Abstract
The transcriptional antiterminator protein LicT regulates the expression of Bacillus subtilis operons involved in beta-glucoside metabolism. It consists of an N-terminal RNA-binding domain (co-antiterminator (CAT)) and two phosphorylatable phosphotransferase system regulation domains (PRD1 and PRD2). In the activated state, each PRD forms a dimeric unit with the phosphorylation sites totally buried at the dimer interface. Here we present the 1.95 A resolution structure of the inactive LicT PRDs as well as the molecular solution structure of the full-length protein deduced from small angle x-ray scattering. Comparison of native (inactive) and mutant (constitutively active) PRD crystal structures shows massive tertiary and quaternary rearrangements of the entire regulatory domain. In the inactive state, a wide swing movement of PRD2 results in dimer opening and brings the phosphorylation sites to the protein surface. This movement is accompanied by additional structural rearrangements of both the PRD1-PRD1 ' interface and the CAT-PRD1 linker. Small angle x-ray scattering experiments indicate that the amplitude of the PRD2 swing might even be wider in solution than in the crystals. Our results suggest that PRD2 is highly mobile in the native protein, whereas it is locked upon activation by phosphorylation.
Highlights
Protein phosphorylation is universally used as a mechanism for signal transduction in all branches of the kingdom of life [1]
LicT from Bacillus subtilis belongs to a family of transcriptional antiterminator proteins that control the expression of genes involved in carbohydrate metabolism
Native and Mutant LicT-PRDs Have Radically Different Structures—Several lines of evidence raised by experiments conducted in solution converged toward the hypothesis that the conformations of wild type inactive LicT and of the constitutive active LicT-H207D/H269D double mutant are radically different [8, 13]
Summary
Protein phosphorylation is universally used as a mechanism for signal transduction in all branches of the kingdom of life [1]. Knowledge of the structure of the phosphorylated state of a protein remains beyond reach. The expression of many catabolic operons in bacteria is controlled by (de)phosphorylation of regulatory proteins as exemplified by transcriptional antitermination. LicT from Bacillus subtilis belongs to a family of transcriptional antiterminator proteins that control the expression of genes involved in carbohydrate metabolism. In the full-length protein, the ability of CAT to bind RNA and prevent the premature arrest of transcription is controlled by two contiguous regulatory domains termed PRD1 and PRD2. These domains respond to regulation signals sent by the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS)
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