Search

Abundant and essential motifs, such as phosphate-binding loops (P-loops), are presumed to be the seeds of modern enzymes. The Walker-A P-loop is absolutely essential in modern NTPase enzymes, in mediating binding, and transfer of the terminal phosphate groups of NTPs. However, NTPase function depends on many additional active-site residues placed throughout the protein's scaffold. Can motifs such as P-loops confer function in a simpler context? We applied a phylogenetic analysis that yielded a sequence logo of the putative ancestral Walker-A P-loop element: a β-strand connected to an α-helix via the P-loop. Computational design incorporated this element into de novo designed β-α repeat proteins with relatively few sequence modifications. We obtained soluble, stable proteins that unlike modern P-loop NTPases bound ATP in a magnesium-independent manner. Foremost, these simple P-loop proteins avidly bound polynucleotides, RNA, and single-strand DNA, and mutations in the P-loop's key residues abolished binding. Binding appears to be facilitated by the structural plasticity of these proteins, including quaternary structure polymorphism that promotes a combined action of multiple P-loops. Accordingly, oligomerization enabled a 55-aa protein carrying a single P-loop to confer avid polynucleotide binding. Overall, our results show that the P-loop Walker-A motif can be implemented in small and simple β-α repeat proteins, primarily as a polynucleotide binding motif., Competing Interests: The authors declare no conflict of interest.

First and foremost: Margaret Dayhoff's 1966 hypothesis on the origin of proteins is now an accepted model for the emergence of large, globular, functional proteins from short, simple peptides. However, the fundamental question of how the first protein(s) emerged still stands. The tools and hypotheses pioneered by Dayhoff, and the over 65 million protein sequences and 12 000 structures known today, enable those who follow in her footsteps to address this question., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)