Electron Spin Resonance Shows Common Structural Features for Different Classes of <i>Eco</i>RI–DNA Complexes

Stone, Katherine M.; Townsend, Jacqueline E.; Sarver, Jessica; Sapienza, Paul J.; Saxena, Sunil; Jen‐Jacobson, Linda

doi:10.1002/ange.200803588

Cited by 15 publications

(26 citation statements)

References 28 publications

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“…Equilibrium association constants (K A ) of Ser180Cys protein and MTSSL-Ser180Cys protein to DNA were determined as described (32). Experiments assessing Cu 2þ enhancement of binding used the same double-strand deoxyoligonucleotide TCGCGAATTCGCG as that in the ESR experiments.…”

Section: Methodsmentioning

confidence: 99%

“…Binding to two different histidines in each subunit of the dimer (e.g., His31-Nε to His147-Nε at 38 Å or His114-Nε to His225-Nε at 36 Å) is intrinsically unlikely because of the symmetry of the homodimer (14), and is in any event excluded by triangulation with Cu 2þ -nitroxide distances (below) and ITC results. (32). The Ser180Cys mutation causes minimal perturbation to the protein structure and the spin-labeled EcoRI retains normal DNA-binding affinity (32).…”

mentioning

confidence: 99%

“…(32). The Ser180Cys mutation causes minimal perturbation to the protein structure and the spin-labeled EcoRI retains normal DNA-binding affinity (32). To selectively measure the Cu 2þ -Ser180Cys distances, DEER signals for the Ser180Cys-DNA-Cu 2þ complex were collected with the pump pulse applied to the nitroxide ESR spectrum and the observer pulses applied to the Cu 2þ ESR spectrum (Fig.…”

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confidence: 99%

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ESR spectroscopy identifies inhibitory Cu ²⁺ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity

Yang¹,

Kurpiewski

Ji³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The relationship between DNA sequence recognition and catalytic specificity in a DNA-modifying enzyme was explored using paramagnetic Cu 2þ ions as probes for ESR spectroscopic and biochemical studies. Electron spin echo envelope modulation spectroscopy establishes that Cu 2þ coordinates to histidine residues in the EcoRI endonuclease homodimer bound to its specific DNA recognition site. The coordinated His residues were identified by a unique use of Cu 2þ -ion based long-range distance constraints. Double electron-electron resonance data yield Cu 2þ -Cu 2þ and Cu 2þ -nitroxide distances that are uniquely consistent with one Cu 2þ bound to His114 in each subunit. Isothermal titration calorimetry confirms that two Cu 2þ ions bind per complex. Unexpectedly, Mg 2þ -catalyzed DNA cleavage by EcoRI is profoundly inhibited by Cu 2þ binding at these hitherto unknown sites, 13 Å away from the Mg 2þ positions in the catalytic centers. Molecular dynamics simulations suggest a model for inhibition of catalysis, whereby the Cu 2þ ions alter critical protein-DNA interactions and water molecule positions in the catalytic sites. In the absence of Cu 2þ , the Mg 2þ -dependence of EcoRI catalysis shows positive cooperativity, which would enhance EcoRI inactivation of foreign DNA by irreparable doublestrand cuts, in preference to readily repaired single-strand nicks. Nonlinear Poisson-Boltzmann calculations suggest that this cooperativity arises because the binding of Mg 2þ in one catalytic site makes the surface electrostatic potential in the distal catalytic site more negative, thus enhancing binding of the second Mg 2þ . Taken together, our results shed light on the structural and electrostatic factors that affect site-specific catalysis by this class of endonucleases.T he biochemical basis of specificity in the interaction of proteins with DNA sites is a major problem of modern molecular genetics. Studies of many protein-DNA complexes by crystallography have elucidated the intermolecular recognition contacts (1), but it is clear that point-to-point contacts cannot fully explain specificity. Solution biochemical and computational studies have shown that a comprehensive view of specificity determination must also include factors such as shape recognition (2), mutual accommodation of the macromolecules through DNA distortion or conformational selection (1, 3-6), and/or DNA-induced protein folding (5, 7). Thermodynamic studies have revealed that specific protein-DNA association is often driven primarily by the favorable entropy increase provided by desolvation of the apposed complementary surfaces (8-10).For those DNA-binding proteins that are also DNA-modifying enzymes (nucleases, methylases, recombinases, repair enzymes, etc.) a key question is the relationship between DNA-binding specificity and catalytic specificity. One exemplary system for addressing specificity determination is the EcoRI endonuclease (3, 11), a 62 kDa homodimer that recognizes the DNA site 5′-GAATTC-3′ and binds as much as 90,000-fold better (3, 12) than a...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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ESR spectroscopy identifies inhibitory Cu ²⁺ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity

Yang¹,

Kurpiewski

Ji³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…49 In the current study, the DEER experiment was performed on the MTSSL-K249C mutant protein using a Bruker EleXsys CW/FT X-band ESR spectrometer with the Bruker X-band ER-MD5 resonator. The MTSSL-K249C sample was prepared in 30% deuterated glycerol, 65% deuterated water, and 5% protonated water resulting in a final protein concentration of 180 µM.…”

Section: Methodsmentioning

confidence: 99%

“…45,48 To shed light on this region, structural constraints were obtained by performing several distance measurements using the DEER experiment on the specific EcoRI-DNA complex. 49 In this work we have leveraged these correlated distance constraints with MD simulations to learn about side chain packing and dynamics of the spin label as well as backbone details at two different spin labeled sites in the EcoRI-DNA complex.…”

Section: Introductionmentioning

confidence: 99%

Simulating the Dynamics and Orientations of Spin-Labeled Side Chains in a Protein–DNA Complex

et al. 2012

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Site-directed spin labeling, wherein a nitroxide side chain is introduced into a protein at a selected mutant site, is increasingly employed to investigate biological systems by electron spin resonance (ESR) spectroscopy. An understanding of the packing and dynamics of the spin label is needed to extract the biologically relevant information about the macromolecule from ESR measurements. In this work, molecular dynamics (MD) simulations were performed on the spin labeled restriction endonuclease, EcoRI in complex with DNA. Mutants of this homodimeric enzyme were previously constructed and distance measurements were performed using the Double Electron Electron Resonance experiment. These correlated distance constraints have been leveraged with MD simulations to learn about side chain packing and preferred conformers of the spin label on sites in an α-helix and a β-strand. We found three dihedral angles of the spin label side chain to be most sensitive to the secondary structure where the spin label was located. Conformers sampled by the spin label differed between secondary structures as well. Cα-Cα distance distributions were constructed and used to extract details about the protein backbone mobility at the two spin labeled sites. These simulation studies enhance our understanding of the behavior of spin labels in proteins and thus expand the ability of ESR spectroscopy to contribute to knowledge of protein structure and dynamics.

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Nucleotide‐Independent Copper(II)‐Based Distance Measurements in DNA by Pulsed ESR Spectroscopy

2017

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As ite-specific Cu 2+ binding motif within aD NA duplex for distance measurements by ESR spectroscopyi s reported. This motif utilizes ac ommercially available 2,2'dipicolylamine (DPA) phosphormadite easily incorporated into any DNAo ligonucleotide during initial DNAs ynthesis. The method only requires the simple post-synthetic addition of Cu 2+ without the need for further chemical modification. Notably,t he label is positioned within the DNAd uplex, as opposed to outside the helical perimeter,f or an accurate measurement of duplex distance.Adistance of 2.7 nm was measured on adoubly Cu 2+ -labeled DNAsequence,whichisin exact agreement with the expected distance from both DNA modeling and molecular dynamic simulations.T his result suggests that with this labeling strategy the ESR measured distance directly reports on backbone DNAd istance,w ithout the need for further modeling.F urthermore,t he labeling strategy is structure-and nucleotide-independent.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: http://dx.

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Electron Spin Resonance Shows Common Structural Features for Different Classes of EcoRI–DNA Complexes

Cited by 15 publications

References 28 publications

ESR spectroscopy identifies inhibitory Cu ²⁺ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity

ESR spectroscopy identifies inhibitory Cu ²⁺ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity

Simulating the Dynamics and Orientations of Spin-Labeled Side Chains in a Protein–DNA Complex

Nucleotide‐Independent Copper(II)‐Based Distance Measurements in DNA by Pulsed ESR Spectroscopy

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Electron Spin Resonance Shows Common Structural Features for Different Classes of EcoRI–DNA Complexes

Cited by 15 publications

References 28 publications

ESR spectroscopy identifies inhibitory Cu 2+ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity

ESR spectroscopy identifies inhibitory Cu 2+ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity

Simulating the Dynamics and Orientations of Spin-Labeled Side Chains in a Protein–DNA Complex

Nucleotide‐Independent Copper(II)‐Based Distance Measurements in DNA by Pulsed ESR Spectroscopy

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ESR spectroscopy identifies inhibitory Cu ²⁺ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity

ESR spectroscopy identifies inhibitory Cu ²⁺ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity