Redefining the Limits of Functional Continuity in the Early Evolution of P-Loop NTPases Demkiv, Andrey O. Toledo-Patiño, Saacnicteh Medina Carmona, Encarnación Berg, Andrej Pinto, Gaspar P. Parracino, Antonietta Sánchez Ruiz, José Manuel Hengge, Alvan C. Laurino, Paola Longo, Liam M. Kamerlin, Shina CL Walker A motif P-loop NTPase Phosphate-binding loop Supplementary materialis available at Molecular Biology and Evolution online.: http://academic.oup.com/mbe/article-lookup/doi/10.1093/molbev/msaf055#supplementary-data At the heart of many nucleoside triphosphatases is a conserved phosphate-binding sequence motif. A current model of early enzyme evolution proposes that this six to eight 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 this provocative model, seven disembodied Walker A-derived peptides were extensively computationally characterized. Although dynamic flickers of nest-like conformations were observed, significant structural similarity between the situated peptide and its disembodied counterpart was not detected. Simulations suggest that phosphate binding is nonspecific, with a 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, 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 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 has been eroded by time. 2025-05-02T08:57:19Z 2025-05-02T08:57:19Z 2025-03-12 journal article Andrey O Demkiv, Saacnicteh Toledo-Patiño, Encarnación Medina-Carmona, Andrej Berg, Gaspar P Pinto, Antonietta Parracino, Jose M Sanchez-Ruiz, Alvan C Hengge, Paola Laurino, Liam M Longo, Shina Caroline Lynn Kamerlin, Redefining the Limits of Functional Continuity in the Early Evolution of P-Loop NTPases, Molecular Biology and Evolution, Volume 42, Issue 4, April 2025, msaf055, [https://doi.org/10.1093/molbev/msaf055] https://hdl.handle.net/10481/103884 10.1093/molbev/msaf055 eng http://creativecommons.org/licenses/by/4.0/ open access Atribución 4.0 Internacional Oxford University Press