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

Abstract

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.

Interaction of calmodulin with the phosphofructokinase target sequence

“…3A) show an increase in pK a for peptide His19 from 6.35 (free) to 7.25 (complex), indicating that the protonated form is stabilized in the complex with Ca 4 CaM. In contrast, the pK a for His107 in Ca 4 CaM decreases by a smaller amount from 6.15 to 5.85 on complex formation with the peptide.…”

“…Initial studies with the P 9-26 peptide showed that the strength of the interaction with Ca 4 CaM is strongly pH-dependent, with K d varying from 0.8 to 300 nM for pH 5.0-8.5 (Skinner, PhD thesis, University of London, 1996). For quantitation of the high affinity 1:1 complex formed with peptide P 1-21 at lower pH values, competition titrations were performed, using spectroscopically ''silent'' peptides (FFFu, K d ¼ 750 ± 120 pM and FFFp, K d ¼ 45 AE 5pM [2]), whose affinity was established to be pH-independent in titrations of the Ca 4 CaMWFFu complex as a function of pH (Fig.…”

“…Ca 4 CaM Á P 1-21 To assess possible structural effects of protonation, changes in 2D 1 H-15 N HSQC NMR spectra of 15 N labeled Ca 4 CaM AE P 1-21 complex were monitored as a function of pH. Fig.…”

“…Fig. 3B shows the superimposition of a series of spectra for the complex and for Ca 4 CaM alone. The HN cross-peak chemical shift changes with pH occur in fast exchange on the NMR time scale, leading to averaged signals.…”

Structures of Anabaena Calcium-binding Protein CcbP

“…7-10). In NMR spectroscopy, metal coordination to a backbone carbonyl can cause deshielding of the backbone nitrogen atom of the succeeding residue (31 We constructed plasmids carrying ccbP genes encoding the wild type protein or mutants at site I under the control of the petE promoter (32). The plasmids were used to transform Anabaena sp., so that the expression of ccbP was inducible with copper.…”