Adaptive and Spandrel-like Constraints at Functional Sites in Protein Folds

Understanding the relationships among amino acid sequences, structures and functions in proteins and how they evolve, remains a central challenge in molecular biology. It is still unclear which sequence elements differentially contribute to structural integrity or molecular function. Even more, there are ongoing debates on whether protein folds emerge as a result of evolution or as a consequence of physical laws. The energy landscapes theory states that proteins are minimally frustrated systems, i.e. they fold by minimising their energetic conflicts. However, some local frustration, believed to be selected for functional reasons, remains in the native state of proteins. Here, we combine reverse folding and structure prediction methods with sequence and local frustration analysis to address the aforementioned ideas. We found that reverse folding techniques are unable to erase evolutionary conserved frustration from certain residues, even when detrimental for structural integrity. We propose that certain frustration hotspots behave like architectural spandrels, not directly shaped by selection but emerging from physical constraints in protein folds which evolution can later co-opt for function. Our results provide a new perspective revealing how sequence variation and functional specificity could evolve from evolutionary, structural and biophysical constraints.