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Mol. Cells 2013; 36(1): 39-46

Published online July 31, 2013

https://doi.org/10.1007/s10059-013-0013-1

© The Korean Society for Molecular and Cellular Biology

Rescue of Deleterious Mutations by the Compensatory Y30F Mutation in Ketosteroid Isomerase

Hyung Jin Cha, Do Soo Jang, Yeon-Gil Kim, Bee Hak Hong, Jae-Sung Woo, Kyong-Tai Kim, and Kwan Yong Choi

1Department of Life Science, Division of Molecular and Life Sciences, Division of Integrative Biosciences and Biotechnology, WCU Program, Pohang University of Science and Technology, Pohang 790-784, Korea, 2Research Institute, Genexine Co., Seongnam 463-400, Korea, 3Pohang Accelerator Laboratory, Pohang University of Science and Technology, Pohang 790-784, Korea, 4Institute for Basic Science, Seoul National University, Seoul 151-742, Korea, 5These authors contributed equally to this work.

Received: January 14, 2013; Revised: April 29, 2013; Accepted: April 30, 2013

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 ? resolution, respectively, and compared with
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.

Keywords active-site recovery, ketosteroid isomerase, more hydrophobic interactions, rescue mechanism, second-site suppressor

Article

Research Article

Mol. Cells 2013; 36(1): 39-46

Published online July 31, 2013 https://doi.org/10.1007/s10059-013-0013-1

Copyright © The Korean Society for Molecular and Cellular Biology.

Rescue of Deleterious Mutations by the Compensatory Y30F Mutation in Ketosteroid Isomerase

Hyung Jin Cha, Do Soo Jang, Yeon-Gil Kim, Bee Hak Hong, Jae-Sung Woo, Kyong-Tai Kim, and Kwan Yong Choi

1Department of Life Science, Division of Molecular and Life Sciences, Division of Integrative Biosciences and Biotechnology, WCU Program, Pohang University of Science and Technology, Pohang 790-784, Korea, 2Research Institute, Genexine Co., Seongnam 463-400, Korea, 3Pohang Accelerator Laboratory, Pohang University of Science and Technology, Pohang 790-784, Korea, 4Institute for Basic Science, Seoul National University, Seoul 151-742, Korea, 5These authors contributed equally to this work.

Received: January 14, 2013; Revised: April 29, 2013; Accepted: April 30, 2013

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 ? resolution, respectively, and compared with
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.

Keywords: active-site recovery, ketosteroid isomerase, more hydrophobic interactions, rescue mechanism, second-site suppressor

Mol. Cells
May 31, 2022 Vol.45 No.5, pp. 273~352
COVER PICTURE
Fe2+ ion depletion-induced expression of BΔGFP at the early stage of leaf development (Choi et al., pp. 294-305).

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