Tandem binding in crystals of a trp represser/operator half-site complex

Lawson, Catherine L.; Carey, Jannette

doi:10.1038/366178a0

Cited by 151 publications

(129 citation statements)

References 25 publications

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“…1D). We chose residue 11 as the amino-terminal boundary of TrpR because this amino acid is the first helical residue in the crystal structure (35). Upon purification, we found that FMN binding and reversible photobleaching are preserved in all constructs (27).…”

Section: Discussionmentioning

confidence: 99%

Light-activated DNA binding in a designed allosteric protein

Strickland

Moffat

Sosnick

2008

Proc. Natl. Acad. Sci. U.S.A.

280

268

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An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an ␣-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical ''allosteric lever arm'' is a general scheme for coupling the function of two proteins.allostery ͉ LOV domain ͉ protein design ͉ trp repressor ͉ alpha helix

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Section: Discussionmentioning

confidence: 99%

Light-activated DNA binding in a designed allosteric protein

Strickland

Moffat

Sosnick

2008

Proc. Natl. Acad. Sci. U.S.A.

280

268

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“…Its structure in both the Trp-free apo-form and the Trp-bound holo-form, and the structure of its DNA complexes, have been determined by both X-ray and NMR analyses (Schevitz et al, 1985;Otwinowski et al, 1988;Lawson & Carey, 1993;Zhao et al, 1993;Zhang et al, 1994). The repressor is known to interact with at least five different operator sequences, i.e., in the trp, trpR, aroH, aroL, and mtr operons Reprint requests to: Oleg Jardetzky, Stanford Magnetic Resonance Laboratory, Stanford University, Stanford, California 94305.5055; e-mail: jardetzky@camis.stanford.edu.…”

Section: " " ~~~mentioning

confidence: 99%

Flexibility of dna binding domain of trp repressor required for recognition of different operator sequences

et al. 1996

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Trp repressor (25 kDa) is a regulatory protein that controls transcription initiation in the tryptophan biosynthetic operon and at least four other operons in Escherichia coli. An alanine to valine mutation (AV77) in the DNA binding domain is known to increase repressor activity at the trp operator in vivo, but not in vitro. We report here the amide proton exchange rates for the DNA-binding domains of both the wild-type and AV77 proteins. We find that the alanine to valine change stabilizes the flexible DNA-binding domain of the repressor. We present in vivo data showing that, although the AV77 repressor is more inhibitory at the trp operator than the wild-type repressor, it does not have increased activity at the aroH or trpR operator; repression at the aroH operator is, in fact, reduced. Our results suggest that the flexibility exhibited by the wild-type repressor allows a broader range of repressor/DNA interactions, whereas the increased rigidity resulting from the AV77 change limits the repressor's effectiveness at some operators.

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“…Structures are known for the corepressor-free and -bound forms of only two other repressors: the tryptophan repressor (TrpR) (27,(31)(32)(33) and the methionine repressor (MetJ) (34 -36). The TrpR and MetJ repressors are similar in that they both bind their corepressors within their DNA-binding domains.…”

Section: Fig 2 Structure Of the Purr-guanine-purf Operator Complexmentioning

confidence: 99%

The X-ray Structure of the PurR-Guanine-purF Operator Complex Reveals the Contributions of Complementary Electrostatic Surfaces and a Water-mediated Hydrogen Bond to Corepressor Specificity and Binding Affinity

Schumacher¹,

Glasfeld²,

Zalkin³

et al. 1997

Journal of Biological Chemistry

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The purine repressor, PurR, is the master regulatory protein of de novo purine nucleotide biosynthesis in Escherichia coli. This dimeric transcription factor is activated to bind to cognate DNA operator sites by initially binding either of its physiologically relevant, high affinity corepressors, hypoxanthine (K d ‫؍‬ 9.3 M) or guanine (K d ‫؍‬ 1.5 M). Here, we report the 2.5-Å crystal structure of the PurR-guanine-purF operator ternary complex and complete the atomic description of 6-oxopurine-induced repression by PurR. As anticipated, the structure of the PurR-guanine-purF operator complex is isomorphous to the PurR-hypoxanthine-purF operator complex, and their protein-DNA and protein-corepressor interactions are nearly identical. The former finding confirms the use of an identical allosteric DNA-binding mechanism whereby corepressor binding 40 Å from the DNA-binding domain juxtaposes the hinge regions of each monomer, thus favoring the formation and insertion of the critical minor groove-binding hinge helices. Strikingly, the higher binding affinity of guanine for PurR and the ability of PurR to discriminate against 2-oxopurines do not result from direct protein-ligand interactions, but rather from a water-mediated contact with the exocyclic N-2 of guanine, which dictates the presence of a donor group on the corepressor, and the better electrostatic complementarity of the guanine base and the corepressor-binding pocket.

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Tandem binding in crystals of a trp represser/operator half-site complex

Cited by 151 publications

References 25 publications

Light-activated DNA binding in a designed allosteric protein

Light-activated DNA binding in a designed allosteric protein

Flexibility of dna binding domain of trp repressor required for recognition of different operator sequences

The X-ray Structure of the PurR-Guanine-purF Operator Complex Reveals the Contributions of Complementary Electrostatic Surfaces and a Water-mediated Hydrogen Bond to Corepressor Specificity and Binding Affinity

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