Conservation of Specificity in Two Low-Specificity Proteins.

Area of Science:

• Biochemistry
• Evolutionary Biology
• Molecular Biology

Background:

• Regulatory proteins often bind short peptide regions of target proteins, modulating their activity.
• The specificity of these interactions is not always clear, with potential roles for both the binding interface and external factors like localization.
• S100A5 and S100A6 are examples of low-specificity regulatory proteins.

Purpose of the Study:

• To investigate the evolutionary basis of peptide-binding specificity in S100A5 and S100A6.
• To determine if binding specificity is an intrinsic property of the S100 peptide interface or dictated by external factors.
• To understand the evolutionary trajectory of paralog specificity.

Main Methods:

• Isothermal titration calorimetry (ITC) to assess peptide binding to human S100A5 and S100A6.
• Analysis of peptide binding properties of S100A5 and S100A6 orthologs from five amniote species.
• Ancestral sequence reconstruction to infer the binding specificity of the ancestral paralogs.
• Site-directed mutagenesis to test the impact of historical mutations on binding specificity.

Main Results:

• Human S100A5 and S100A6 exhibit distinct yet overlapping peptide-binding profiles.
• Peptide binding specificity has been conserved across amniote lineages for at least 320 million years.
• The inferred ancestral protein bound all peptides recognized by modern S100A5 and S100A6, indicating subfunctionalization.
• A single historical mutation in S100A5 was sufficient to confer binding to an S100A6-specific peptide, demonstrating evolvability.

Conclusions:

• Strong evolutionary constraints exist on the peptide-binding specificity of S100 proteins.
• Despite low intrinsic specificity, the peptide-binding interface of S100 proteins plays a significant role in biological function.
• Paralog specificity likely evolved through subfunctionalization from a broadly binding ancestor.