Inducer responses of BenM, a LysR-type transcriptional regulator from Acinetobacter baylyi ADP1.

BenM and CatM control transcription of a complex regulon for aromatic compound degradation. These Acinetobacter baylyi paralogues belong to the largest family of prokaryotic transcriptional regulators, the LysR-type proteins. Whereas BenM activates transcription synergistically in response to two effectors, benzoate and cis,cis-muconate, CatM responds only to cis,cis-muconate. Here, site-directed mutagenesis was used to determine the physiological significance of an unexpected benzoate-binding pocket in BenM discovered during structural studies. Residues in BenM were changed to match those of CatM in this hydrophobic pocket. Two BenM residues, R160 and Y293, were found to mediate the response to benzoate. Additionally, alteration of these residues caused benzoate to inhibit activation by cis,cis-muconate, positioned in a separate primary effector-binding site of BenM. The location of the primary site, in an interdomain cleft, is conserved in diverse LysR-type regulators. To improve understanding of this important family, additional regulatory mutants were analysed. The atomic-level structures were characterized of the effector-binding domains of variants that do not require inducers for activation, CatM(R156H) and BenM(R156H,T157S). These structures clearly resemble those of the wild-type proteins in their activated muconate-bound complexes. Amino acid replacements that enable activation without effectors reside at protein interfaces that may impact transcription through effects on oligomerization.

Distinct effector-binding sites enable synergistic transcriptional activation by BenM, a LysR-type regulator.

BenM, a bacterial transcriptional regulator, responds synergistically to two effectors, benzoate and cis,cis-muconate. CatM, a paralog with overlapping function, responds only to muconate. Structures of their effector-binding domains revealed two effector-binding sites in BenM. BenM and CatM are the first LysR-type regulators to be structurally characterized while bound with physiologically relevant exogenous inducers. The effector complexes were obtained by soaking crystals with stabilizing solutions containing high effector concentrations and minimal amounts of competing ions. This strategy, including data collection with fragments of fractured crystals, may be generally applicable to related proteins. In BenM and CatM, the binding of muconate to an interdomain pocket was facilitated by helix dipoles that provide charge stabilization. In BenM, benzoate also bound in an adjacent hydrophobic region where it alters the effect of muconate bound in the primary site. A charge relay system within the BenM protein appears to underlie synergistic transcriptional activation. According to this model, Glu162 is a pivotal residue that forms salt-bridges with different arginine residues depending on the occupancy of the secondary effector-binding site. Glu162 interacts with Arg160 in the absence of benzoate and with Arg146 when benzoate is bound. This latter interaction enhances the negative charge of muconate bound to the adjacent primary effector-binding site. The redistribution of the electrostatic potential draws two domains of the protein more closely towards muconate, with the movement mediated by the dipole moments of four alpha helices. Therefore, with both effectors, BenM achieves a unique conformation capable of high level transcriptional activation.

Oligomerization of BenM, a LysR-type transcriptional regulator: structural basis for the aggregation of proteins in this family.

LysR-type transcriptional regulators comprise the largest family of homologous regulatory DNA-binding proteins in bacteria. A problematic challenge in the crystallization of LysR-type regulators stems from the insolubility and precipitation difficulties encountered with high concentrations of the full-length versions of these proteins. A general oligomerization scheme is proposed for this protein family based on the structures of the effector-binding domain of BenM in two different space groups, P4(3)22 and C222(1). These structures used the same oligomerization scheme of dimer-dimer interactions as another LysR-type regulator, CbnR, the full-length structure of which is available [Muraoka et al. (2003), J. Mol. Biol. 328, 555-566]. Evaluation of packing relationships and surface features suggests that BenM can form infinite oligomeric arrays in crystals through these dimer-dimer interactions. By extrapolation to the liquid phase, such dimer-dimer interactions may contribute to the significant difficulty in crystallizing full-length members of this family. The oligomerization of dimeric units to form biologically important tetramers appears to leave unsatisfied oligomerization sites. Under conditions that favor association, such as neutral pH and concentrations appropriate for crystallization, higher order oligomerization could cause solubility problems with purified proteins. A detailed model by which BenM and other LysR-type transcriptional regulators may form these arrays is proposed.

Incompatibility with formin Cdc12p prevents human profilin from substituting for fission yeast profilin: insights from crystal structures of fission yeast profilin.

Expression of human profilin-I does not complement the temperature-sensitive cdc3-124 mutation of the single profilin gene in fission yeast Schizosaccharomyces pombe, resulting in death from cytokinesis defects. Human profilin-I and S. pombe profilin have similar affinities for actin monomers, the FH1 domain of fission yeast formin Cdc12p and poly-L-proline (Lu, J., and Pollard, T. D. (2001) Mol. Biol. Cell 12, 1161-1175), but human profilin-I does not stimulate actin filament elongation by formin Cdc12p like S. pombe profilin. Two crystal structures of S. pombe profilin and homology models of S. pombe profilin bound to actin show how the two profilins bind to identical surfaces on animal and yeast actins even though 75% of the residues on the profilin side of the interaction differ in the two profilins. Overexpression of human profilin-I in fission yeast expressing native profilin also causes cytokinesis defects incompatible with viability. Human profilin-I with the R88E mutation has no detectable affinity for actin and does not have this dominant overexpression phenotype. The Y6D mutation reduces the affinity of human profilin-I for poly-l-proline by 1000-fold, but overexpression of Y6D profilin in fission yeast is lethal. The most likely hypotheses to explain the incompatibility of human profilin-I with Cdc12p are differences in interactions with the proline-rich sequences in the FH1 domain of Cdc12p and wider "wings" that interact with actin.

CatM regulation of the benABCDE operon: functional divergence of two LysR-type paralogs in Acinetobacter baylyi ADP1.

Two LysR-type transcriptional regulators, BenM and CatM, control benzoate consumption by the soil bacterium Acinetobacter baylyi ADP1. These homologs play overlapping roles in the expression of multiple genes. This study focuses on the benABCDE operon, which initiates benzoate catabolism. At this locus, BenM and CatM each activate transcription in response to the catabolite cis,cis-muconate. BenM, but not CatM, additionally responds to benzoate as an effector. Regulation by CatM alone is insufficient for growth on benzoate as the sole carbon source. However, three point mutations independently increased CatM-activated benA transcription and enabled growth on benzoate without BenM. Two mutations generate variants with one amino acid change in the 303-residue CatM, CatM(V158M) and CatM(R156H). These substitutions affected regulation of benA differently than that of catB, another CatM-regulated gene involved in benzoate catabolism. In relation to CatM, CatM(V158M) increased cis,cis-muconate-dependent transcription of benA but decreased that of catB. CatM(R156H) increased effector-independent expression of catB compared to CatM. In contrast, cis,cis-muconate was required with CatM(R156H) to activate unusually high benA expression. Thus, induction by cis,cis-muconate depends on both the sequence of CatM and the promoter. A point mutation at position -40 of the benA promoter enhanced CatM-activated gene expression and altered regulation by CatM(R156H). BenM and CatM bound to the same locations on ben region DNA. The frequency with which spontaneous mutations allow CatM to substitute for BenM might predict that one regulator would be sufficient for controlling benzoate consumption. This prediction is discussed in light of current and previous studies of the BenM-CatM regulon.