title: Symmetrical stabilization of bound Ca2+ ions in a cooperative pair of EF-hands through hydrogen bonding of coordinating water molecules in calbindin D(9k).
abstract: Water molecules are found to complete the Ca2+ coordination sphere when a protein fails to provide enough ligating oxygens. Hydrogen bonding of these water molecules to the protein backbone or side chains may contribute favorably to the Ca2+ affinity, as suggested in an earlier study of two calbindin D(9k) mutants [E60D and E60Q; Linse et al. (1994) Biochemistry 33, 12478-12486]. To investigate the generality of this conclusion, another side chain, Gln 22, which hydrogen bonds to a Ca2+-coordinating water molecule in calbindin D(9k), was mutated. Two calbindin D(9k) mutants, (Q22E+P43M) and (Q22N+P43M), were constructed to examine the interaction between Gln 22 and the water molecule in the C-terminal calcium binding site II. Shortening of the side chain, as in (Q22N+P43M), reduces the affinity of binding two calcium ions by a factor of 18 at low ionic strength, whereas introduction of a negative charge, as in (Q22E+P43M), leads to a 12-fold reduction. In 0.15 M KCl, a 7-fold reduction in affinity was observed for both mutants. The cooperativity of Ca2+ binding increases for (Q22E+P43M), while it decreases for (Q22N+P43M). The rates of Ca2+ dissociation are 5.5-fold higher for the double mutants than for P43M at low ionic strength. For both mutants, reduced strength of hydrogen bonding to calcium-coordinating water molecules is a likely explanation for the observed effects on Ca2+ affinity and dissociation. In the apo forms, the (Q22E+P43M) mutant has lower stability toward urea denaturation than (Q22N+P43M) and P43M. 2D (1)H NMR and crystallographic experiments suggest that the structure of (Q22E+P43M) and (Q22N+P43M) is unchanged relative to P43M, except for local perturbations in the loop regions.
authors.affiliation: Department of Physical Chemistry 2 and of Molecular Biophysics, P.O. Box 124, Lund University, SE-221 00 Lund, Sweden. jonas.fast@fkem2.lth.se
authors.forename: J
authors.initials: J
authors.lastname: Fast
authors.forename: M
authors.initials: M
authors.lastname: Håkansson
authors.forename: A
authors.initials: A
authors.lastname: Muranyi
authors.forename: G P
authors.initials: GP
authors.lastname: Gippert
authors.forename: E
authors.initials: E
authors.lastname: Thulin
authors.forename: J
authors.initials: J
authors.lastname: Evenäs
authors.forename: L A
authors.initials: LA
authors.lastname: Svensson
authors.forename: S
authors.initials: S
authors.lastname: Linse
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D064026:Calbindins; D002118:Calcium / Q000737:chemistry* / Q000494:pharmacology; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006860:Hydrogen Bonding; D007477:Ions; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D064030:S100 Calcium Binding Protein G / Q000737:chemistry*; D013816:Thermodynamics; D013997:Time Factors; D014508:Urea / Q000494:pharmacology; D014867:Water / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D064026:Calbindins; D002614:Chelating Agents; D007477:Ions; D064030:S100 Calcium Binding Protein G; D014867:Water; D011189:Potassium Chloride; D014508:Urea; D002118:Calcium