Kinetic tuning of the EF-hand calcium binding motif: the gateway residue independently adjusts (i) barrier height and (ii) equilibrium.
In EF-hand calcium binding sites of known structure, the side chain provided by the ninth EF-loop position lies at the entrance of the shortest pathway connecting the metal binding cavity to solvent. The location of this residue suggests that it could serve as a "gateway", providing steric and electrostatic control over the kinetics of Ca2+ binding and dissociation. To test this hypothesis, the present study has engineered the putative gateway side chain of a model EF-hand site and determined the resulting effects on metal ion affinity and dissociation kinetics. The model site chosen was that of the Escherichia coli galactose binding protein (GBP), in which the putative gateway is a Gln side chain. Nine engineered GBP molecules were generated and isolated, each exhibiting native-like activity and global conformation. Two control substitutions at the fourth EF-loop position, a noncoordinating surface residue, had no significant effect on either the equilibrium or the kinetics of the model site. The remaining seven proteins, which possessed unique substitutions at the ninth EF-loop position (Glu, Asn, Asp, Thr, Ser, Gly, Ala), in each case significantly altered the affinity or dissociation kinetics of the site for Tb3+, used as a probe metal ion. Neutral side chains at the gateway position yielded a 590-fold range of Tb3+ dissociation kinetics but only a 3-fold range of Tb3+ affinities, indicating that the size or polarity of these substitutions alters the transition state barrier for metal binding and release without substantially shifting the equilibrium. In contrast, acidic side chains yielded as much as a 34-fold decrease in the Tb3+ off-rate and a 33-fold increase in Tb3+ affinity, suggesting that a negative charge at the gateway position increases the equilibrium stability of the bound metal ion and thereby slows metal release. Thus, kinetic tuning by the gateway side chain exhibits both transition state and ground state tuning mechanisms. In natural EF-hand sequences, different gateway side chains are used by slow buffering sites and rapid signaling sites, providing evidence that the gateway position is an important physiological determinant of metal binding kinetics.
Drake
S K
SK
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Falke
J J
JJ
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D015202:Protein Engineering; D013725:Terbium / Q000378:metabolism
D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; D013725:Terbium; D005690:Galactose