title: X-ray structure of a Rex-family repressor/NADH complex insights into the mechanism of redox sensing.
abstract: The redox-sensing repressor Rex regulates transcription of respiratory genes in response to the intra cellular NADH/NAD(+) redox poise. As a step toward elucidating the molecular mechanism of NADH/NAD(+) sensing, the X-ray structure of Thermus aquaticus Rex (T-Rex) bound to effector NADH has been determined at 2.9 A resolution. The fold of the C-terminal domain of T-Rex is characteristic of NAD(H)-dependent enzymes, whereas the N-terminal domain is similar to a winged helix DNA binding motif. T-Rex dimerization is primarily mediated by "domain-swapped" alpha helices. Each NADH molecule binds to the C-terminal domain near the dimer interface. In contrast to NAD(H)-dependent enzymes, the nicotinamide is deeply buried within a hydrophobic pocket that appears to preclude substrate entry. We show that T-Rex binds to the Rex operator, and NADH but not NAD(+) inhibits T-Rex/DNA binding activity. A mechanism for redox sensing by Rex family members is proposed by analogy with domain closure of NAD(H)-dependent enzymes.
authors.affiliation: Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205, USA.
authors.forename: E Allen
authors.initials: EA
authors.lastname: Sickmier
authors.forename: Dimitris
authors.initials: D
authors.lastname: Brekasis
authors.forename: Shanthi
authors.initials: S
authors.lastname: Paranawithana
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Bonanno
authors.forename: Mark S B
authors.initials: MS
authors.lastname: Paget
authors.forename: Stephen K
authors.initials: SK
authors.lastname: Burley
authors.forename: Clara L
authors.initials: CL
authors.lastname: Kielkopf
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D016370:Gene Products, rex / Q000737:chemistry / Q000235:genetics*; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D009876:Operon; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013824:Thermus / Q000737:chemistry
chemical_list: D016370:Gene Products, rex; D012097:Repressor Proteins; D009243:NAD; D004247:DNA