Identification and characterization of aspartate residues that play key roles in the allosteric regulation of a transcription factor: aspartate 274 is essential for inducer binding in lac repressor

Chang, Wen-I; Barrera, Pamela; Matthews, Kathleen S.

doi:10.1021/bi00178a018

Cited by 15 publications

(21 citation statements)

References 58 publications

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“…Mutations of D88, which anchors K84 in the wild‐type induced conformation, also decrease inducibility of the repressor (Chang et al 1994; Suckow et al 1996). Mutating D88 would deprive the K84 cation of a negative anchor in the induced state.…”

Section: Resultsmentioning

confidence: 99%

Allosteric transition pathways in the lactose repressor protein core domains: Asymmetric motions in a homodimer

Flynn

Swint-Kruse

Kong³

et al. 2003

Protein Science

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The crystal structures of lactose repressor protein (LacI) provide static endpoint views of the allosteric transition between DNA-and IPTG-bound states. To obtain an atom-by-atom description of the pathway between these two conformations, motions were simulated with targeted molecular dynamics (TMD). Strikingly, this homodimer exhibited asymmetric dynamics. All asymmetries observed in this simulation are reproducible and can begin on either of the two monomers. Asymmetry in the simulation originates around D149 and was traced back to the pre-TMD equilibrations of both conformations. In particular, hydrogen bonds between D149 and S193 adopt a variety of configurations during repetitions of this process. Changes in this region propagate through the structure via noncovalent interactions of three interconnected pathways. The changes of pathway 1 occur first on one monomer. Alterations move from the inducer-binding pocket, through the N-subdomain ␤-sheet, to a hydrophobic cluster at the top of this region and then to the same cluster on the second monomer. These motions result in changes at (1) side chains that form an interface with the DNA-binding domains and (2) K84 and K84Ј, which participate in the monomer-monomer interface. Pathway 2 reflects consequent reorganization across this subunit interface, most notably formation of a H74-H74Ј -stacking intermediate. Pathway 3 extends from the rear of the inducer-binding pocket, across a hydrogen-bond network at the bottom of the pocket, and transverses the monomer-monomer interface via changes in H74 and H74Ј. In general, intermediates detected in this study are not apparent in the crystal structures. Observations from the simulations are in good agreement with biochemical data and provide a spatial and sequential framework for interpreting existing genetic data.Keywords: Gene regulation; allosteric mechanism; structural flexibility; conformational transition pathway; targeted molecular dynamics Supplemental material: See www.proteinscience.org Since its discovery in 1961, the lactose (lac) operon has been the prototypic model for studying genetic and allosteric regulation (Jacob and Monod 1961;Monod et al. 1965;Matthews and Nichols 1998;Matthews et al. 2000). The central component of this regulatory system-lactose repressor protein (LacI)-binds to three operator sites within the lac operon to repress gene transcription (Matthews 1992). Inducer sugars, such as allolactose (Jobe and Bourgeois 1972) These authors made equal contributions to this work. Article and publication are at http://www.proteinscience.org/cgi

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Section: Resultsmentioning

confidence: 99%

Allosteric transition pathways in the lactose repressor protein core domains: Asymmetric motions in a homodimer

Flynn

Swint-Kruse

Kong³

et al. 2003

Protein Science

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“…Whether this interaction provides only affinity and specificity, or is also important to the coupling between cytidine binding and CytR⅐CRP cooperativity, cannot be determined from our data. However, we note that in LacI, only affinity is affected (46).…”

Section: Inducibility Of Wild-type-cid Heterodimers In Vivo-mentioning

confidence: 88%

Allosteric Mechanism of Induction of CytR-regulated Gene Expression

Barbier¹,

Short²,

Senear³

1997

Journal of Biological Chemistry

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Transcription from cistrons of the Escherichia coliOther CytR mutants that do not exhibit the CID phenotype were found to bind cytidine with affinity similar to wild-type CytR. The rate of transcription regulated by heterodimeric CytR composed of one CytR281N and one wild-type subunit was compared with that regulated by wild-type CytR under inducing conditions. The data support the conclusion that the first cytidine binding step alone is sufficient to induce.

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“…These authors likewise report that Q248R in a wild-type background is insensitive to inducer (Kleina and Miller 1990). D274 also contacts IPTG (Friedman et al 1995;Lewis et al 1996), and D274N/wild type cannot bind inducer in vitro (Chang et al 1994). D274G also has an inducer-insensitive phenotype, as does T276A (Suckow et al, 1996).…”

Section: Local Structural Effects: M223i/t N246s Q248r D274n/g T2mentioning

confidence: 98%

Plasticity of quaternary structure: Twenty‐two ways to form a LacI dimer

Swint-Kruse

2001

Protein Science

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The repressor proteins of the LacI/GalR family exhibit significant similarity in their secondary and tertiary structures despite less than 35% identity in their primary sequences. Furthermore, the core domains of these oligomeric repressors, which mediate dimerization, are homologous with the monomeric periplasmic binding proteins, extending the issue of plasticity to quaternary structure. To elucidate the determinants of assembly, a structure-based alignment has been created for three repressors and four periplasmic binding proteins. Contact maps have also been constructed for the three repressor interfaces to distinguish any conserved interactions. These analyses show few strict requirements for assembly of the core N-subdomain interface. The interfaces of repressor core C-subdomains are well conserved at the structural level, and their primary sequences differ significantly from the monomeric periplasmic binding proteins at positions equivalent to LacI 281 and 282. However, previous biochemical and phenotypic analyses indicate that LacI tolerates many mutations at 281. Mutations at LacI 282 were shown to abrogate assembly, but for Y282D this could be compensated by a second-site mutation in the core N-subdomain at K84 to L or A. Using the link between LacI assembly and function, we have further identified 22 second-site mutations that compensate the Y282D dimerization defect in vivo. The sites of these mutations fall into several structural regions, each of which may influence assembly by a different mechanism. Thus, the 360-amino acid scaffold of LacI allows plasticity of its quaternary structure. The periplasmic binding proteins may require only minimal changes to facilitate oligomerization similar to the repressor proteins.Keywords: Structural plasticity; quaternary structure; lactose repressor protein; purine repressor protein; periplasmic binding protein; trehalose repressor protein One fascinating aspect of protein folding is that a wide range of primary sequences can generate very similar secondary and tertiary structures. For example, Thornton and her colleagues have demonstrated that many sequences with less than 35% identity have the same folding topology (Swindells et al. 1998; this reference also provides a very helpful review of the various structural classification schemes currently available). Plasticity of folding is well illustrated by the LacI/GalR family of repressor proteins. Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ ps.35801. † The Escherichia coli LacI has long served as a prototype for proteins that negatively control transcription. In vivo, LacI binds tightly to a specific operator DNA site, preventing transcription of downstream lactose metabolic genes. If lactose becomes the predominant energy source, the bacteria first produce the metabolite allolactose, which binds to LacI and induces a conformational switch to a protein structure with only nonspecific DNA-binding affinity. The large excess of genomic bacterial DNA

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Identification and characterization of aspartate residues that play key roles in the allosteric regulation of a transcription factor: aspartate 274 is essential for inducer binding in lac repressor

Cited by 15 publications

References 58 publications

Allosteric transition pathways in the lactose repressor protein core domains: Asymmetric motions in a homodimer

Allosteric transition pathways in the lactose repressor protein core domains: Asymmetric motions in a homodimer

Allosteric Mechanism of Induction of CytR-regulated Gene Expression

Plasticity of quaternary structure: Twenty‐two ways to form a LacI dimer

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