Conformational Priming of RepA-WH1 for Functional Amyloid Conversion Detected by NMR Spectroscopy

Abstract

How proteins with a stable globular fold acquire the amyloid state is still largely unknown. RepA is a versatile plasmidic DNA binding protein, functional either as a transcriptional repressor or as an initiator or inhibitor of DNA replication, the latter through the assembly of an amyloidogenic oligomer. Its N-terminal domain (WH1) is responsible for discrimination between these functional abilities by undergoing hitherto unknown structural changes. Furthermore, when expressed alone, RepA-WH1 behaves as a synthetic prion-like protein causing an amyloid proteinopathy in bacteria. RepA-WH1 is a stable dimer whose conformational dynamics had not been explored. Here we have studied it through NMR {1H}-15N relaxation and H/D exchange kinetics measurements. The N- and the C- terminal -helices, which lock the WH1 fold in each subunit of the dimer, as well as an internal amyloidogenic loop, show reduced stability and are partially unfolded in solution. S4-indigo, a small molecule ligand known to interfere with the amyloid assembly of RepA-WH1, binds to and tethers the N-terminal -helix and a {beta}-hairpin that is involved in dimerization, thus providing evidence for a priming role of fraying ends and dimerization switches in the amyloidogenesis of folded proteins.