Compensatory Evolution of a WW Domain Variant Lacking the Strictly Conserved Trp Residue |
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Authors: | Hayato Yanagida Tomoaki Matsuura Tetsuya Yomo |
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Affiliation: | (1) Graduate School of Frontier Biosciences, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan;(2) Department of Bioinformatics Engineering, Graduate School of Information Science and Technology, Osaka University, Osaka, Japan;(3) Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Tokyo, Japan;(4) Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency (JST), Tokyo, Japan |
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Abstract: | Replacement of conserved amino acid residues during evolution of proteins can lead to divergence and the formation of new families with novel functions, but is often deleterious to both protein structure and function. Using the WW domain, we experimentally examined whether and to what degree second-site mutations can compensate for the reduction of function and loss of structure that accompany substitution of a strictly conserved amino acid residue. The W17F mutant of the WW domain, with substitution of the most strictly conserved Trp residue, is known to lack a specific three-dimensional structure and shows reduced binding affinity in comparison to the wild type. To obtain second-site revertants, we performed a selection experiment based on the proline-rich peptide (PY ligand) binding affinity using the W17F mutant as the initial sequence. After selection by ribosome display, we were able to select revertants that exhibited a maximum ninefold higher affinity to the PY ligand than the W17F mutant and showed an even better affinity than the wild type. In addition, we found that the functional restoration resulted in increased binding specificity in selected revertants, and the structures were more compact, with increased amounts of secondary structure, in comparison to the W17F mutant. Our results suggest that the defective structure and function of the proteins caused by mutations in highly conserved residues occurring through divergent evolution not only can be restored but can be further improved by compensatory mutations. |
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Keywords: | WW domain Conserved residue Divergent evolution Directed evolution Compensatory mutation Second-site revertant Unfolded protein Ribosome display |
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