The engineering of binding affinity at metal ion binding sites for the stabilization of proteins: subtilisin as a test case

Abstract

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)

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

“…A similar effect with divalent cations (and negative charges) has been noted in other protein systems (Voordouw et al, 1976;Filimonov et al, 1978;Bashford et al, 1986;Linse et al, 1988;Pantoliano et al, 1988). The effect of phosphate above its second pK, is expected to be essentially the same as sulfate.…”

A step towards understanding the folding mechanism of horseradish peroxidase

“…The effect of Ca2+ on the stability of the apo-HRP indicates that either the acidic conditions used to remove the haem resulted in protonation of the acidic groups that led to a loss of Ca2+ from the protein, or there is an additional Caz+-binding site in the apo-HRP molecule. Evidence for additional, but much weaker, binding sites was first obtained for subtilisin when the effect of higher concentrations of Ca2+ on the kinetic thermal stability of subtilisin was studied (Pantoliano et al, 1988). Interestingly, Phelps et al (1971) compared the titration results of native and apo-HRP and they discovered that the apo-HRP had eight exposed carboxyl groups after comparing it with the native protein.…”

Complex between the subtilisin from a mesophilic bacterium and the Leech inhibitor eglin-C

“…This structure was subsequently refined at 1.8 A resolution with phenyl methyl sulfonyl fluoride (PMSF) inhibitor covalently bound (Bott, Ultsch, Kossiakoff, Graycar, Katz & Power, 1988), in complex with Streptomyces inhibitor at 2.6 A (Hirono, Akagawa, Mitsui & Iitaka, 1984) and at 2.1 A as a complex with the chymotrypsin inhibitor 2 from barley seed (McPhalen, Svendsen, Jonassen & James, 1985;McPhalen & James, 1988). High-resolution structures of site-directed mutants of SBPN (Pantoliano et al, 1987(Pantoliano et al, , 1988 have also been reported. The three-dimensional structures of other Bacilli subtilisins have been published: the SBCARL-eglin-C complex at 1.8/k (McPhalen, Schnebli & Jamesl 1985;McPhalen & James, 1988) and independently at a nominal resolution of 1.2 A (Bode, Papamokos, Musil, Seemfiller & Fritz, 1986;Bode, Papamokos & Musil, 1987), and native SBCARL at 2.5A (Neidhart & Petsko, 1988).…”

“…The first ion, Cal, is tightly bound and cannot be completely removed with EDTA under non-denaturing conditions, whilst the second, Ca2, is more loosely bound and can be removed with EDTA (Voordouw et al, 1976;Pantoliano et al, 1988). The effective overall negative charge of the subtilisins given in Table 4 is changed by +4 by the binding of the two calcium ions, providing both sites are fully occupied.…”

“…Genetically engineered mutants of subtilisin BPN' have been extensively investigated (e.g. Russell, Thomas & Fersht, 1987;Pantoliano, Ladner, Bryan, Rollence, Wood & Poulos, 1987;Pantoliano, Whitlow, Wood, Rollence, Finzel, Gilliland, Poulos & Bryan, 1988). In addition the subtilisins have extensive practical uses, mainly as additives to laundry detergents to improve their cleaning power by hydrolysis of protein stains.…”