Metal-dependent allosteric activation and inhibition on the same molecular scaffold: the copper sensor CopY from <i>Streptococcus pneumoniae</i>

Glauninger, Hendrik; Zhang, Yifan; Higgins, Khadine A.; Jacobs, Alexander; Martin, Julia; Fu, Yue; Coyne, rd H. Jerome; Bruce, Kevin E.; Maroney, Michael J.; Clemmer, David E.; Capdevila, Daiana A.; Giedroc, David P.

doi:10.1039/c7sc04396a

Cited by 31 publications

(58 citation statements)

References 54 publications

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“…hirae copper repressor and the upstream binding repeats are highly similar to S . pneumoniae (32, 33), and contain the 10-base sequence, E . hirae operators upon initial observation lacked the extended regions flanking this sequence in pneumococcus (Figure 1B) (22).…”

Section: Resultsmentioning

confidence: 99%

“…Previous studies showed that CopY specifically bound to the cop operon operator in a sequence and metal specific manner as disrupting the operator bases or adding copper disrupted CopY binding, while adding manganese or iron had no detectable effect (32). These studies were done with only one full operator intact (32, 33). Thus, using an electric mobility shift assay (EMSA), we qualitatively tested CopY binding to DNA to the two-operator 61-base dsDNA fragment.…”

Section: Resultsmentioning

confidence: 99%

“…These motifs can also bind zinc with a stoichiometry of two CxC motifs needed to bind one zinc (33). Copper binding causes a conformational change in the copper repressor leading to the DNA-binding release of what was thought to be the full cop operon operator, T/GACAnnTGTA (where n represents any nucleotide), while zinc binding leads to tighter cop operon operator binding (32, 33). However, the atomic and protein structural detail of how binding metal directly leads to the conformational changes associated with DNA binding is currently unknown.…”

Section: Introductionmentioning

confidence: 99%

“…Multiple studies regarding the cop operon have been performed in S . pneumoniae (11, 17, 27, 32, 33, 35, 36). The pneumococcal cop operon contains, copY as the repressor, cupA as a membrane-associated copper chaperone, and copA as the copper-specific exporter (16, 27, 32).…”

Section: Introductionmentioning

confidence: 99%

“…CupA copper chelation allows for the recycling of CopY and its apo- or zinc-bound form to return to the cop operator to repress the operon. Pneumococcal CopY is homologous to several known antibiotic resistance repressors including BlaI, a Staphylococcus aureus MecI homolog that represses the gene for a β - lactamase (32, 33, 38). Like CopY, BlaI and MecI interact with a known operator sequence, TACA/TGTA, form a homodimer, and are mostly helical in secondary structure (32, 38).…”

Section: Introductionmentioning

confidence: 99%

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Characterization of consensus operator site for Streptococcus pneumoniae copper repressor, CopY

O’Brien

Alvin

Menghani

et al. 2019

Preprint

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Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems (cop operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repression. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, these homologs have been characterized to bind a 10-base consensus sequence T/GACAnnTGTA. Here, we bioinformatically and empirically characterize these operator sites across species using S. pneumoniae CopY as a guide for binding. By examining the 21-base repeat operators for the pneumococcal cop operon and comparing binding of recombinant CopY to this, and the operator sites found in Enterococcus hirae, we show using biolayer interferometry that the T/GACAnnTGTA sequence is essential to binding, but it is not sufficient. We determine a more comprehensive S. pneumoniae CopY operator sequence to be RnYKACAAATGTARnY (where “R” is purine, “Y” is pyrimidine, and “K” is either G or T) binding with an affinity of 28 nM. We further propose that the cop operon operator consensus site of pneumococcal homologs be RnYKACAnnYGTARnY. This study illustrates the necessity to explore bacterial operator sites further to better understand bacterial gene regulation.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Characterization of consensus operator site for Streptococcus pneumoniae copper repressor, CopY

O’Brien

Alvin

Menghani

et al. 2019

Preprint

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SAXSMoW 2.0: Online calculator of the molecular weight of proteins in dilute solution from experimental SAXS data measured on a relative scale

et al. 2018

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Knowledge of molecular weight, oligomeric states, and quaternary arrangements of proteins in solution is fundamental for understanding their molecular functions and activities. We describe here a program SAXSMoW 2.0 for robust and quick determination of molecular weight and oligomeric state of proteins in dilute solution, starting from a single experimental small‐angle scattering intensity curve, I(q), measured on a relative scale. The first version of this calculator has been widely used during the last decade and applied to analyze experimental SAXS data of many proteins and protein complexes. SAXSMoW 2.0 exhibits new features which allow for the direct input of experimental intensity curves and also automatic modes for quick determinations of the radius of gyration, volume, and molecular weight. The new program was extensively tested by applying it to many experimental SAXS curves downloaded from the open databases, corresponding to proteins with different shapes and molecular weights ranging from ~10 kDa up to about ~500 kDa and different shapes from globular to elongated. These tests reveal that the use of SAXSMoW 2.0 allows for determinations of molecular weights of proteins in dilute solution with a median discrepancy of about 12% for globular proteins. In case of elongated molecules, discrepancy value can be significantly higher. Our tests show discrepancies of approximately 21% for the proteins with molecular shape aspect ratios up to 18.

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Unique underlying principles shaping copper homeostasis networks

Novoa‐Aponte

Argüello

2022

J Biol Inorg Chem

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Copper is essential in cells as a cofactor for key redox enzymes. Bacteria have acquired molecular components that sense, uptake, distribute, and expel copper ensuring that cuproenzymes are metallated and steady-state metal levels are maintained. Toward preventing deleterious reactions, proteins bind copper ions with high affinities and transfer the metal via ligand exchange, warranting that copper ions are always complexed. Consequently, the directional copper distribution within cell compartments and across cell membranes requires specific dynamic interactions and metal exchange between cognate holo-apo protein partners. These metal exchange reactions are determined by thermodynamic and kinetics parameters and influenced by mass action. Then, copper distribution can be conceptualized as a molecular system of singular interacting elements that maintain a physiological copper homeostasis. This review focuses on the impact of copper high-affinity binding and exchange reactions on the homeostatic mechanisms, the conceptual models to describe the cell as a homeostatic system, the various molecule functions that contribute to copper homeostasis, and the alternative system architectures responsible for copper homeostasis in model bacteria. Graphical Abstract

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Metal-dependent allosteric activation and inhibition on the same molecular scaffold: the copper sensor CopY from Streptococcus pneumoniae

Abstract: The dynamics and marginal stability of CopY enable allosteric activation of DNA binding by Zn(ii) and inhibition by Cu(i).

Cited by 31 publications

References 54 publications

Characterization of consensus operator site for Streptococcus pneumoniae copper repressor, CopY

Characterization of consensus operator site for Streptococcus pneumoniae copper repressor, CopY

SAXSMoW 2.0: Online calculator of the molecular weight of proteins in dilute solution from experimental SAXS data measured on a relative scale

Unique underlying principles shaping copper homeostasis networks

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