response Regulator PhoB

“…The two PhoB N dimers have been extensively characterized both structurally and biochemically (26 -29). As expected, a dimer along the ␣4-␤5-␣5 surface (here termed dimer 1) forms in the presence of a phosphoryl group analog (29) and represents the functional, phosphorylation-mediated dimer. Unexpectedly, metal binding alone supports a modified version of dimer 1 (27).…”

“…Thus, we sought an alternative method of assessing communication between PhoB N active sites by examining binding of a phosphoryl group analog. BeF 3 Ϫ binds to the active site of response regulators and mimics the Asp-phosphate linkage of phosphorylated response regulators (22,29,47). A plot of the quench in PhoB N tryptophan fluorescence versus BeF 3 Ϫ concentration (Fig.…”

Structure and dynamics of the polymyxin-resistance-associated response regulator PmrA in complex with the promoter DNA

“…Several DBD-DNA complex structures revealed that DBDs bind to promoter DNA sequences in a head-to-tail manner [13][14][15] . It was also suggested that the flexible linker connecting the REC and DBD should allow the DBD to bind to DNA in any orientation, as dictated by the DNA sequence or protein-protein interaction specifically between DBDs 16 . However, the flexible linker may introduce high mobility of the complex and hamper crystal structure study of activated response regulators.…”