Effects of metal-binding loop mutations on ligand binding to calcium- and integrin-binding protein 1. Evolution of the EF-hand?
Calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous, multifunctional regulatory protein consisting of four helix-loop-helix EF-hand motifs. Neither EF-I nor EF-II binds divalent metal ions; however, EF-III is a mixed Mg2+/Ca2+-binding site, and EF-IV is a higher-affinity Ca2+-specific site. Through the generation of several CIB1 mutant proteins, we have investigated the importance of the last (-Z) metal-coordinating position of EF-III (D127) and EF-IV (E172) with respect to the binding of CIB1 to Mg2+, Ca2+, and its biological target, the cytoplasmic domain of the platelet alphaIIb integrin. A D127N mutant had reduced Mg2+ and Ca2+ affinity at EF-III but retained affinity for the alphaIIb domain. A D127E mutant had increased Mg2+ and Ca2+ affinity at EF-III, but unexpectedly, the affinity for the alphaIIb domain was too low for binding to be observed. E172Q and E172D mutants showed no and weak Mg2+ binding at EF-IV, respectively, and each mutant had reduced Ca2+ affinity at EF-IV and showed moderate metal-dependent differences in affinity for the alphaIIb domain. Finally, a D127Q mutant bound Mg2+ and Ca2+ in a manner similar to that of D127N, but like that of D127E, the affinity for the alphaIIb domain was reduced below the detection limit. These data, combined with a NMR-based structural comparison of the Mg2+- and Ca2+-loaded CIB1-alphaIIb peptide complexes, suggest that the D127E and D127Q mutations have a disruptive effect on alphaIIb binding since they expand the metal-binding loop and change the alpha-helix positions in EF-III. Conversely, upon replacement of the ancestral Glu with Asp at the -Z position of EF-III, CIB1 gained affinity for alphaIIb, and the Ca2+ affinity of CIB1 shifted into a range where the protein is able to act as an intracellular Ca2+ sensor.
Yamniuk
Aaron P
AP
Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Calgary, AB, Canada T2N 1N4.
Gifford
Jessica L
JL
Linse
Sara
S
Vogel
Hans J
HJ
D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002151:Calorimetry; D003593:Cytoplasm / Q000378:metabolism; D020832:EF Hand Motifs; D008024:Ligands; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D013050:Spectrometry, Fluorescence
D002135:Calcium-Binding Proteins; D008024:Ligands; D008670:Metals