bi6b00919_si_002.mpg (3.84 MB)
Metal Ion Binding at the Catalytic Site Induces Widely Distributed Changes in a Sequence Specific Protein–DNA Complex
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posted on 2016-10-27, 13:34 authored by Kaustubh Sinha, Sahil S. Sangani, Andrew D. Kehr, Gordon S. Rule, Linda Jen-JacobsonMetal
ion cofactors can alter the energetics and specificity of
sequence specific protein–DNA interactions, but it is unknown
if the underlying effects on structure and dynamics are local or dispersed
throughout the protein–DNA complex. This work uses EcoRV endonuclease
as a model, and catalytically inactive lanthanide ions, which replace
the Mg2+ cofactor. Nuclear magnetic resonance (NMR) titrations
indicate that four Lu3+ or two La3+ cations
bind, and two new crystal structures confirm that Lu3+ binding
is confined to the active sites. NMR spectra show that the metal-free
EcoRV complex with cognate (GATATC) DNA is structurally distinct from
the nonspecific complex, and that metal ion binding sites are not
assembled in the nonspecific complex. NMR chemical shift perturbations
were determined for 1H–15N amide resonances,
for 1H–13C Ile-δ-CH3 resonances, and for stereospecifically assigned Leu-δ-CH3 and Val-γ-CH3 resonances. Many chemical
shifts throughout the cognate complex are unperturbed, so metal binding
does not induce major conformational changes. However, some large
perturbations of amide and side chain methyl resonances occur as far
as 34 Å from the metal ions. Concerted changes in specific residues
imply that local effects of metal binding are propagated via a β-sheet
and an α-helix. Both amide and methyl resonance perturbations
indicate changes in the interface between subunits of the EcoRV homodimer.
Bound metal ions also affect amide hydrogen exchange rates for distant
residues, including a distant subdomain that contacts DNA phosphates
and promotes DNA bending, showing that metal ions in the active sites,
which relieve electrostatic repulsion between protein and DNA, cause
changes in slow dynamics throughout the complex.
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Many chemical shiftsGATATCCatalytic Site Induces Widelymetal ionsBound metal ionsLeu -δ-CH 3metal ion binding sitesamide hydrogen exchange ratesmetal Ion BindingNMR spectra showmethyl resonance perturbationsEcoRVside chain methyl resonancesmetal bindingproteincontacts DNA phosphatesVal -γ-CH 3 resonancesNMR chemical shift perturbations
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