The DNA Binding Arm of λ Repressor: Critical Contacts from a Flexible Region

Clarke, Neil D.; Beamer, Lesa J.; Goldberg, Harry R.; Berkower, Carol; Pabo, Carl O.

doi:10.1126/science.1833818

Cited by 91 publications

(50 citation statements)

References 10 publications

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“…Figure 1B shows the crystal structure of a 92-residue fragment that can still dimerize and bind to DNA. 15,16 Based on this structure, Huang and Oas selected an 80-residue fragment (λ ) that favors the monomeric state Figure 1A), making it appropriate for folding studies. 17 In the 92-residue fragment, helix five is extended by seven residues and forms important packing interactions between the two members of the dimer.…”

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Atomistic Folding Simulations of the Five-Helix Bundle Protein λ₆₋₈₅

2010

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Protein folding is a classic grand challenge that is relevant to numerous human diseases, such as protein misfolding diseases like Alzheimer's. Solving the folding problem will ultimately require a combination of theory, simulation, and experiment; with theory and simulation providing an atomically-detailed picture of both the thermodynamics and kinetics of folding and experimental tests grounding these models in reality. However, theory and simulation generally fall orders of magnitude short of biologically relevant timescales. Here we report significant progress towards closing this gap: an atomistic model of the folding of an 80-residue fragment of the λ repressor protein with explicit solvent that captures dynamics on 10 millisecond timescales. In addition, we provide a number of predictions that warrant further experimental investigation. For example, our model's native state is a kinetic hub and biexponential kinetics arise from the presence of many free energy basins separated by barriers of different heights rather than a single low barrier along one reaction coordinate (the previously proposed incipient downhill scenario).Understanding protein folding is a long-standing problem with important medical applications, such as elucidating the role of protein misfolding in diseases like Alzheimer's. Solving the folding problem will ultimately require a combination of theory, simulation, and experiment; with theory and simulation providing an atomically-detailed picture of both the thermodynamics and kinetics of folding and experimental tests grounding these models in reality. However, modeling long timescale dynamics (e.g. microseconds, milliseconds, and beyond) with sufficient statistical accuracy and chemical detail to make a quantitative connection with experiments is extremely challenging. Much progress has been made with small, fast-folding proteins (less than 40 residues and one millisecond folding timescales 1 ) but can the methods used scale to larger, slower systems? Here we report significant progress in this direction: an atomistic model of the folding of an 80-residue fragment of the λ repressor protein (λ 6-85 ) with explicit solvent that captures dynamics on a 10 millisecond timescale.This advance builds on a growing body of work on describing molecular kinetics with network models called Markov state models (MSMs). MSMs are discrete time master equation models that essentially serve as maps of a molecule's conformational space. [1][2][3] The states in an MSM come from kinetic clustering of atomistic simulations (i.e. conformations that can interconvert rapidly are grouped together into what is called a metastable state). Thus, these models are an important advance over previous approaches, like diffusion-collision models, 4,5 as an MSM's states are derived from dynamics in detailed pande@stanford.edu. Supporting Information Available: Methods, Figure S1 to S14, To test whether the MSM approach can scale to larger systems, we have built MSMs for the D14A mutant of λ 6-85 ( Figure 1A). 12...

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Atomistic Folding Simulations of the Five-Helix Bundle Protein λ₆₋₈₅

2010

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“…1C), a Lys (K4, where the numbering does not include the removed fMet), forms H bonds in the major groove to the O6 positions of two consecutive Gs in the operator DNA. Any substitutions at K4 (e.g., with Gln, yielding K4Q in CI) greatly reduce binding despite the fact that the HTH motif is unchanged (5,18). To test whether the N-terminal region of Lrp has a functional role similar to that of N-terminal CI, we generated the corresponding K6Q substitution in EcoLrp (Fig.…”

Section: Comparative Effects Of An N-terminal Tail Substitution In Lrmentioning

confidence: 99%

“…The alignment also shows the similarity to the N terminus of the CI activator/repressor from phage. The "actives" are substitutions in this region of CI that retained the ability to maintain lysogeny (18,23,36). Letters in red are Lrp residues differing from EcoLrp.…”

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Recognition of DNA by the Helix-Turn-Helix Global Regulatory Protein Lrp Is Modulated by the Amino Terminus

et al. 2011

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The AsnC/Lrp family of regulatory proteins links bacterial and archaeal transcription patterns to metabolism. In Escherichia coli, Lrp regulates approximately 400 genes, over 200 of them directly. In earlier studies, lrp genes from Vibrio cholerae, Proteus mirabilis, and E. coli were introduced into the same E. coli background and yielded overlapping but significantly different regulons. These differences were seen despite amino acid sequence identities of 92% (Vibrio) and 98% (Proteus) to E. coli Lrp, including complete conservation of the helix-turn-helix motifs. The N-terminal region contains many of the sequence differences among these Lrp orthologs, which led us to investigate its role in Lrp function. Through the generation of hybrid proteins, we found that the N-terminal diversity is responsible for some of the differences between orthologs in terms of DNA binding (as revealed by mobility shift assays) and multimerization (as revealed by gel filtration, dynamic light scattering, and analytical ultracentrifugation). These observations indicate that the N-terminal tail plays a significant role in modulating Lrp function, similar to what is seen for a number of other regulatory proteins.

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“…Structural data are available for each of these classes of DNA-binding domains (18). In addition to these highly conserved motifs, an additional element, adjacent to the motif, can contribute to binding by making direct base pair contacts, as in the lambda repressor and in the homeodomains (8,56). These adjacent regions are more difficult to identify, since they generally contribute less to the binding than the conserved motif and do not have a well-defined secondary structure in either the absence or the presence of DNA (8,37,40,56).…”

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A mutation outside the two zinc fingers of ADR1 can suppress defects in either finger.

Camier¹,

Kacherovsky²,

Young³

1992

Mol. Cell. Biol.

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A second-site mutation that restored DNA binding to ADR1 mutants altered at different positions in the two zinc fingers was identified. This mutation (called IS1) was a conservative change of arginine 91 to lysine in a region amino terminal to the two zinc fingers and known from previous experiments to be necessary for DNA binding. IS1 increased binding to the UAS1 sequence two-to sevenfold for various ADR1 mutants and twofold for wild-type ADR1. The change of arginine 91 to glycine decreased binding twofold, suggesting that this arginine is involved in DNA binding in the wild-type protein. The increase in binding by IS1 did not involve protein-protein interactions between the two ADR1 monomers, nor did it require the presence of the sequences flanking UAS1. However, the effect of IS1 was influenced by the sequence of the first finger, suggesting that interactions between the region amino terminal to the fingers and the fingers themselves could exist. A model for the role of the amino-terminal region based on these results and sequence homologies with other DNA-binding motifs is proposed.Recognition of specific DNA sequences is a critical step in many events of cell life. The combination of genetic analysis and structural studies has allowed the identification and characterization of a large number of DNA-binding domains responsible for recognizing and binding specific DNA sequences (45). These domains generally behave as independent units which can be swapped between different proteins. For each unit, the binding often occurs between an alphahelical structure and the major groove of DNA. The specificity of binding is achieved primarily by the formation of hydrogen bonds between the amino acids and the bases. Interactions with atoms of the sugar phosphate backbone are also important in positioning the domains to allow them to align correctly with the bases. Several structures that have DNA-binding properties have now been identified. Among them are the helix-turn-helix motifs found in prokaryotic proteins, such as the lambda repressor (43), and in eukaryotic homeodomains (27,40); the three classes of zinc fingers (3); the leucine zipper, which is characterized by its dimerization motif (36); and the beta-ribbon motif found in some prokaryotic proteins (48). Structural data are available for each of these classes of . In addition to these highly conserved motifs, an additional element, adjacent to the motif, can contribute to binding by making direct base pair contacts, as in the lambda repressor and in the homeodomains (8, 56). These adjacent regions are more difficult to identify, since they generally contribute less to the binding than the conserved motif and do not have a well-defined secondary structure in either the absence or the presence of DNA (8,37,40,56).In this report, we describe a mutation outside the zinc finger region of ADR1 which enhances the DNA-binding activity of the protein. ADR1 ADH2 promoter, the UAS1 sequence. Even though single molecules of ADR1 bind independently to each half of UAS1,...

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The DNA Binding Arm of λ Repressor: Critical Contacts from a Flexible Region

Cited by 91 publications

References 10 publications

Atomistic Folding Simulations of the Five-Helix Bundle Protein λ₆₋₈₅

Atomistic Folding Simulations of the Five-Helix Bundle Protein λ₆₋₈₅

Recognition of DNA by the Helix-Turn-Helix Global Regulatory Protein Lrp Is Modulated by the Amino Terminus

A mutation outside the two zinc fingers of ADR1 can suppress defects in either finger.

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The DNA Binding Arm of λ Repressor: Critical Contacts from a Flexible Region

Cited by 91 publications

References 10 publications

Atomistic Folding Simulations of the Five-Helix Bundle Protein λ6−85

Atomistic Folding Simulations of the Five-Helix Bundle Protein λ6−85

Recognition of DNA by the Helix-Turn-Helix Global Regulatory Protein Lrp Is Modulated by the Amino Terminus

A mutation outside the two zinc fingers of ADR1 can suppress defects in either finger.

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Atomistic Folding Simulations of the Five-Helix Bundle Protein λ₆₋₈₅

Atomistic Folding Simulations of the Five-Helix Bundle Protein λ₆₋₈₅