Journal of Molecular Biology
Distinct Effector-binding Sites Enable Synergistic Transcriptional Activation by BenM, a LysR-type Regulator
Introduction
The BenM and CatM transcriptional regulators of the soil bacterium Acinetobacter baylyi ADP1 are paralogs with overlapping functions.1 BenM has the distinctive feature of activating transcription synergistically in response to two effectors, benzoate and cis,cis-muconate (hereafter designated muconate).2 In contrast, CatM responds only to muconate. As reported here, comparisons of these LysR-type transcriptional regulators (LTTRs), which are 59% identical in sequence, reveal the structural basis of their response to effectors. Despite the prevalence of the LTTR family,3 structural analyses of protein−effector interactions have been hampered by the inability to crystallize these regulators bound to their cognate small-molecule inducers.
LTTRs are the most common type of transcriptional regulator in proteobacteria.4 For example, strains of Acinetobacter, Agrobacterium, Escherichia, Pseudomonas, and Sinorhizobium each have genomes predicted to encode approximately 40 to 120 family members. LTTRs regulate all types of metabolic function including amino acid biosynthesis, aromatic compound degradation, oxidative stress, and virulence. The most conserved LTTR region is the N-terminal DNA-binding domain.3 In this domain, a winged-helix-turn-helix motif was confirmed in CbnR, the sole LTTR for which a full-length structure is known.5 The DNA-binding domain of CbnR connects to a two-domain regulatory region resembling periplasmic binding proteins.6 This fold is conserved in the structures of several LTTR regulatory domains despite great sequence variability.[7], [8], 9.
The regulatory domain structures, when interpreted with respect to genetic studies, suggest that an interdomain hinge region serves as an effector-binding site. The binding of small-molecule effectors most likely causes structural changes that alter DNA binding/bending and contact with RNA polymerase. Nevertheless, effector-mediated conformational changes in LTTRs remain unclear. The structures of the inactive and active forms of the OxyR regulatory domain were characterized.7 However, this is a rare case where the LTTR does not bind an effector but instead responds to oxidation-state changes via disulfide bond formation.
Problems with low solubility make structural studies of LTTRs notoriously difficult. X-ray crystallographic studies have succeeded by removing DNA-binding domains and using high-salt buffers. Such buffers create alternative problems by establishing competition for protein binding between ions in the crystallization buffer and the natural effectors. In studies of DntR and CysB, the presumed effector-binding sites contained thiocyanate, acetate and/or sulfate ions that may mimic the natural ligands.[8], 9., 10. Here, we report the direct visualization of biologically relevant ligands with the effector-binding domains (EBDs) of BenM and CatM.
BenM and CatM control a complex regulon for aromatic compound degradation in A. baylyi ADP1 (Figure 1). Benzoate consumption requires transcriptional activation by BenM and CatM at four loci where the relative importance of each regulator varies. Additionally, during growth on benzoate, BenM and CatM repress genes used to consume alternative aromatic compounds.11 Their functional overlap reflects sequence similarity that is 85% overall and 98% in the DNA binding domains. Both regulators respond to muconate. Nevertheless, the ability to respond to benzoate is unique to BenM. At the benA promoter, benzoate or muconate alone activates BenM-mediated transcription. Together, they yield a BenM-dependent level of transcriptional activation that is higher than the sum of their individual effects.2,12 This physiologically important type of regulation enables the rapid integration of cellular signals.2,12,13 Here, comparisons of BenM-EBD and CatM-EBD provide a model for the structural basis of transcriptional synergism.
Section snippets
Structural analysis of BenM-EBD, CatM-EBD and their ligand complexes
The EBDs of CatM and BenM lack 80 N-terminal residues including the DNA binding domain.14 A histidine tag at the C terminus, for purification, does not interfere with BenM function in vivo or effector-binding in vitro.2,13 To confirm that the tag does not interfere with CatM function, an allele encoding the modified protein (catM5550) was introduced into the A. baylyi chromosome. The resulting mutant grew at wild-type rates with benzoate as the carbon source (data not shown). Thus, the
Conformational changes in BenM- and CatM-EBD associated with transcriptional regulation
Overall, BenM- and CatM-EBD are similar to the known structures of LTTR regulatory domains.5,[7], [8], 9. Two of these, DntR and CbnR, share similar EBD sequences with BenM and CatM and belong to the same LTTR subclass involved in aromatic compound degradation.16 In contrast, there is minimal sequence similarity between the regulatory regions of CysB or OxyR and the ADP1 regulators. Sequence identity between BenM-EBD and the comparable regions of CysB and OxyR are only 12 and 21%, respectively (
Chemicals
Reagent grade chemicals and 18 M Ohm cm−1 Infinity Nanopure UF water were used. The Fluka puris grade (>99.9%) imidazole used for protein purification had UV absorbent impurities evident at 280 nm. Muconate was purchased from Acros or provided as a gift from Celgene.
Histidine-tagged derivatives of CatM
The effect of the hexahistidine tag of CatM was tested by overlap-extension PCR, as for BenM.2 Acinetobacter strains are derived from A. baylyi ADP1, formerly designated Acinetobacter sp. or A. calcoaceticus.25 A modified catM
Acknowledgements
X-ray data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source (APS), Argonne National Laboratory with the help of John Rose and Zhi-Jie Liu and at the Structural Biology Center Collaborative Access Team (SBC-CAT) 19-BM beamline with the help of Santiago Lima and Michelle Momany. The assistance of staff at both beamlines is greatly appreciated. APS use was supported by the U.S. Department of Energy, Office of Science, Office of
References (43)
- et al.
Crystal structure of a full-length LysR-type transcriptional regulator, CbnR: unusual combination of two subunit forms and molecular bases for causing and changing DNA bend
J. Mol. Biol.
(2003) - et al.
Structural basis of the redox switch in the OxyR transcription factor
Cell
(2001) - et al.
Development of a bacterial biosensor for nitrotoluenes: the crystal structure of the transcriptional regulator DntR
J. Mol. Biol.
(2004) - et al.
The structure of the cofactor-binding fragment of the LysR family member, CysB: a familiar fold with a surprising subunit arrangement
Structure
(1997) - et al.
Constitutive mutations of the OccR regulatory protein affect DNA bending in response to metabolites released from plant tumors
J. Biol. Chem.
(2002) - et al.
Functional dissection of the LysR-type CysB transcriptional regulator. Regions important for DNA binding, inducer response, oligomerization, and positive control
J. Biol. Chem.
(2001) - et al.
Deciphering the molecular basis of multidrug recognition: crystal structures of the Staphylococcus aureus multidrug binding transcription regulator QacR
Res. Microbiol.
(2003) - et al.
Processing of X-ray diffraction data collected in oscillation mode
Methods Enzymol.
(1997) SOLVE and RESOLVE: automated structure solution and density modification
Methods Enzymol.
(2003)- et al.
The benPK operon, proposed to play a role in transport, is part of a regulon for benzoate catabolism in Acinetobacter sp. strain ADP1
Microbiology
(2002)
Synergistic transcriptional activation by one regulatory protein in response to two metabolites
Proc. Natl. Acad. Sci. USA
Molecular biology of the LysR family of transcriptional regulators
Annu. Rev. Microbiol.
ExtraTrain: a database of extragenic regions and transcriptional information in prokaryotic organisms
BMC Microbiol.
Atomic structure and specificity of bacterial periplasmic receptors for active transport and chemotaxis: variation of common themes
Mol. Microbiol.
Solution of the structure of the cofactor-binding fragment of CysB: a struggle against non-isomorphism
Acta Crystallog. sect. D
Transcriptional cross-regulation of the catechol and protocatechuate branches of the beta-ketoadipate pathway contributes to carbon source-dependent expression of the Acinetobacter sp. strain ADP1 pobA gene
Appl. Environ. Microbiol.
Regulation of benzoate degradation in Acinetobacter sp. strain ADP1 by BenM, a LysR-type transcriptional activator
J. Bacteriol.
Benzoate decreases the binding of cis,cis-muconate to the BenM regulator despite the synergistic effect of both compounds on transcriptional activation
J. Bacteriol.
Crystallization of the effector-binding domains of BenM and CatM, LysR-type transcriptional regulators from Acinetobacter sp. ADP1
Acta Crystallog. sect. D
Automated MAD and MIR structure solution
Acta Crystallog. sect. D
Bacterial transcriptional regulators for degradation pathways of aromatic compounds
Microbiol. Mol. Biol. Rev.
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O. E. and S. H. made equal scientific contributions to this article.
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Present addresses: O. C. Ezezika, Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA; T. J. Clark, Logistics Health Inc., 1319 St. Andrew Street, La Crosse, WI 54603, USA.