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.
Bibliographical noteFunding Information:
This research was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009503), Rural Development Administration, Korea and by a grant from the National R & D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea ( 1320240). We thank the staff at beam-lines 6B and 6C, Pohang Accelerator Laboratory, Korea, for help with data collection, and Mr. Han Seop Shin for technical assistance. We also thank Dr. Ki Joon Cho (Korea University, Korea) for helpful discussion.
- Active-site recovery
- Ketosteroid isomerase
- More hydrophobic interactions
- Rescue mechanism
- Second-site suppressor
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology