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

Summary

  • 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.[5] Covariance NMR resolves additional cross peaks, some arising from long-range constraints, from otherwise overlapped regions of a 4D FT NOESY spectrum.

    Chemical shift analysis

    Overlay of the [1H,15N]-HSQC spectra for CBD1 in the Ca2+ bound (red) and apo (blue) states


    Chemical shift differences




    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.

    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.
    Ca2+ bound

    Ca2+ bound


    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

    Conclusions

  • 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.

    Acknowledgments

    This work was supported by the NSF Grant O621482.
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