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 language | English |
|---|---|
| Pages (from-to) | 39-46 |
| Number of pages | 8 |
| Journal | Molecules and cells |
| Volume | 36 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 2013 Jul 1 |
| Externally published | Yes |
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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 journal › Article
}
TY - JOUR
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
PY - 2013/7/1
Y1 - 2013/7/1
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
UR - http://www.scopus.com/inward/record.url?scp=84893698071&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84893698071&partnerID=8YFLogxK
U2 - 10.1007/s10059-013-0013-1
DO - 10.1007/s10059-013-0013-1
M3 - Article
C2 - 23740430
AN - SCOPUS:84893698071
VL - 36
SP - 39
EP - 46
JO - Molecules and Cells
JF - Molecules and Cells
SN - 1016-8478
IS - 1
ER -