Functional changes during the evolution of secondarily single sHsp in Erwiniaceae.

(A) Schematic phylogeny of sHsps in Enterobacterales. Gene duplication resulting in IbpA + IbpB two-protein system is marked with a star, while the loss of ibpB gene in Erwiniaceae clade is marked with a cross; AncA0 – reconstructed last common ancestor of IbpA from Erwiniaceae and Enterobacteriaceae, expressed as a part of two-protein system; AncA1 – reconstructed last common ancestor of secondarily single IbpA from Erwiniaceae. (B) Extant sHsps’ ability to stimulate luciferase refolding. sHsps were present during the luciferase thermal denaturation step. Refolding of denatured luciferase was performed by the Hsp70-Hsp100 chaperone system. (C) Binding of extant and ancestral sHsps to heat-aggregated E. coli proteins. E. coli proteins were heat aggregated and immobilized on a BLI sensor. sHsps were heat activated before the binding step. (D) Sequestrase activity of extant and ancestral sHsps; Luciferase was heat denatured in the presence of different concentrations of sHsps and size of formed sHsps – substrate assemblies was measured by DLS; results are shown as average hydrodynamic radius ± SD. (E) Extant and ancestral sHsps’ ability to stimulate luciferase refolding.

Substitutions at positions 66 and 109 that occurred between nodes A0 and A1 are crucial for ancestral sHsps to work as a single protein.

Luciferase refolding assay was performed as in 1B. (A) Schematic phylogeny of Enterobacterales IbpA showing increased ratio of nonsynonymous to synonymous substitutions (ω) on the branch between nodes AncA0 and AncA1. Loss of cooperating IbpB is marked on a tree. (B) Identification of substitutions necessary for AncA0 to obtain AncA1 – like activity in luciferase disaggregation; seven candidate mutations were introduced into AncA0 (AncA0+7); subsequently, in series of six mutants, each of the candidate positions was reversed to a more ancestral state (AncA0 + 6* variants) (C) Effect of substitutions at positions 66 and 109 on the ability of AncA0 and AncA1 to stimulate luciferase refolding.

Substitutions at positions 66 and 109 decreased the affinity of AncA0 ACD to C-terminal peptide and aggregated substrate.

(A) Structural model of complex formed by AncA0 Q66H G109D a-crystallin domain dimer (purple and lilac) and AncA0 C-terminal peptide (orange). (B) Effect of Q66H G109D substitutions on AncA0 ACD’s affinity to C-terminal peptide. Binding of ACDs of AncA0 or AncA0 Q66H G109D to C-terminal peptide was analyzed by BLI. Biolayer thickness at the end of association step is shown for each concentration as a fraction of thickness at saturating concentration. (C) Effect of Q66H G109D substitutions on AncA0 ACD’s affinity to aggregated E. coli proteins bound to BLI sensor. Analysis was performed as in 1C.

Differences at positions 66 and 109 determine functional differences between extant IbpA proteins from E. coli and E. amylovora.

(A) Effect of substitutions at position 66 and 109 (and homologous) on the ability of IbpA from E. amylovora and E. coli to stimulate luciferase refolding. Assay was performed as in 1B. (B, C) Effect of substitutions at analyzed positions on binding of IbpA from E. coli (A)and E. amylovora (B) to aggregated luciferase; assay was performed as in 1C. (D-H) Effect of substitutions at analyzed positions on inhibition of Hsp 70 system binding to aggregates by extant sHsps (D) Experimental scheme. (E-H) Aggregate-bound sHsps differently inhibit Hsp70 binding. BLI sensor with immobilized aggregated luciferase and aggregate bound sHsps was incubated with Hsp70 or buffer (spontaneous dissociation curve). Grey traces present Hsp70 binding to immobilized aggregates in the absence of sHsps.