Architectonic Spandrels in the Origin of Enzymatic Function

How new molecular functions emerge during protein evolution remains a fundamental question in molecular biology. The energy landscape theory states that proteins are minimally frustrated, i.e. they have minimized their internal conflicts, to allow robust folding. Yet, not all energetic conflicts are eliminated, with functional regions such as catalytic residues and ligand-binding sites being often enriched in frustrated interactions, trading localized stability for biological activity. However, it is still uncertain whether this functional frustration is an evolutionary adaptation, positively selected despite its energetic cost or an inevitable physical byproduct of the fold architecture. Here, we combine reverse folding, structure prediction, and sequence analysis with local frustration profiling to address this long-standing question. Unexpectedly, we found that reverse folding algorithms are unable to energetically minimize evolutionary conserved frustration at specific residues, even when detrimental to overall structural stability. We propose that these frustration hotspots act as architectural spandrels, inherent physical constraints of the fold that evolution subsequently co-opts for function. Our findings connect biophysical constraints and evolutionary selection, providing a new framework to understand how functional specificity emerges in protein landscapes.