Distinct Effector-binding Sites Enable Synergistic Transcriptional Activation by BenM, a LysR-type Regulator

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Abstract

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.

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

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    O. E. and S. H. made equal scientific contributions to this article.

    2

    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.

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