Perturbation from a Distance:  Mutations that Alter LacI Function through Long-Range Effects

Swint-Kruse, Liskin; Zhan, Haifei; Fairbanks, Bonnie M.; Maheshwari, Atul; Matthews, Kathleen S.

doi:10.1021/bi035116x

Cited by 41 publications

(80 citation statements)

References 64 publications

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“…In wild-type repressor and many other variants, this value is ∼2 (30,49). The ratio decreases slightly in V52A, V52F, V52Q, and V52R, whereas this value is increased in V52H, V52S, and V52W.…”

Section: Iptg Binding Of V52 Mutantsmentioning

confidence: 96%

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Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

2006

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A significant number of eukaryotic regulatory proteins are predicted to have disordered regions. Many of these proteins bind DNA, which may serve as a template for protein folding. Similar behavior is seen in the prokaryotic LacI/GalR family of proteins that couple hinge-helix folding with DNA binding. These hinge regions form short α-helices when bound to DNA, but appear to be disordered in other states. An intriguing question is whether and to what degree intrinsic helix propensity contributes to the function of these proteins. In addition to its interaction with operator DNA, the LacI hinge helix interacts with the hinge helix of the homodimer partner as well as to the surface of the inducer-binding domain. To explore the hierarchy of these interactions, we made a series of substitutions in the LacI hinge helix at position 52, the only site in the helix that does not interact with DNA and/or the inducer-binding domain. The substitutions at V52 have significant effects on operator binding affinity and specificity, and several substitutions also impair functional communication with the inducer-binding domain. Results suggest that helical propensity of amino acids in the hinge region alone does not dominate function; helix-helix packing interactions appear to also contribute. Further, the data demonstrate that variation in operator sequence can overcome side chain effects on hinge helix folding and/or hinge•hinge interactions. Thus, this system provides a direct example whereby an extrinsic interaction (DNA binding) guides internal events that influence folding and functionality. KeywordsLacI; helical propensity; allostery; DNA binding Protein folding has long been a fundamental issue in biological sciences. Beyond the general question of how a polypeptide sequence adopts a three-dimensional structure, coupled folding and binding have been identified as a key feature of partially unstructured proteins in multiple regulatory processes (e.g., transcription regulation, signal transduction, membrane transport and signaling (6-9)). Indeed, more and more intrinsically disordered proteins are found to fold upon binding to their target partners, ligands, or even small ions (10-13). In the case of transcription regulation, mutual cooperative folding of both protein and DNA has been † This work was supported by NIH Grant GM22441 and Robert A. Welch Grant C-576 to Kathleen Shive Matthews. Liskin Swint-Kruse is supported by NIH Grant P20 RR17708 from the Institutional Development Award program of the National Center for Research Resources.*To whom correspondence should be addressed. Telephone: 713−348−4871; Fax: 713−348−6149; Email: E-mail: ksm@bioc.rice.edu.. observed (6,11,14,15). In this paper, coupled folding is explored in greater detail for the hinge helix of the lactose repressor protein (LacI) 1 . NIH Public AccessLacI is a premier model for the regulation of gene expression and allosteric transition in biological systems (2) 2 . This 150-kDa homotetramer is a dimer of functionally independent dimers, with ...

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Section: Iptg Binding Of V52 Mutantsmentioning

confidence: 96%

“…To further examine the effects of substitutions on the allosteric transition of this protein, operator release experiments were conducted. Nitrocellulose filter binding was used to monitor decreased levels of bound DNA as a function of inducer concentration (30,31,38,39). Results are presented in Table 3 and Figure 4.…”

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confidence: 99%

Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

2006

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“…With the published structures, site-directed mutagenesis has been used to test a number of structure/function theories (e.g. [91]), sometimes with outcomes that contradict structural predictions (e.g. [92]).…”

Section: Biophysical Analysis Of Laci Mutational Variantsmentioning

confidence: 99%

The lactose repressor system: paradigms for regulation, allosteric behavior and protein folding

Wilson

Zhan

Swint-Kruse

et al. 2006

Cell. Mol. Life Sci.

156

145

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In 1961, Jacob and Monod proposed the operon model for gene regulation based on metabolism of lactose in Escherichia coli. This proposal was followed by an explication of allosteric behavior by Monod and colleagues. The operon model rationally depicted how genetic mechanisms can control metabolic events in response to environmental stimuli via coordinated transcription of a set of genes with related function (e.g. metabolism of lactose). The allosteric response found in the lactose repressor and many other proteins has been extended to a variety of cellular signaling pathways in all organisms. These two models have shaped our view of modern molecular biology and captivated the attention of a surprisingly broad range of scientists. More recently, the lactose repressor monomer was used as a model system for experimental and theoretical explorations of protein folding mechanisms. Thus, the lac system continues to advance our molecular understanding of genetic control and the relationship between sequence, structure and function.

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“…The second flexible region in the LacI/GalR proteins is a three-stranded "pivot" between the N-and C-subdomains of the regulatory domains (20,21). Changes at this pivot occur when small, allosteric ligands bind the regulatory domain.…”

Section: Laci/galr Proteinsmentioning

confidence: 99%

Flexibility and Disorder in Gene Regulation: LacI/GalR and Hox Proteins

Bondos

Swint-Kruse

Matthews

2015

Journal of Biological Chemistry

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Perturbation from a Distance: Mutations that Alter LacI Function through Long-Range Effects

Cited by 41 publications

References 64 publications

Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

The lactose repressor system: paradigms for regulation, allosteric behavior and protein folding

Flexibility and Disorder in Gene Regulation: LacI/GalR and Hox Proteins

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