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, Kwan Yong Choi

Research output: Contribution to journalArticle

8 Citations (Scopus)

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 A 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 6.4C, 5.1C and 10.0C, 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.

Original languageEnglish
Pages (from-to)39-46
Number of pages8
JournalMolecules and cells
Volume36
Issue number1
DOIs
Publication statusPublished - 2013 Jul 1
Externally publishedYes

Fingerprint

Ketosteroids
Isomerases
Mutation
Catalytic Domain
Hydrophobic and Hydrophilic Interactions
Thermodynamics
Hydroxyl Radical
Freezing
Proteins
Temperature

Keywords

  • Active-site recovery
  • Ketosteroid isomerase
  • More hydrophobic interactions
  • Rescue mechanism
  • Second-site suppressor

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Rescue of deleterious mutations by the compensatory Y30F mutation in ketosteroid isomerase. / Cha, Hyung Jin; Jang, Do Soo; Kim, Yeon Gil; Hong, Bee Hak; Woo, Jae Sung; Kim, Kyong Tai; Choi, Kwan Yong.

In: Molecules and cells, Vol. 36, No. 1, 01.07.2013, p. 39-46.

Research output: Contribution to journalArticle

Cha, Hyung Jin ; Jang, Do Soo ; Kim, Yeon Gil ; Hong, Bee Hak ; Woo, Jae Sung ; Kim, Kyong Tai ; Choi, Kwan Yong. / Rescue of deleterious mutations by the compensatory Y30F mutation in ketosteroid isomerase. In: Molecules and cells. 2013 ; Vol. 36, No. 1. pp. 39-46.
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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 A 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 6.4C, 5.1C and 10.0C, 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.",
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T1 - Rescue of deleterious mutations by the compensatory Y30F mutation in ketosteroid isomerase

AU - Cha, Hyung Jin

AU - Jang, Do Soo

AU - Kim, Yeon Gil

AU - Hong, Bee Hak

AU - Woo, Jae Sung

AU - Kim, Kyong Tai

AU - Choi, Kwan Yong

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N2 - 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 A 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 6.4C, 5.1C and 10.0C, 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.

AB - 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 A 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 6.4C, 5.1C and 10.0C, 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.

KW - Active-site recovery

KW - Ketosteroid isomerase

KW - More hydrophobic interactions

KW - Rescue mechanism

KW - Second-site suppressor

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