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.

P-loop Walker A 基序是数百个结合三磷酸核苷酸 (NTP) 和介导磷酰基转移 (P-loop NTPase) 的必需酶家族的基础，包括最早的 DNA/RNA 解旋酶、转位酶和重组酶。这些酶的原始前体是什么？这些大而复杂的蛋白质能从简单的多肽中产生吗？之前，我们展示了嵌入简单 βα 重复蛋白中的 P 环与 NTP 结合，但出乎意料的是，ssDNA 和 RNA 也是如此。在这里，我们超越了配体结合的纯生物物理功能，以展示基本的解旋酶样活动。我们进一步构建了仅包含一个 β-(P-loop)-α 元件的简单 40 个残基多肽。尽管它们很简单，但这些 P 环原型赋予了诸如链分离和交换等功能。首先，这些多肽解开 dsDNA，并在添加 NTP 或无机多磷酸盐后，释放结合的 ssDNA 链，以重新形成 dsDNA。结合动力学和低分辨率结构分析表明，活性是由从二聚体到高阶组装的寡聚形式介导的。后者让人想起现存的 P 环重组酶，如 RecA。总的来说，这些 P-loop 原型构成了对最早 P-loop NTPase 的序列、结构和功能的合理描述。他们还表明，多功能性和动态组装是赋予短多肽复杂的进化相关功能的关键。结合动力学和低分辨率结构分析表明，活性是由从二聚体到高阶组装的寡聚形式介导的。后者让人想起现存的 P 环重组酶，如 RecA。总的来说，这些 P-loop 原型构成了对最早 P-loop NTPase 的序列、结构和功能的合理描述。他们还表明，多功能性和动态组装是赋予短多肽复杂的进化相关功能的关键。结合动力学和低分辨率结构分析表明，活性是由从二聚体到高阶组装的寡聚形式介导的。后者让人想起现存的 P 环重组酶，如 RecA。总的来说，这些 P-loop 原型构成了对最早 P-loop NTPase 的序列、结构和功能的合理描述。他们还表明，多功能性和动态组装是赋予短多肽复杂的进化相关功能的关键。