Ca2+ coordination to backbone carbonyl oxygen atoms in calmodulin and other EF-hand proteins: 15N chemical shifts as probes for monitoring individual-site Ca2+ coordination.
Examination of the NMR 15N chemical shifts of a number of EF-hand proteins shows that the shift value for the amido nitrogen of the residue in position 8 of a canonical EF-hand loop (or position 10 of a pseudo EF-hand loop) provides a good indication of metal occupation of that site. The NH of the residue in position 8 is covalently bonded to the carbonyl of residue 7, the only backbone carbonyl that coordinates to the metal ion in a canonical EF-hand loop. Upon metal coordination to this carbonyl, there is an appreciable deshielding of the 15N nucleus at position 8 (+4 to +8 ppm) due to the polarization of the O(7)=C(7)-N(8) amido group and the corresponding reduction in the electron density of the nitrogen atom. This deshielding effect is effectively independent of the binding of metal to the other site of an EF-hand pair, allowing the 15N shifts to be used as probes for site-specific occupancy of metal binding sites. In addition, a Ca2+-induced change in side-chain Halpha-Calpha-Cbeta-Hbeta torsion angle for isoleucine or valine residues in position 8 can also contribute to the deshielding of the amide 15N nucleus. This conformational effect occurs only in sites I or III and takes place upon binding a Ca2+ ion to the other site of an EF-hand pair (site II or IV) regardless of whether the first site is occupied. The magnitude of this effect is in the range +5 to +7 ppm. A Ca2+ titration of 15N-labeled apo-calmodulin was performed using 2D 1H-15N HSQC NMR spectra. The changes in the 15N chemical shifts and intensities for the peaks corresponding to the NH groups of residues in position 8 of the EF-hand loops allowed the amount of metal bound at sites II, III and IV to be monitored directly at partial degrees of saturation. The peak corresponding to site I could only be monitored at the beginning and end of the titration because of line broadening effects in the intermediate region of the titration. Sites III and IV both titrate preferentially and the results demonstrate clearly that sites in either domain fill effectively in parallel, consistent with a significant positive intradomain cooperativity of calcium binding.
Biekofsky
R R
RR
Molecular Structure Division, Physical Biochemistry Division, National Institute for Medical Research, London, U.K.
Martin
S R
SR
Browne
J P
JP
Bayley
P M
PM
Feeney
J
J
D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002264:Carboxylic Acids / Q000737:chemistry; D004331:Drosophila melanogaster; D007532:Isoleucine / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D011487:Protein Conformation; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D009418:S100 Proteins / Q000378:metabolism; D017186:Titrimetry; D019209:Troponin C / Q000378:metabolism; D014633:Valine / Q000378:metabolism
D064026:Calbindins; D002147:Calmodulin; D002264:Carboxylic Acids; D009587:Nitrogen Isotopes; D064030:S100 Calcium Binding Protein G; D009418:S100 Proteins; D019209:Troponin C; D002104:Cadmium; D007532:Isoleucine; D007930:Leucine; D014633:Valine; D002118:Calcium