Long-range allosteric effects - How adenine nucleotides shape the conformational landscape of the Ski2-like RNA helicase, Brr2

Brr2 is an essential Ski2-like RNA helicase that exhibits a unique structure among the spliceosomal helicases. Brr2 harbors a catalytically active N-terminal helicase cassette and a structurally similar, but enzymatically inactive C-terminal helicase cassette. Both cassettes contain a nucleotide binding pocket. Here we use biophysical and computational methods to delineate the functional connectivity between the cassettes and how occupancy of the nucleotide binding sites may influence each other. Our results show that Brr2 exhibits high specificity for adenine nucleotides with both cassettes binding ADP tighter than ATP. Adenine nucleotide affinity for the inactive C-terminal cassette is more than two orders of magnitude higher than that of the active N-terminal cassette, largely determined by slow nucleotide release. Mutations at the inter-cassette surfaces and in the connecting linker diminish the affinity of adenine nucleotides for both cassettes. Abrogation of nucleotide binding at the C-terminal cassette reduces nucleotide binding at the N-terminal cassette, 70 Å away. Molecular dynamics simulations identified structural communication lines that likely mediate the long-range allosteric effects. Together, our results reveal intricate networks of intra-molecular interactions in the complex Brr2 RNA helicase, which fine-tune its nucleotide affinities and which could be exploited for regulating the enzyme during splicing.