Compensatory Evolution of a WW Domain Variant Lacking the Strictly Conserved Trp Residue

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.

A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function

“…ε pro abx Þ. However, the mean epistasis score was also unable to separate all three known stabilizing hYAP65 WW domain mutations present in our dataset from the bulk of the mutations as well as two known activityenhancing mutations (19)(20)(21)(22) (Fig. 2C).…”

Comprehensive Analysis of the Effects of Escherichia coli ORFs on Protein Translation Reaction

“…Although we are still at the starting point of investigating the precise roles of the modifier proteins on the reaction, combining the experimental data with the PPI network may provide insight into how these proteins interact with the translation machinery. Moreover our data may be useful not only for technologies utilizing in vitro translation systems (19,48,49) but also for designing in vitro translation systems with significantly improved performance than those available at present and also for protein production in vivo, such as by coexpressing beneficial components together with a protein of interest.…”

In Vitro Selection of Proteins that Undergo Covalent Labeling with Small Molecules by Thiol‐Disulfide Exchange by Using Ribosome Display

“…The P4S peptide contains the P4S mutation (Pro4!Ser) in the first position of the known binding motif (PPxY) of the WW domain, and thus has lower binding-affinity to the WW domain (K d > 1 mm). [12] Thus, we set the conditions such that selection based on peptide (noncovalent) binding would not occur easily. To broaden the explorable sequence space, an initial library was prepared by mixing the genes encoding wild-type and W17F-mutant WW-domains in a 1:1 ratio, and by introducing random mutations into the full length of the genes by error-prone PCR.…”

“…Next, to investigate peptide sequence specificity, we replaced biotin-P4S with two different peptides, ePY and P4W (peptide sequences are shown in Figure 3). These two peptides were chosen as they are representative of peptides with affinities known to be higher and lower than that of P4S, [12] respectively. Comparison of the binding to biotin-P4S with that for the two other targets showed that biotin-P4S gave the strongest signal; this indicated that the f7 mutant had specificity for the peptide sequence of the binding target used in the selection experiment.…”

“…The W17F (Trp17!Phe) mutant is known to have as low an affinity to P4S peptide as the wild-type, but broader specificity, presumably due to its unstructured conformation. [12,13] We generated an initial library harboring an average of 1.0 amino acid mutations per clone, with a theoretical diversity of at least 10 8 (see the Experimental Section). Note that 10 8 was the diversity of the initial pool, and significantly larger numbers (up to 10 12 ) were screened because mutations were introduced in the amplification processes during subsequent rounds of selection.…”