Residues that contribute Ca2+ binding ligands are represented as red triangles.
Largest chemical shift changes cluster in four regions.
Each region contributes one or more Ca2+ binding ligands.
Conformational Dynamics of the Na+/Ca2+ Exchanger
Eric Johnson,1 Lei Brüschweiler-Li,1 Mark Hilge,2 Geerten Vuister 2 & Rafael Brüschweiler 1
1 Department of Chemistry and Biochemistry, Florida State University and National High Magnetic Field Laboratory (NHMFL), Tallahassee, FL
2 Radboud University, Nijmegen, The Netherlands
The Na+/Ca2+ exchanger (NCX) plays a critical role in the cardiac cycle.
The NCX is regulated by CBD1, a 16 kDa intracellular Ca2+ binding domain.
CBD1 displays an immunoglobulin fold consisting of two antiparallel beta sheets.
The CBD1 fold is very different from that of the EF hand proteins (eg. calmodulin, calbindin, troponin).
RDC and 15N relaxation experiments performed at 800 MHz reveal systematic differences between the Ca2+ bound and apo (Ca2+ free) states of CBD1
This poster demonstrates how 4D covariance NMR provides substantial resolution enhancement to 4D NOESY spectra. Covariance NMR resolves additional cross peaks, some arising from long-range constraints, from otherwise overlapped regions of a 4D FT NOESY spectrum.
Chemical shift analysisOverlay of the [1H,15N]-HSQC spectra for CBD1 in the Ca2+ bound (red) and apo (blue) states
Chemical shift differences
Residual dipolar coupling analysis
The Ca2+ bound structure has been solved by NMR and X-ray crystallography (2.5 Å resolution).
There is no structure available for the apo state.
Correlation plots of the RDCs measured experimentally (Dexp) and the values back-calculated (Dcalc) from the crystal structure for the Ca2+ bound (left) and apo (right) states.
RDCs for both states are largely consistent with the crystal structure.
Linear correlation coefficients for the Ca2+ bound and apo states are r = 0.93 and r = 0.83.
Q values for the Ca2+ bound and apo states are QCa = 0.30 and Qapo = 0.46 (Q = rms(Dcalc-Dexp)/rms(Dexp)).
Agreement for individual residues color-mapped onto the 3D structure for the Ca2+ bound (left) and apo (right) RDC data.
Agreement between Dexp and Dcalc evaluated by the quantity |(Dexp- Dcalc)/Dexp|
yellow: low values, good agreement
red: high values, poor agreement
Large RDC differences are observed for the binding site of the apo state
Backbone dynamics from 15N relaxation:
NOE values (upper) and generalized order parameters S2 (bottom) plotted as a function of residue number.
Changes are observed in the acidic segment that contributes several Ca2+ binding ligands.
For an axially symmetric tensor:
Ca2+ bound: tau_c = 12.2 ns, anisotropy Dpar/Dperp = 2.0
Apo: tau_c = 10.9 ns, anisotropy Dpar/Dperp = 1.6
High quality structural dynamics information can be obtained at 800 MHz for a 16 kDa Ca2+ regulator.
The effects of Ca2+ binding on the structure and dynamics of CBD1 have been investigated using 15N relaxation, RDCs, and chemical shifts.
Large segments of the molecule are similar by these measures in the Ca2+ bound and apo states.
Significant changes are consistently observed near the binding sites.
This work was supported by the NSF Grant O621482.
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