“Protein Un‐Design”: Evolutionary Origins of Native DNA‐Binding Architectures in Unfolded Polypeptides

Abstract

We seek to test the hypothesis that the evolutionary origins of globular proteins, however exquisite in their structural organization, lie within the conformational repertoire of unstructured polypeptides. A model is provided by the high‐mobility‐group (HMG) box, an L‐shaped a‐helical motif of sharp DNA bending that represents a primordial innovation at the root of Eukarya. Whereas the modern HMG box exhibits conserved architectural features (such as an “aromatic buttress” underlying its angular protein surface; Racca, J., et al. J. Biol. Chem. E‐pub (2014)), we demonstrate that natively unfolded variants with defective buttresses can, as a seeming paradox, exhibit native DNA binding and DNA bending. Canonical structure is regained on specific DNA binding as inferred from CD and 1H‐15N NMR. Coupled DNA‐dependent protein folding and DNA bending suggest that primordial boxes could readily have evolved from unstructured precursors, driven by selection for architectural gene regulation. Functional “proof of principle” is provided by unfolded variants of human testis‐determining factor SRY. In a rat embryonic gonadal‐ridge cell line such variant HMG boxes are able to regulate the principal target gene in the nascent testis (Sox9), activating a male transcriptional program. We envisage that progressive stabilization of nascent folds represents a fundamental mechanism for the rapid evolution of autonomous protein structures, yielding the modern landscape of DNA‐binding architectures. In this view extant unstructured DNA‐ and RNA‐binding motifs represent relics of the transition between RNA and protein worlds.Acknowledgements. We thank L. Labeots, A. Jansco‐Radek, J. Radek, and G. Chan for their participation in the early stages of this work.