The engineering of binding affinity at metal ion binding sites for the stabilization of proteins: subtilisin as a test case.
A weak Ca2+ binding site in the bacterial serine protease subtilisin BPN' (EC 3.4.21.14) was chosen as a model to explore the feasibility of stabilizing a protein by increasing the binding affinity at a metal ion binding site. The existence of this weak Ca2+ binding site was first discovered through a study of the rate of thermal inactivation of wild-type subtilisin BPN' at 65 degrees C as a function of the free [Ca2+]. Increasing the [Ca2+] in the range 0.10-100 mM caused a 100-fold decrease in the rate of thermal inactivation. The data were found to closely fit a theoretical titration curve for a single Ca2+ specific binding site with an apparent log Ka = 1.49. A series of refined X-ray crystal structures (R less than or equal to 0.15, 1.7 A) of subtilisin in the presence of 0.0, 25.0, and 40.0 mM CaCl2 has allowed a detailed structural characterization of this Ca2+ binding site. Negatively charged side chains were introduced in the vicinity of the bound Ca2+ by changing Pro 172 and Gly 131 to Asp residues through site-directed and random mutagenesis techniques, respectively. These changes were found to increase the affinity of the Ca2+ binding site by 3.4- and 2-fold, respectively, when compared with the wild-type protein (ionic strength = 0.10). X-ray studies of these new variants of subtilisin revealed the carboxylate side chains to be 6.8 and 13.2 A, respectively, from the bound Ca2+. These distances and the degree of enhanced binding are consistent with simple electrostatic theory.(ABSTRACT TRUNCATED AT 250 WORDS)
Pantoliano
M W
MW
Genex Corporation, Gaithersburg, Maryland 20877.
Whitlow
M
M
Wood
J F
JF
Rollence
M L
ML
Finzel
B C
BC
Gilliland
G L
GL
Poulos
T L
TL
Bryan
P N
PN
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D004563:Electrochemistry; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D011487:Protein Conformation; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013816:Thermodynamics; D014961:X-Ray Diffraction
D008670:Metals; D013381:Subtilisins; D002118:Calcium