Long-range allostery mediates cooperative adenine nucleotide binding by the Ski2-like RNA helicase Brr2
| Autor: | Marina V. Rodnina, K. Vester, Dmitry Burakovskiy, Markus C. Wahl, Pohl Milón, Eva Absmeier, Tahereh Ghane, Petra Imhof, Karine F. Santos |
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| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
SF2 superfamily 2 intramolecular regulation sn small nuclear T1 truncation 1 IG immunoglobulin-like domain Biochemistry Substrate Specificity HB helical bundle domain pre-mRNA precursor messenger RNA Protein structure Tris tris(hydroxymethyl)aminomethane Adenosine Triphosphate Adenine nucleotide enzyme kinetics Nucleotide CC C-terminal cassette Amino Acids chemistry.chemical_classification biology Chemistry Adenine Nucleotides Editors' Pick NTR N-terminal region Ribonucleoproteins Small Nuclear RNA Helicase A MD molecular dynamics Adenosine Diphosphate superfamily 2 helicase RNA splicing HLH helix–loop–helix domain wt wild type 500 Naturwissenschaften und Mathematik::540 Chemie::540 Chemie und zugeordnete Wissenschaften RNA Helicases Research Article RNA helicase NC N-terminal cassette Allosteric regulation NTPase nucleic-acid-dependent nucleotide triphosphatase Molecular Dynamics Simulation DSF differential scanning fluorimetry SEM standard error of the mean FRET fluorescence resonance energy transfer 03 medical and health sciences h human Protein Domains WH winged-helix domain protein conformation Humans Molecular Biology RNP ribonucleoprotein complex Binding Sites 030102 biochemistry & molecular biology Helicase Cell Biology allosteric regulation v/v volume/volume Kinetics 030104 developmental biology DTT dithiothreitol ss single-stranded Mutation pre-mRNA splicing biology.protein Biophysics BAC bacterial artificial chromosome mant methylanthraniloyl Structural communication pre mRNA splicing |
| Zdroj: | The Journal of Biological Chemistry Journal of Biological Chemistry |
| DOI: | 10.17169/refubium-31866 |
| Popis: | 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 connected by a linker region. Both cassettes contain a nucleotide-binding pocket, but it is unclear whether nucleotide binding in these two pockets is related. Here we use biophysical and computational methods to delineate the functional connectivity between the cassettes and determine whether occupancy of one nucleotide-binding site may influence nucleotide binding at the other cassette. 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, as determined by slow nucleotide release. Mutations at the intercassette surfaces and in the connecting linker diminish the affinity of adenine nucleotides for both cassettes. Moreover, we found that 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 these long-range allosteric effects, predominantly across the intercassette interface. Together, our results reveal intricate networks of intramolecular interactions in the complex Brr2 RNA helicase, which fine-tune its nucleotide affinities and which could be exploited to regulate enzymatic activity during splicing. |
| Databáze: | OpenAIRE |
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