Single-Molecule Dynamics and Mechanisms of Metalloregulators and Metallochaperones

Chen, Peng; Keller, Aaron M.; Joshi, Chandra P.; Martell, Danya J.; Andoy, Nesha May; Benítez, Jaime J.; Chen, Tai-Yen; Santiago, Ace George; Yang, Feng

doi:10.1021/bi400597v

Cited by 15 publications

(18 citation statements)

References 149 publications

(304 reference statements)

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“…Both of these processes likely involve a ternary complex as a common intermediate, in which two protein molecules each partially bind to the recognition site ( Fig. 5c ), as we proposed previously 37 . This ternary complex is possible because of the homodimeric nature of CueR and ZntR: they can form bivalent contacts with DNA in which their two DNA-binding domains bind to the two halves of their dyad-symmetric recognition sequences ( Fig.…”

Section: Discussionsupporting

confidence: 65%

“…Therefore, both apo and holo forms of CueR and ZntR have concentration-dependent unbinding kinetics from chromosomal recognition sites, where the dependence likely results from the assisted dissociation or direct substitution mechanism that we discovered for CueR−DNA interactions in vitro 12 (see Discussion and Supplementary Fig. 41b and Supplementary Note 18.2 ) 15 37 38 39 40 .…”

Section: Resultsmentioning

confidence: 83%

“…5e ). Although no evidence exists for higher-order oligomeric CueR or ZntR complexes on regular DNA, our previous in vitro study has revealed a related ternary complex in CueR interactions with an engineered DNA Holliday junction 37 45 . Relatedly, ternary complexes involving multivalent contacts with DNA were also proposed to rationalize the concentration-dependent unbinding from DNA of nonspecific chromosomal organization proteins 38 39 that are dimeric and of a single-strand-DNA-binding protein 15 that has multiple DNA-binding domains.…”

Section: Discussionmentioning

confidence: 81%

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Concentration- and chromosome-organization-dependent regulator unbinding from DNA for transcription regulation in living cells

et al. 2015

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Binding and unbinding of transcription regulators at operator sites constitute a primary mechanism for gene regulation. While many cellular factors are known to regulate their binding, little is known on how cells can modulate their unbinding for regulation. Using nanometer-precision single-molecule tracking, we study the unbinding kinetics from DNA of two metal-sensing transcription regulators in living Escherichia coli cells. We find that they show unusual concentration-dependent unbinding kinetics from chromosomal recognition sites in both their apo- and holo-forms. Unexpectedly, their unbinding kinetics further varies with the extent of chromosome condensation, and more surprisingly, varies in opposite ways for their apo-repressor vs. holo-activator forms. These findings suggest likely broadly relevant mechanisms for facile switching between transcription activation and deactivation in vivo and in coordinating transcription regulation of resistance genes with the cell cycle.

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Section: Discussionsupporting

confidence: 65%

Section: Resultsmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 81%

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Concentration- and chromosome-organization-dependent regulator unbinding from DNA for transcription regulation in living cells

et al. 2015

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“…It forms homodimers with three domains: a N-terminal DNA-binding domain, a central dimerization helix, and a C-terminal region where two cysteines are responsible for metal binding (12,17). CueR homologs bind Cu ϩ with 10 Ϫ19 -10 Ϫ21 M affinities (12,19) and both forms of the regulator, CueR apo and CueR holo , bind the cognate promoter regions (20,21). The high binding affinity of the sensor has led to assume that there is no "free" cytoplasmic Cu ϩ (12).…”

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confidence: 99%

The interplay of the metallosensor CueR with two distinct CopZ chaperones defines copper homeostasis in Pseudomonas aeruginosa

Novoa‐Aponte

Ramírez

Argüello

2019

Journal of Biological Chemistry

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Edited by Chris WhitfieldCopper homeostasis in pathogenic bacteria is critical for cuproprotein assembly and virulence. However, in vivo biochemical analyses of these processes are challenging, which has prevented defining and quantifying the homeostatic interplay between Cu ؉ -sensing transcriptional regulators, chaperones, and sequestering molecules. The cytoplasm of Pseudomonas aeruginosa contains a Cu ؉ -sensing transcriptional regulator, CueR, and two homologous metal chaperones, CopZ1 and CopZ2, forming a unique system for studying Cu ؉ homeostasis. We found here that both chaperones exchange Cu ؉ , albeit at a slow rate, reaching equilibrium after 3 h, a time much longer than P. aeruginosa duplication time. Therefore, they appeared as two separate cellular Cu ؉ pools. Although both chaperones transferred Cu ؉ to CueR in vitro, experiments in vivo indicated that CopZ1 metallates CueR, eliciting the translation of Cu ؉ efflux transporters involved in metal tolerance. Although this observation was consistent with the relative Cu ؉ affinities of the three proteins (CopZ1 < CueR < CopZ2), in vitro and in silico analyses also indicated a stronger interaction between CopZ1 and CueR that was independent of Cu ؉ . In contrast, CopZ2 function was defined by its distinctly high abundance during Cu 2؉ stress. Under resting conditions, CopZ2 remained largely in its apo form. Metal stress quickly induced CopZ2 expression, and its holo form predominated, reaching levels commensurate with the cytoplasmic Cu ؉ levels. In summary, these results show that CopZ1 acts as chaperone delivering Cu ؉ to the CueR sensor, whereas CopZ2 functions as a fast-response Cu ؉ -sequestering storage protein. We propose that equivalent proteins likely play similar roles in most bacterial systems. by guest on July 4, 2020 http://www.jbc.org/ Downloaded from Figure 1. Structural differences of CopZ1 and CopZ2. Conserved Cu ϩ binding motifs of (A) CopZ1-like and (B) CopZ2-like proteins. C, native PAGE gel of purified CopZ1 and CopZ2 in the absence (left) and presence of equimolar amounts of Cu ϩ (right). The vertical dividing line in panel C indicates where the image has been spliced; all signals were from an identical original image and have not been altered. Arrow indicates multimeric structures of CopZ2. Cytoplasmic Cu ؉ distribution in P. aeruginosa J. Biol. Chem. (2019) 294(13) 4934 -4945 4935 by guest on July 4, 2020 http://www.jbc.org/ Downloaded from Figure 10. Model of Cu ؉ homeostasis via CopZ1, CopZ2, and CueR interplay in the cytoplasm of P. aeruginosa. Three different landscapes are represented. Resting conditions (left) in the absence of Cu-stress, where CopZ1 (yellow) and CopZ2 (blue) appear to be at similar levels and CopZ2 is only partially metallated. Early response (central panel) takes place within 1-3 min of external Cu 2ϩ exposure. Once Cu enters into the cytoplasm, CopZ1 metallates the sensor CueR (orange) leading to transcriptional activation of the CueR regulon genes (dotted box), whereas CopZ2 acts as an early Cu ϩ stor...

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“…2e, lower) 10, 11 . The rates of both pathways depend linearly on the free protein concentration, and both likely occur through a common ternary protein 2 -DNA complex, in which the two homodimeric proteins each use one DNA-binding domain to bind to half of the dyad recognition sequence 5, 24 . As Zur is also a homodimer, Zur also could form this ternary complex and undergo assisted dissociation or direct substitution, leading to its concentration-facilitated unbinding from DNA.…”

Section: Resultsmentioning

confidence: 99%

Biphasic unbinding of Zur from DNA for transcription (de)repression in Live Bacteria

2018

Preprint

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11Transcription regulator on-off binding to DNA constitutes a mechanistic paradigm in gene 12 regulation, in which the repressors/activators bind to operator sites tightly while the corresponding non-13repressors/non-activators do not. Another paradigm regards regulator unbinding from DNA to be a 14 unimolecular process whose kinetics is independent of regulator concentration. Using single-molecule 15 single-cell measurements, we find that the behaviors of the zinc-responsive uptake regulator Zur 16 challenges these paradigms. Apo-Zur, a non-repressor and presumed non-DNA binder, can bind to 17 chromosome tightly in live E. coli cells, likely at non-consensus sequence sites. Moreover, the 18 unbinding from DNA of its apo-non-repressor and holo-repressor forms both show a biphasic, 19repressed-followed-by-facilitated kinetics with increasing cellular protein concentrations. The 20 facilitated unbinding likely occurs via a ternary complex formation mechanism; the repressed unbinding 21is first-of-its-kind and likely results from protein oligomerization on chromosome, in which an inter-22 protein salt-bridge plays a key role. This biphasic unbinding could provide functional advantages in 23Zur's facile switching between repression and derepression. 24 25

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Single-Molecule Dynamics and Mechanisms of Metalloregulators and Metallochaperones

Cited by 15 publications

References 149 publications

Concentration- and chromosome-organization-dependent regulator unbinding from DNA for transcription regulation in living cells

Concentration- and chromosome-organization-dependent regulator unbinding from DNA for transcription regulation in living cells

The interplay of the metallosensor CueR with two distinct CopZ chaperones defines copper homeostasis in Pseudomonas aeruginosa

Biphasic unbinding of Zur from DNA for transcription (de)repression in Live Bacteria

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