Reengineering Cro Protein Functional Specificity with an Evolutionary Code

Hall, Branwen M.; Vaughn, Erin; Begaye, Adrian R.; Cordes, Matthew

doi:10.1016/j.jmb.2011.08.056

Cited by 5 publications

(14 citation statements)

References 49 publications

(74 reference statements)

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“…For comparison, ϕKO2 Cro, which has ∼90% sequence identity with N15 Cro and an identical cognate O R 3 sequence, binds O R 3 with apparent K D = 44 nM by EMSA under somewhat different conditions, and binds ϕKO2 O R 2 and O R 1 very weakly ( K D ∼ 1–2 μM) (45). The cognate affinities of N15/ϕKO2 Cro for O R 3 DNA are both weaker than that of λ Cro, which binds λ O R 3 with apparent K D = 3.6 ± 0.5 nM in under EMSA conditions comparable to those used here for N15 Cro (21).…”

Section: Resultsmentioning

confidence: 55%

“…For electrophoretic mobility shift assays of binding to the O R 3 site, two oligonucleotides (5′-GCAAAATTATAGCCAGCTATAAAGAGCG-3′ and its reverse complement) were obtained from Integrated DNA Technologies (Coralville, IA) and purified by urea-denatured polyacrylamide gel electrophoresis. End-labeling of one strand with 32 P and annealing of the two strands to form a duplex were then performed as described previously for λ Cro (21), resulting in a 28 base-pair singly 32 P- labeled duplex consisting of the 16 base-pair N15 O R 3 site flanked at each end by four base pairs of the natural flanking DNA sequence (18), and then further flanked at each end by two GC base pairs. O R 2 and O R 1 sites were constructed similarly using the same flanking DNA as the O R 3 site.…”

Section: Methodsmentioning

confidence: 99%

“…Electrophoretic mobility shift assays for N15 Cro were performed and imaged as described for λ Cro (21). At each concentration of Cro protein, the apparent fraction of DNA bound by Cro was measured as the intensity of the band representing the bound form divided by the total intensity measured for that lane of the gel.…”

Section: Methodsmentioning

confidence: 99%

“…The three pairings in λ Cro correspond to direct protein–DNA contacts observed in the λ Cro–DNA complex (20). We previously used the code to re-engineer the functional specificity of λ Cro (21).…”

Section: Introductionmentioning

confidence: 99%

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Extreme divergence between one-to-one orthologs: the structure of N15 Cro bound to operator DNA and its relationship to the λ Cro complex

Hall

Roberts

Cordes

2019

Nucleic Acids Research

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The gene cro promotes lytic growth of phages through binding of Cro protein dimers to regulatory DNA sites. Most Cro proteins are one-to-one orthologs, yet their sequence, structure and binding site sequences are quite divergent across lambdoid phages. We report the cocrystal structure of bacteriophage N15 Cro with a symmetric consensus site. We contrast this complex with an orthologous structure from phage λ, which has a dissimilar binding site sequence and a Cro protein that is highly divergent in sequence, dimerization interface and protein fold. The N15 Cro complex has less DNA bending and smaller DNA-induced changes in protein structure. N15 Cro makes fewer direct contacts and hydrogen bonds to bases, relying mostly on water-mediated and Van der Waals contacts to recognize the sequence. The recognition helices of N15 Cro and λ Cro make mostly nonhomologous and nonanalogous contacts. Interface alignment scores show that half-site binding geometries of N15 Cro and λ Cro are less similar to each other than to distantly related CI repressors. Despite this divergence, the Cro family shows several code-like protein–DNA sequence covariations. In some cases, orthologous genes can achieve a similar biological function using very different specific molecular interactions.

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Section: Resultsmentioning

confidence: 55%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Extreme divergence between one-to-one orthologs: the structure of N15 Cro bound to operator DNA and its relationship to the λ Cro complex

Hall

Roberts

Cordes

2019

Nucleic Acids Research

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“…To examine the effect of the crystal environment on protein conformation, we consider the Cro transcription factor from bacteriophage λ as a model system. λ Cro functions as a homodimer and is a paradigm system for studying protein‐DNA interactions, gene regulation, and protein fold evolution . In the dimer, the relatively rigid subunits display an α + β fold and are connected by a plastic β‐sheet region that permits a hinge‐like motion between the two domains [Fig.…”

Section: Introductionmentioning

confidence: 99%

Packing interface energetics in different crystal forms of the λ Cro dimer

Ahlstrom

Miyashita

2013

Proteins

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Variation among crystal structures of the λ Cro dimer highlights conformational flexibility. The structures range from a wild type closed to a mutant fully open conformation, but it is unclear if each represents a stable solution state or if one may be the result of crystal packing. Here we use molecular dynamics (MD) simulation to investigate the energetics of crystal packing interfaces and the influence of site-directed mutagenesis on them, in order to examine the effect of crystal packing on wild type and mutant Cro dimer conformation. Replica exchange MD of mutant Cro in solution shows that the observed conformational differences between the wild type and mutant protein are not the direct consequence of mutation. Instead, simulation of Cro in different crystal environments reveals that mutation affects the stability of crystal forms. Molecular Mechanics Poisson-Boltzmann Surface Area binding energy calculations reveal the detailed energetics of packing interfaces. Packing interfaces can have diverse properties in strength, energetic components, and some are stronger than the biological dimer interface. Further analysis shows that mutation can strengthen packing interfaces by as much as ~5 kcal/mol in either crystal environment. Thus, in the case of Cro, mutation provides an additional energetic contribution during crystal formation that may stabilize a fully open higher energy state. Moreover, the effect of mutation in the lattice can extend to packing interfaces not involving mutation sites. Our results provide insight into possible models for the effect of crystallization on Cro conformational dynamics and emphasize careful consideration of protein crystal structures.

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Bacteriophage Transcription Factor Cro Regulates Virulence Gene Expression in Enterohemorrhagic Escherichia coli

Hernandez-Doria

Sperandio

2018

Cell Host & Microbe

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Bacteriophage-encoded genetic elements control bacterial biological functions. Enterohemorrhagic Escherichia coli (EHEC) strains harbor lambda-phages encoding the Shiga-toxin (Stx), which is expressed during the phage lytic cycle and associated with exacerbated disease. Phages also reside dormant within bacterial chromosomes through their lysogenic cycle, but how this impacts EHEC virulence remains unknown. We find that during lysogeny the phage transcription factor Cro activates the EHEC type III secretion system (T3SS). EHEC lambdoid phages are lysogenic under anaerobic conditions when Cro binds to and activates the promoters of T3SS genes. Interestingly, the Cro sequence varies among phages carried by different EHEC outbreak strains, and these changes affect Cro-dependent T3SS regulation. Additionally, infecting mice with the related pathogen C. rodentium harboring the bacteriophage cro from EHEC results in greater T3SS gene expression and enhanced virulence. Collectively, these findings reveal the role of phages in impacting EHEC virulence and their potential to affect outbreak strains.

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Reengineering Cro Protein Functional Specificity with an Evolutionary Code

Cited by 5 publications

References 49 publications

Extreme divergence between one-to-one orthologs: the structure of N15 Cro bound to operator DNA and its relationship to the λ Cro complex

Extreme divergence between one-to-one orthologs: the structure of N15 Cro bound to operator DNA and its relationship to the λ Cro complex

Packing interface energetics in different crystal forms of the λ Cro dimer

Bacteriophage Transcription Factor Cro Regulates Virulence Gene Expression in Enterohemorrhagic Escherichia coli

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