Abstract
The cyclooxygenase-2 is a pro-inflammatory and cancer marker, whose mRNA stability and translation is regulated by the CUG-binding protein 2 interacting with AU-rich sequences in the 3′ untranslated region. Here, we present the solution NMR structure of CUG-binding protein 2 RRM3 in complex with 5′-UUUAA-3′ originating from the COX-2 3′-UTR. We show that RRM3 uses the same binding surface and protein moieties to interact with AU- and UG-rich RNA motifs, binding with low and high affinity, respectively. Using NMR spectroscopy, isothermal titration calorimetry and molecular dynamics simulations, we demonstrate that distinct sub-states characterized by different aromatic side-chain conformations at the RNA-binding surface allow for high- or low-affinity binding with functional implications. This study highlights a mechanism for RNA discrimination possibly common to multiple RRMs as several prominent members display a similar rearrangement of aromatic residues upon binding their targets.
Highlights
The cyclooxygenase-2 is a pro-inflammatory and cancer marker, whose mRNA stability and translation is regulated by the CUG-binding protein 2 interacting with AU-rich sequences in the 3′ untranslated region
Scalar coupling measurements and molecular dynamics (MD) simulations demonstrated that the binding surface of CUG-BP2 RRM3 exists in multiple states and that a conformational switch of aromatic side-chains is at the origin of the fine tuning of the affinity for different targets
RRM3 binds to AU-rich motifs found in the cyclooxygenase 2 (COX-2) mRNA 3′-untranslated region (UTR)
Summary
The cyclooxygenase-2 is a pro-inflammatory and cancer marker, whose mRNA stability and translation is regulated by the CUG-binding protein 2 interacting with AU-rich sequences in the 3′ untranslated region. RRMs are about 90 amino acids long and adopt a βαββαβ secondary structure forming an antiparallel β-sheet stacked onto two α-helices[1] They bind two to three nucleotides on the β-sheet through the RNPs. RNA-binding proteins such as CUG-BP2 contain multiple RRM copies and are involved in several regulatory processes. At the β-sheet RNA-binding interface of both domains, the UG dinucleotide shares structural features with the left-handed Z-RNA helix and only the last two uracils stack with the conserved phenylalanines of the RNPs. All four nucleotides are recognized by hydrogen-bonding to the protein backbone and side-chains. The solution structure of CUG-BP1 RRM3 in complex with (UG)[3] shows that four out of six nucleotides stack on aromatic residues of the β-sheet surface and specific recognition is ensured by a dense network of hydrogen-bonds between RNA moieties and the protein backbone and polar side-chains. Scalar coupling measurements and molecular dynamics (MD) simulations demonstrated that the binding surface of CUG-BP2 RRM3 exists in multiple states and that a conformational switch of aromatic side-chains is at the origin of the fine tuning of the affinity for different targets
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