Structural Basis of the Transcriptional Regulation of the Proline Utilization Regulon by Multifunctional PutA

https://doi.org/10.1016/j.jmb.2008.05.084Get rights and content

Abstract

The multifunctional Escherichia coli proline utilization A (PutA) flavoprotein functions both as a membrane-associated proline catabolic enzyme and as a transcriptional repressor of the proline utilization genes putA and putP. To better understand the mechanism of transcriptional regulation by PutA, we have mapped the put-regulatory region, determined a crystal structure of the PutA ribbon–helix–helix domain (PutA52, a polypeptide corresponding to residues 1–52 of E. coli PutA) complexed with DNA, and examined the thermodynamics of DNA binding to PutA52. Five operator sites, each containing the sequence motif 5′-GTTGCA-3′, were identified using gel-shift analysis. Three of the sites are shown to be critical for repression of putA, whereas the two other sites are important for repression of putP. The 2.25-Å-resolution crystal structure of PutA52 bound to one of the operators (operator 2; 21 bp) shows that the protein contacts a 9-bp fragment corresponding to the GTTGCA consensus motif plus three flanking base pairs. Since the operator sequences differ in flanking bases, the structure implies that PutA may have different affinities for the five operators. This hypothesis was explored using isothermal titration calorimetry. The binding of PutA52 to operator 2 is exothermic, with an enthalpy of − 1.8 kcal/mol and a dissociation constant of 210 nM. Substitution of the flanking bases of operator 4 into operator 2 results in an unfavorable enthalpy of 0.2 kcal/mol and a 15-fold-lower affinity, showing that base pairs outside of the consensus motif impact binding. Structural and thermodynamic data suggest that hydrogen bonds between Lys9 and bases adjacent to the GTTGCA motif contribute to transcriptional regulation by fine-tuning the affinity of PutA for put control operators.

Introduction

Proline is used as a source of carbon, nitrogen, and energy through two oxidative steps catalyzed by proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH).1, 2, 3, 4, 5, 6, 7 In enteric bacteria such as Escherichia coli, proline utilization requires two genes, putP and putA. The former encodes the PutP high-affinity Na +-proline transporter, and the latter encodes the multifunctional flavoprotein proline utilization A (PutA).8, 9 PutA is unique in that it functions both as a transcriptional repressor of the put genes and as a membrane-associated bifunctional proline catabolic enzyme.2, 10, 11, 12 The enzymatic and transport functions of the putA and putP genes, respectively, are conserved among different Gram-negative bacteria, whereas the genetic organization and regulatory mechanisms that control the expression of these genes are highly divergent.7, 10, 11, 12, 13, 14, 15, 16, 17, 18 The focus of this work is to provide a molecular and structural understanding of the regulation of put genes in E. coli by PutA.

PutA from E. coli combines PRODH, P5CDH, and transcriptional regulatory activities into a single polypeptide of 1320 amino acids.2, 19 Insights into the organization of the functional domains in PutA have been gained from molecular dissection and characterization of truncated PutA proteins. The PRODH and P5CDH active sites are located within residues 261–612 and 650–1130, respectively, with the PRODH active site utilizing a FAD cofactor and with P5CDH activity requiring NAD +. Structural studies have shown that the PRODH domain forms a unique (βα)8 barrel,20, 21 and that reduction by dithionite causes dramatic conformational changes in the FAD ribityl chain.22 Molecular dissection studies showed that the DNA-binding domain is contained in residues 1–47.23 Subsequently, the crystal structure of PutA52 (a polypeptide corresponding to residues 1–52 of E. coli PutA) was solved, showing that PutA is a member of the ribbon–helix–helix (RHH) family of transcriptional regulators.23, 24

While knowledge of PutA structure and function continues to build, a considerable gap in our understanding of critical PutA–DNA interactions in the put control DNA region remains. To further understand the regulation of proline metabolism in E. coli, we have identified the PutA binding sites in the put-regulatory region, elucidated the roles of these operators in repressing the expression of putA and putP, determined the crystal structure of PutA52 bound to one of the identified operators, and investigated the thermodynamics of DNA binding to PutA52 using isothermal titration calorimetry (ITC).

Section snippets

Identification of PutA binding sites

Initial localization of PutA binding sites in the put control DNA region was performed by gel mobility shift assays using different fragments of the 419-bp put control DNA. Systematic evaluation of different regions of the put control DNA indicated that PutA does not bind to the 1- to 170-bp region immediately downstream of putP (Fig. 1a, lanes 3 and 4). However, PutA was observed by gel mobility shift assays to bind to regions 183–231 and 342–412 of the put control DNA (data not shown).

Transcriptional regulation of the put regulon

Based on the arrangement of the five PutA–DNA binding sites, PutA most likely represses the put genes by hindering the σ70-dependent binding of E. coli RNA polymerase to the putA and putP promoter regions.32 We did not find additional PutA consensus binding sites in the coding regions of putA and putP, indicating that PutA binds only to the put intergenic region. Previous reports suggested that proline, via PutA, regulates the expression of putA more tightly than putP.25, 27 Here we have shown

Materials

Chemicals and buffers were purchased from Fisher Scientific and Sigma-Aldrich, Inc., unless otherwise stated. Restriction endonucleases and T4 DNA ligase were purchased from Fermentas and Invitrogen, respectively. BCA reagents used for protein quantitation were obtained from Pierce. Goat anti-rabbit secondary antibody was purchased from Amersham, Inc. E. coli strains XL-blue and BL21 DE3 pLysS were purchased from Stratagene. E. coli strain JT31 putA lacZ was a generous gift from J. Wood

Acknowledgements

This research was supported by National Institutes of Health grants GM065546 (J.J.T.) and GM061068 (D.F.B.), and National Science Foundation grant MCB0091664 (D.F.B.). C.A.B. was supported by a postdoctoral fellowship from the National Library of Medicine (2-T15-LM07089-14). We thank Damian Coventry and Paul Bourke for providing the computer code used in penetration depth calculations. Part of this research was performed at the Advanced Light Source, which is supported by the Director, Office

References (53)

  • P. Chakrabarti et al.

    C–H·O hydrogen bond involving proline residues in alpha-helices

    J. Mol. Biol.

    (1998)
  • K. Mattison et al.

    Structure of FitAB from Neisseria gonorrhoeae bound to DNA reveals a tetramer of toxin–antitoxin heterodimers containing pin domains and ribbon–helix–helix motifs

    J. Biol. Chem.

    (2006)
  • B.W. Matthews

    Solvent content of protein crystals

    J. Mol. Biol.

    (1968)
  • J.L.A. Abrahamson et al.

    Proline dehydrogenase from Escherichia K12, properties of the membrane-associated enzyme

    Eur. J. Biochem.

    (1983)
  • P.F. Straub et al.

    Isolation, DNA sequence analysis, and mutagenesis of a proline dehydrogenase gene (putA) from Bradyrhizobium japonicum

    Appl. Environ. Microbiol.

    (1996)
  • N. Krishnan et al.

    Characterization of a bifunctional PutA homologue from Bradyrhizobium japonicum and identification of an active site residue that modulates proline reduction of the flavin adenine dinucleotide cofactor

    Biochemistry

    (2005)
  • N. Krishnan et al.

    Oxygen reactivity of PutA from Helicobacter species and proline-linked oxidative stress

    J. Bacteriol.

    (2006)
  • P. Ostrovsky De Spicer et al.

    Regulation of proline utilization in Salmonella typhimurium: a membrane-associated dehydrogenase binds DNA in vitro

    J. Bacteriol.

    (1991)
  • S. Vílchez et al.

    Proline catabolism by Pseudomonas putida: cloning, characterization, and expression of the put genes in the presence of root exudates

    J. Bacteriol.

    (2000)
  • L.-M. Chen et al.

    Regulation of proline utilization in enteric bacteria: cloning and characterization of the Klebsiella put control region

    J. Bacteriol.

    (1991)
  • S. Vílchez et al.

    Control of expression of divergent Pseudomonas putida put promoters for proline catabolism

    Appl. Environ. Microbiol.

    (2000)
  • B. Keuntje et al.

    Expression of the putA gene encoding proline dehydrogenase from Rhodobacter capsulatus is independent of NtrC regulation but requires an Lrp-like activator protein

    J. Bacteriol.

    (1995)
  • Y. Nakada et al.

    Divergent structure and regulatory mechanism of proline catabolic systems: characterization of the putAP proline catabolic operon of Pseudomonas aeruginosa PAO1 and its regulation by PruR, an AraC/XylS family protein

    J. Bacteriol.

    (2002)
  • J.H. Lee et al.

    Coactivation of Vibrio vulnificus putAP operon by cAMP receptor protein and PutR through cooperative binding to overlapping sites

    Mol. Microbiol.

    (2006)
  • M. Zhang et al.

    Structures of the Escherichia coli PutA proline dehydrogenase domain in complex with competitive inhibitors

    Biochemistry

    (2004)
  • Y.H. Lee et al.

    Structure of the proline dehydrogenase domain of the multifunctional PutA flavoprotein

    Nat. Struct. Biol.

    (2003)
  • Cited by (0)

    The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

    Y.Z. and J.D.L. contributed equally to this research.

    View full text