Glycine-Proline driven flexibility and interface-dependent conservation of six-residue Ω-loops in protein complexes

Ω-loops are six- to sixteen-residue, non-regular structural motifs that occur in protein structures, connecting regular regions and forming a well-defined, ordered structural segment. They are functionally important because of their strategic location on the protein surface, where they are often associated with molecular recognition, ligand binding, and catalytic activity. Their non-regular nature enables them to adopt different conformations, which makes them important in protein dynamics and allosteric communication. In this work, we have curated six-residue Ω-loops from diverse protein-protein complexes of varying stability and found them to be abundant across all. We identified 1205 hydrogen-bonded and 6518 non-hydrogen-bonded Ω-loops, leading to an average of ~1.7 Ω-loops per structure. Glycine and proline were the most enriched residues in the loop, with proline preferentially positioned at the fourth position and glycine distributed across multiple positions, supporting their roles in chain reversal and conformational flexibility. Using ProteinMPNN-based sequence-design modelling and Rate4Site evolutionary-rate analysis, we show that Ω-loops occupy a relatively constrained sequence space, with interface loops in obligate homodimers and heterodimers more conserved than non-interface loops; the opposite trend was observed in transient and crystal dimers. Mapping known pathogenic and benign mutations onto the Ω-loop present in human proteins, together with a case study of the TEM1 β-lactamase Ω-loop, further illustrates how small sequence changes within these loops can produce crucial structural and functional consequences, leading to altered enzyme activity and antibiotic resistance. Altogether, we report that Ω-loops are not merely connectors of secondary structure but are structurally constrained, evolutionarily significant elements that contribute to protein folding, stability and function.