Redefining the Limits of Functional Continuity in the Early Evolution of P-Loop NTPases

Abstract

At the heart of many nucleoside triphosphatases is a conserved sequence motif that binds phosphate. A current model of early enzyme evolution proposes that this 6-8 residue motif could have sparked the emergence of the very first nucleoside triphosphatases - a striking example of evolutionary continuity from simple beginnings, if true. To test whether this provocative evolutionary model holds for the ancient and ubiquitous P-Loop NTPases, the properties of seven disembodied Walker A-derived peptides were extensively characterized by Hamiltonian replica exchange molecular dynamics simulations. Although dynamic flickers of nest-like conformations were observed, significant structural similarity between the situated peptide and its disembodied counterpart was not detected - even in the presence of orthophosphate or a nucleotide. Simulations suggest that phosphate binding is non-specific, with a slight preference for GTP over orthophosphate. Control peptides with the same amino acid composition but different sequences and situated conformations behaved similarly to the Walker A peptides with respect to conformational dynamics and phosphate binding, revealing no indication that the Walker A sequence is privileged as a disembodied peptide. We conclude that the evolutionary history of the P-Loop NTPase family is unlikely to have started with a disembodied Walker A peptide in an aqueous environment. The limits of evolutionary continuity for this protein family, and the environmental context within which it emerged, must be reconsidered. Finally, we argue that motifs such as the Walker A motif may represent incomplete or fragmentary molecular fossils - the true nature of which have been eroded by time. Significance StatementThe first proteins were undoubtedly small, but when did those seeds emerge, and what did they look like? It is widely believed that the Walker A P-loop motif is a seed for the emergence of phosphate binding proteins, snugly binding phosphate in a structurally conserved nest. We probe this hypothesis through detailed computational characterization of disembodied Walker A and control peptides, showing that any nest formation is transient, and phosphate binding is weak and non-specific. Thus, we do not find structural continuity represented in the conserved Walker A motif, and current models of early P-loop evolution require revision. Further, care is required when interpreting highly conserved sequence fragments more broadly, as these may merely represent eroded molecular fossils.