Metal Ion Binding at the Catalytic Site Induces Widely
Distributed Changes in a Sequence Specific Protein–DNA Complex
Kaustubh Sinha
Sahil S. Sangani
Andrew D. Kehr
Gordon S. Rule
Linda Jen-Jacobson
10.1021/acs.biochem.6b00919.s001
https://acs.figshare.com/articles/journal_contribution/Metal_Ion_Binding_at_the_Catalytic_Site_Induces_Widely_Distributed_Changes_in_a_Sequence_Specific_Protein_DNA_Complex/4106331
Metal
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 Mg<sup>2+</sup> cofactor. Nuclear magnetic resonance (NMR) titrations
indicate that four Lu<sup>3+</sup> or two La<sup>3+</sup> cations
bind, and two new crystal structures confirm that Lu<sup>3+</sup> 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 <sup>1</sup>H–<sup>15</sup>N amide resonances,
for <sup>1</sup>H–<sup>13</sup>C Ile-δ-CH<sub>3</sub> resonances, and for stereospecifically assigned Leu-δ-CH<sub>3</sub> and Val-γ-CH<sub>3</sub> 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.
2016-10-27 13:35:01
Many chemical shifts
GATATC
Catalytic Site Induces Widely
metal ions
Bound metal ions
Leu -δ-CH 3
metal ion binding sites
amide hydrogen exchange rates
metal Ion Binding
NMR spectra show
methyl resonance perturbations
EcoRV
side chain methyl resonances
metal binding
protein
contacts DNA phosphates
Val -γ-CH 3 resonances
NMR chemical shift perturbations