title: Rescue of deleterious mutations by the compensatory Y30F mutation in ketosteroid isomerase.
abstract: Proteins have evolved to compensate for detrimental mutations. However, compensatory mechanisms for protein defects are not well understood. Using ketosteroid isomerase (KSI), we investigated how second-site mutations could recover defective mutant function and stability. Previous results revealed that the Y30F mutation rescued the Y14F, Y55F and Y14F/Y55F mutants by increasing the catalytic activity by 23-, 3- and 1.3-fold, respectively, and the Y55F mutant by increasing the stability by 3.3 kcal/mol. To better understand these observations, we systematically investigated detailed structural and thermodynamic effects of the Y30F mutation on these mutants. Crystal structures of the Y14F/Y30F and Y14F/Y55F mutants were solved at 2.0 and 1.8 previoulsy solved structures of wild-type and other mutant KSIs. Structural analyses revealed that the Y30F mutation partially restored the active-site cleft of these mutant KSIs. The Y30F mutation also increased Y14F and Y14F/Y55F mutant stability by 3.2 and 4.3 kcal/mol, respectively, and the melting temperatures of the Y14F, Y55F and Y14F/Y55F mutants by 6.4°C, 5.1°C and 10.0°C, respectively. Compensatory effects of the Y30F mutation on stability might be due to improved hydrophobic interactions because removal of a hydroxyl group from Tyr30 induced local compaction by neighboring residue movement and enhanced interactions with surrounding hydrophobic residues in the active site. Taken together, our results suggest that perturbed active-site geometry recovery and favorable hydrophobic interactions mediate the role of Y30F as a secondsite suppressor.
authors.affiliation: Department of Life Science, WCU Program, Pohang University of Science and Technology, Pohang, 790-784, Korea.
authors.forename: Hyung Jin
authors.initials: HJ
authors.lastname: Cha
authors.forename: Do Soo
authors.initials: DS
authors.lastname: Jang
authors.forename: Yeon-Gil
authors.initials: YG
authors.lastname: Kim
authors.forename: Bee Hak
authors.initials: BH
authors.lastname: Hong
authors.forename: Jae-Sung
authors.initials: JS
authors.lastname: Woo
authors.forename: Kyong-Tai
authors.initials: KT
authors.lastname: Kim
authors.forename: Kwan Yong
authors.initials: KY
authors.lastname: Choi
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D055162:Biocatalysis / Q000187:drug effects; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004795:Enzyme Stability / Q000187:drug effects; D006860:Hydrogen Bonding / Q000187:drug effects; D007536:Isomerism; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D017510:Protein Folding / Q000187:drug effects; D016958:Pseudomonas putida / Q000201:enzymology*; D013251:Steroid Isomerases / Q000235:genetics*; D014508:Urea / Q000494:pharmacology
chemical_list: D050505:Mutant Proteins; D014508:Urea; D013251:Steroid Isomerases