Metamorphic proteins are unexpected paradigms of the protein folding problem, as their sequences encode two alternative folds, which reversibly interconvert within biologically relevant timescales to trigger different cellular responses. A quintessential example of a metamorphic protein is found in the universally conserved family of NusG-like factors, which bind to RNA polymerase (RNAP) to support processive RNA synthesis and to couple transcription to ongoing cellular processes. The specialized bacterial NusG paralog, RfaH, undergoes an all-α to all-β fold-switch of a whole protein domain to bind to RNAP and activate the expression of virulence and conjugation genes in bacterial pathogens.
Given that its fold-switch is directly involved in the regulation of its biological function, there is great interest in understanding its refolding mechanism, its effect on RNAP upon binding and how these two structures are encoded in a single sequence. Here, we describe our experimental and computational efforts to characterize the fold-switch, function, and coevolution of RfaH. By combining molecular dynamics and hydrogen-deuterium exchange mass spectrometry, a consistent structural interconversion pathway is conceived, in which at least two intermediates enable RfaH to switch back and forth between its native states. These methods also enable to elucidate the effects of RfaH binding to RNAP, largely inaccessible for most structural biology methods. Moreover, coevolutionary analysis of thousands of RfaH sequences enables to determine key residue pairs encoding both RfaH folds and predict their structures from sequence information alone.
The experimental insights that unveiled the fold-switch of RfaH can be further exploited in state-of-the-art protein structure prediction methods to distinguish between metamorphic and non-metamorphic homologs, constituting one of the many ongoing efforts to find signatures and general properties to ultimately describe the protein metamorphome.