Structural and functional basis of transcriptional regulation by TetR family protein CprB from S. coelicolor A3(2).
Bottom Line: Binding of the DNA results in the restructuring of the dimeric interface of CprB, inducing a pendulum-like motion of the helix-turn-helix motif that inserts into the major groove.Experiments performed on a subset of DNA sequences from Streptomyces coelicolor A3(2) suggest that CprB is most likely a pleiotropic regulator.Apart from serving as an autoregulator, it is potentially a part of a network of proteins that modulates the γ-butyrolactone synthesis and antibiotic regulation pathways in S. coelicolor A3(2).
Affiliation: Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, Maharashtra, India IITB-Monash Research Academy, Mumbai 400076, Maharashtra, India.Show MeSH
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Mentions: In contrast to the apo form of the protein, which is a dimeric unit, the CprB–CS complex was found to be a dimer of dimers. Both the dimeric units are bound at opposite sides of the 22-bp CS and there are no interactions between the two dimers. The center-to-center distance between the two monomers of a homodimer is 38.2 Å (measured from amide nitrogen atom of G44 from both the recognition helices α3 of homodimer), as shown in Figure 1A. The CprB consists of 215 amino acids; however, due to the weak and/or no electron density observed for the residues 1–4, 113, 114, 165–175 and 212–215 in monomer A, 1–4, 166–169 and 213–215 in monomer B, 1–4, 168–174 and 213–215 in monomer C and 1–7, 77–79, 118, 119, 167–173 and 213–215 in monomer D, they were not included in the final refined structure. Similar to the apo form of the CprB, within a dimeric unit of the CprB–CS complex, the two monomers possess a pseudo two-fold symmetry axis. The monomers of a homodimer are covalently connected via a disulfide linkage between cysteine residues at position 159. In the apo form of CprB, the nature of this disulfide bond is LH (left handed) spiral with strain energy of 4.18 kcal/mol. There is no conformational change in the disulfide bond of CprB upon binding to the CS; however, there is an increase in strain energy upon binding (5.12 kcal/mol for monomers A and B and 6.5 kcal/mol for monomers C and D; analysis of disulfide bond was done using web server, http://126.96.36.199/python/disulfideanalysis/search.html). In general, the LH spiral disulfide bonds are known to confer structural stability (55–57). Similar scenario was also observed in another TetR-FTR protein, SbtR (Thermus thermophilus HB8) where the disulfide bond possess a LH spiral geometry and partakes in stabilizing the dimeric unit (57). To shed light on the role played by the disulfide bond in CprB, cysteine 159 was mutated to serine. The mutant form (C159S) could not be expressed in appreciable amounts to conduct further experiments. The dramatically reduced expression level indicates that the disulfide bond is important for structural stability of the protein. Comparative analysis of the CprB–CS complex and the apo form of CprB was performed using LBD domain as a reference frame (Figure 1B). Binding of the CS to CprB induces a general pendulum-like movement in the overall protein structure as shown in Figure 1C. A twist in the dimeric interface results in a coordinated motion of the DNA binding HTH motif about the connector helix α4. This motion facilitates a snug fit of helix α3 into the major groove of the CS, thereby aiding DNA binding event. A minimal shift of 1 Å in the position of the disulfide bond was observed (as shown in Figure 1D) for the dimeric unit formed by monomers C and D, thereby highlighting the fact that it acts like a tether between the two subunits. It is possible that the two CprB dimers utilize the disulfide bond as a fulcrum to rotate between conformational states.
Affiliation: Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, Maharashtra, India IITB-Monash Research Academy, Mumbai 400076, Maharashtra, India.