Biology subjects

Santos, K. F.

Publications and source records attributed to Santos, K. F..

3 recordsLinked to original sources

bioRxiv2021

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 [A] 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.

biochemistry↗

bioRxiv2019

Positive and negative intra-molecular modulation in a dual-cassette RNA helicase

RNA helicase Brr2 is required for the activation of the spliceosome prior to the first catalytic step of splicing. Brr2 represents a distinct subgroup of Ski2-like nucleic acid helicases whose members comprise tandem helicase cassettes. Only the N-terminal cassette of Brr2 is an active ATPase and can unwind substrate RNAs. The C-terminal cassette represents a pseudo-enzyme that can stimulate RNA-related activities of the N-terminal cassette. However, the molecular mechanisms, by which the C-terminal cassette modulates the activities of the N-terminal unit remain elusive. Here, we show that N- and C-terminal cassettes adopt vastly different relative orientations in a crystal structure of Brr2 in complex with an activating domain of the spliceosomal Prp8 protein as compared to the crystal structure of isolated Brr2. Likewise, the cassettes occupy different relative positions and engage in different inter-cassette contacts during different stages of splicing. Engineered disulfide bridges that lock the cassettes in two different relative orientations have opposite effects on RNA-related activities of the N-terminal cassette compared to the unrestrained protein. Moreover, different relative positioning of the cassettes strongly influences ATP hydrolysis by the N-terminal cassette. Our results demonstrate that the inactive C-terminal cassette of Brr2 can exert both positive and negative influence on the active N-terminal helicase unit from a distance.

biochemistry↗

bioRxiv2019

Molecular Mechanism Underlying Inhibition of Intrinsic ATPase Activity in a Ski2-like RNA Helicase

RNA-dependent NTPases can act as RNA/RNA-protein remodeling enzymes and typically exhibit low NTPase activity in the absence of RNA/RNA-protein substrates. How futile intrinsic NTP hydrolysis is prevented is frequently not known. The ATPase/RNA helicase Brr2 belongs to the Ski2-like family of nucleic acid-dependent NTPases and is an integral component of the spliceosome. Comprehensive nucleotide binding and hydrolysis studies are not available for a member of the Ski2-like family. We present crystal structures of Chaetomium thermophilum Brr2 in the apo, ADP-bound and ATPyS-bound states, revealing nucleotide-induced conformational changes and a hitherto unknown ATPyS binding mode. Our results in conjunction with Brr2 structures in other molecular contexts reveal multiple molecular mechanisms that contribute to the inhibition of intrinsic ATPase activity, including an N-terminal region that restrains the RecA-like domains in an open conformation and exclusion of an attacking water molecule, and suggest how RNA substrate binding can lead to ATPase stimulation.\n\nHIGHLIGHTSO_LICrystal structures of Brr2 in complex with different adenine nucleotides.\nC_LIO_LIThe Brr2 N-terminal region counteracts conformational changes induced by ATP binding.\nC_LIO_LIBrr2 excludes an attacking water molecule in the absence of substrate RNA.\nC_LIO_LIDifferent helicase families resort to different NTPase mechanisms.\nC_LI

biochemistry↗