Helicase-like functions in phosphate loop containing beta-alpha polypeptides

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 118; no. 16; pp. 1 - 9
• Main Authors: Vyas, Pratik, Trofimyuk, Olena, Longo, Liam M., Deshmukh, Fanindra Kumar, Sharon, Michal, Tawfik, Dan S.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 20-04-2021
• Subjects: Binding; Biological Sciences; Deoxyribonucleic acid; Dimers; DNA; DNA helicase; Nucleotides; Polypeptides; Polyphosphates; Proteins; Prototypes; RecA protein; Ribonucleic acid; RNA; Structure-function relationships; polyphosphate; P-loop; protein evolution; Walker A; multifunctionality
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2016131118

The P-loop Walker A motif underlies hundreds of essential enzyme families that bind nucleotide triphosphates (NTPs) and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/RNA helicases, translocases, and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple βα repeat proteins bind NTPs but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one β-(P-loop)-α element. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. The latter are reminiscent of extant P-loop recombinases such as RecA. Overall, these P-loop prototypes compose a plausible description of the sequence, structure, and function of the earliest P-loop NTPases. They also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.