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Effects of site-directed mutagenesis in the N-terminal domain of thermolysin on its stabilization.

ABSTRACT: The thermolysin variant G8C/N60C/S65P in which the triple mutation in the N-terminal domain, Gly8?Cys/Asn60?Cys/Ser65?Pro, is undertaken increases stability [Yasukawa, K. and Inouye, K. (2007) Improving the activity and stability of thermolysin by site-directed mutagenesis. Biochim. Biophys. Acta 1774, 1281-1288] and its mechanism is examined in this study. The apparent denaturing temperatures based on ellipticity at 222 nm of the wild-type thermolysin (WT), G8C/N60C, S65P and G8C/N60C/S65P were 85, >95, 88 and >95°C, respectively. The first-order rate constants, k(obs), of the thermal inactivation of WT and variants at 10 mM CaCl? increased with increasing thermal treatment temperatures (70-95°C), and those at 80°C decreased with increasing CaCl? concentrations (1-100 mM). The k(obs) values were in the order of WT > S65P > G8C/N60C?G8C/N60C/S65P at all temperatures and CaCl? concentrations. These results indicate that the mutational combination, Gly8?Cys/Asn60?Cys and Ser65?Pro, increases stability only as high as Gly8?Cys/Asn60?Cys does. Assuming that irreversible inactivation of thermolysin occurs only in the absence of calcium ions, the dissociation constants, K(d), to the calcium ions of WT, G8C/N60C, S65P and G8C/N60C/S65P were 47, 8.9, 17 and 7.2 mM, respectively, suggesting that Gly8?Cys/Asn60?Cys and Ser65?Pro stabilize thermolysin by improving its affinity to calcium ions, most probably the one at the Ca²?-binding site III in the N-terminal domain.

SUBMITTER: Kawasaki Y

PROVIDER: S-EPMC3528001 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Effects of site-directed mutagenesis in the N-terminal domain of thermolysin on its stabilization.

Kawasaki Yuichi Y Yasukawa Kiyoshi K Inouye Kuniyo K

Journal of biochemistry 20121019 1

The thermolysin variant G8C/N60C/S65P in which the triple mutation in the N-terminal domain, Gly8→Cys/Asn60→Cys/Ser65→Pro, is undertaken increases stability [Yasukawa, K. and Inouye, K. (2007) Improving the activity and stability of thermolysin by site-directed mutagenesis. Biochim. Biophys. Acta 1774, 1281-1288] and its mechanism is examined in this study. The apparent denaturing temperatures based on ellipticity at 222 nm of the wild-type thermolysin (WT), G8C/N60C, S65P and G8C/N60C/S65P were ...[more]

PMID: 23087322

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Project description:BackgroundThe mglA gene from the bacterium Myxococcus xanthus encodes a 22kDa protein related to the Ras superfamily of monomeric GTPases. MglA is required for the normal function of A-motility (adventurous), S-motility (social), fruiting body morphogenesis, and sporulation. MglA and its homologs differ from all eukaryotic and other prokaryotic GTPases because they have a threonine (Thr78) in place of the highly conserved aspartate residue of the consensus PM3 (phosphate-magnesium binding) region. To identify residues critical for MglA function or potential protein interactions, and explore the function of Thr78, the phenotypes of 18 mglA mutants were characterized.ResultsNine mutants, with mutations predicted to alter residues that bind the guanine base or coordinate magnesium, did not produce detectable MglA. As expected, these mutants were mot- dev- because MglA is essential for these processes. Of the remaining nine mutants, seven showed a wild-type distribution pattern for MglA but fell into two categories with regard to function. Five of the seven mutants exhibited mild phenotypes, but two mutants, T78D and P80A, abolished motility and development. The localization pattern of MglA was abolished in two mutants that were mot- spo- and dev-. These two mutants were predicted to alter surface residues at Asp52 and Thr54, which suggests that these residues are critical for proper localization and may define a protein interaction site. Improving the consensus match with Ras at Thr78 abolished function of MglA. Only the conservative serine substitution was tolerated at this position. Merodiploid constructs revealed that a subset of alleles, including mglAD52A, were dominant and also illustrated that changing the balance of MglA and its co-transcribed partner, MglB, affects A-motility.ConclusionOur results suggest that GTP binding is critical for stability of MglA because MglA does not accumulate in mutants that cannot bind GTP. The threonine in PM3 of MglA proteins represents a novel modification of the highly conserved GTPase consensus at this position. The requirement for a hydroxyl group at this position may indicate that MglA is subject to modification under certain conditions. Proper localization of MglA is critical for both motility and development and likely involves protein interactions mediated by residues Asp52 and Thr54.

Dataset Information

Effects of site-directed mutagenesis in the N-terminal domain of thermolysin on its stabilization.

Publications

Effects of site-directed mutagenesis in the N-terminal domain of thermolysin on its stabilization.

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