Analyzing protein structure and function using ancestral gene reconstruction

Abstract

Protein families with functionally diverse members can illuminate the structural determinants of protein function and the process by which protein structure and function evolve. To identify the key amino acid changes that differentiate one family member from another, most studies have taken a horizontal approach, swapping candidate residues between present-day family members. This approach has often been stymied, however, by the fact that shifts in function often require multiple interacting mutations; chimeric proteins are often nonfunctional, either because one lineage has amassed mutations that are incompatible with key residues that conferred a new function on other lineages, or because it lacks mutations required to support those key residues. These difficulties can be overcome by using a vertical strategy, which reconstructs ancestral genes and uses them as the appropriate background in which to study the effects of historical mutations on functional diversification. In this review, we discuss the advantages of the vertical strategy and highlight several exemplary studies that have used ancestral gene reconstruction to reveal the molecular underpinnings of protein structure, function, and evolution.

Figure 1

Dissecting the sequence determinants of function within a family that has an ancestral function (filled circle) and a derived function (open circle). The functional change was caused by a subset of the sequence changes along branch C (black box). In the scenario shown here, permissive mutations on branch B (star) were required to allow the protein to tolerate the function-switching mutations; restrictive mutations incompatible with the ancestral function accumulated on branch D (cross). Swapping residues between modern proteins (arrow) is inefficient because the sequences differ by all mutations along A, B, C, and D. Further, protein X does not have the permissive mutations and cannot take on the derived function. Protein Y has restrictive mutations that do not allow it tolerate the ancestral function.

Figure 2

Permissive and restrictive mutations shaped GR evolution. GRs and MRs result from an ancient gene duplication (arrow). ancCR was activated by aldosterone and cortisol (filled square, filled triangle). Cortisol specificity (open square, filled triangle) evolved between ancGR1 and ancGR2. The mutations that led to the functional change (black box) were preceded by permissive mutations (star) and followed by restrictive mutations (X). MRs do not have the permissive mutations and cannot tolerate the mutations in the black box. Human and fish GRs have restrictive mutations that do not allow reversion of the mutations in the black box to the ancestral state.

Figure 3

Structural basis for the evolution of cortisol specificity. A) Structure of AncCR (white, 2Q1H) with bound aldosterone (orange) overlaid on the structure of AncGR2 (blue, 3GN8) with bound DEX (purple), a cortisol analog. Mutation S106P disrupts a helix cap and introduces a kink at the N-terminus of helix 7, repositioning helix 7 so that Gln-111 of AncGR2 can form a cortisol-specific hydrogen bond. B) Schematic representation of restrictive mutations G114Q and L197M. The derived Gln-Met pair in AncGR2 can be tolerated in the derived conformation (right) but clash when helix 7 is in its ancestral position (left). The hydrogen-bond of Gln-111 with cortisol (purple) is shown in red.