Correlation of fitness landscapes from three orthologous TIM barrels originates from sequence and structure constraints

Abstract

Sequence divergence of orthologous proteins enables adaptation to environmental stresses and promotes evolution of novel functions. Limits on evolution imposed by constraints on sequence and structure were explored using a model TIM barrel protein, indole-3-glycerol phosphate synthase (IGPS). Fitness effects of point mutations in three phylogenetically divergent IGPS proteins during adaptation to temperature stress were probed by auxotrophic complementation of yeast with prokaryotic, thermophilic IGPS. Analysis of beneficial mutations pointed to an unexpected, long-range allosteric pathway towards the active site of the protein. Significant correlations between the fitness landscapes of distant orthologues implicate both sequence and structure as primary forces in defining the TIM barrel fitness landscape and suggest that fitness landscapes can be translocated in sequence space. Exploration of fitness landscapes in the context of a protein fold provides a strategy for elucidating the sequence-structure-fitness relationships in other common motifs.

RheoScale: A tool to aggregate and quantify experimentally determined substitution outcomes for multiple variants at individual protein positions

“…To develop the RheoScale calculator, we used both simulated data (Figure and ) and experimental data previously published for the LacI/GalR paralogs (Meinhardt et al., ). We further tested the calculator on published data for pyruvate kinase, an enzyme for which multiple experimental functional parameters are available (Ishwar et al., ; Tang et al., ), and three TIM barrel orthologs that were characterized using deep mutational scanning (Chan et al., ). Finally, we considered the scores in light of known structural features to identify potential features of rheostat positions.…”

“…Although the assay output is (i) a combination of all possible changes in stability and function, and (ii) sensitive to the threshold of the biological competition assay (Mavor et al., ), data for a given position usually report results for all possible amino acid substitutions and thus may provide information about rheostat positions. Here, we have used RheoScale to analyze results for three TIM barrel isozymes that were exhaustively substituted in the central β barrel and parts of the flanking loops (Figure A and B) (Chan et al., ). This region is known to be important for TIM barrel stability and contains the enzyme active site (Chan et al., ; Gangadhara, Laine, Kathuria, Massi, & Matthews, ).…”

“…Here, we have used RheoScale to analyze results for three TIM barrel isozymes that were exhaustively substituted in the central β barrel and parts of the flanking loops (Figure A and B) (Chan et al., ). This region is known to be important for TIM barrel stability and contains the enzyme active site (Chan et al., ; Gangadhara, Laine, Kathuria, Massi, & Matthews, ). This data set allowed us to test the score calculations with assay output common to deep mutational scanning (i.e., a biological fitness score) and to compare and contrast rheostat positions in three proteins with structural and functional similarities (albeit with low sequence identities in the range of 30–40%).…”

“…(A) Side and (B) top views of the ribbon diagram for a TIM barrel structure. The substituted regions (Chan et al., ) are colored with alternating green and magenta. Each of these regions comprise a β strand and parts of the flanking N‐ and C‐loops; these eight strands form the central beta barrel of the protein.…”

“…(C) RheoScale scores were derived from the experimental data of Chan et al. (). Structurally equivalent positions are shown on the X ‐axis using the numbering for the Ss isozyme.…”