Conformational Stability Adaptation of a Double-Stranded RNA-Binding Domain to Transfer RNA Ligand

Abstract

The double-stranded RNA-binding domain (dsRBD) is a broadly distributed domain among RNA-maturing enzymes. Although this domain recognizes dsRNA's structures via a conserved canonical structure adopting an α 1 −β 1 β 2 β 3 −α 2 topology, several dsRBDs can accommodate discrete structural extensions expanding further their functional repertoire. How these structural elements engage cooperative communications with the canonical structure and how they contribute to the dsRBD's overall folding are poorly understood. Here, we addressed these issues using the dsRBD of human dihydrouridine synthase-2 (hDus2) (hDus2−dsRBD) as a model. This dsRBD harbors N-and C-terminal extensions, the former being directly involved in the recognition of tRNA substrate of hDus2. These extensions engage residues that form a long-range hydrophobic network (LHN) outside the RNA-binding interface. We show by coarse-grain Brownian dynamics that the Nt-extension and its residues F359 and Y364 rigidify the major folding nucleus of the canonical structure via an indirect effect. hDus2−dsRBD unfolds following a two-state cooperative model, whereas both F359A and Y364A mutants, designed to destabilize this LHN, unfold irreversibly. Structural and computational analyses show that these mutants are unstable due to an increase in the dynamics of the two extensions favoring solvent exposure of α2-helix and weakening the main folding nucleus rigidity. This LHN appears essential for maintaining a thermodynamic stability of the overall system and eventually a functional conformation for tRNA recognition. Altogether, our findings suggest that functional adaptability of extended dsRBDs is promoted by a cooperative hydrophobic coupling between the extensions acting as effectors and the folding nucleus of the canonical structure.

Dihydrouridine synthesis in tRNAs is under reductive evolution in Mollicutes

“…These structures have confirmed the common domain architecture of Dus enzymes consisting of an N-terminal domain adopting a TIM-BARREL fold wherein the binding site of the FMN redox coenzyme lies in its centre, and a C-terminal helical domain formed by 4-helix bundle, which is dedicated to tRNA recognition [16,18]. Apart from these two strictly conserved protein domains, there are additional domains that are exclusively observed among certain members of eukaryotic Dus [15,22,23].…”

Dihydrouridine in the Transcriptome: New Life for This Ancient RNA Chemical Modification

“…We have recently shown that the recognition mechanism of the tRNA substrate by Homo sapiens Dus2 (hDus2 or Dus2L) is much more complex than that observed in bacterial enzymes (Figure ). Indeed, hDus2 has a structural insertion within the TIM-barrel and an additional double-stranded RNA binding domain (dsRBD) that is appended to the polypeptide just after the HD, both playing a role in tRNA recognition (Figures and ). ,, The dsRBD is a double-stranded RNA (dsRNA) recognition module and is mainly found in proteins involved in mRNA transport, processing or editing. − Our structures of the hDus2 dsRBD in complex with a dsRNA (Figure C), as well as in-depth investigations by site-directed mutagenesis, nuclear magnetic resonance (NMR), and small-angle X-ray scattering (SAXS) of the interaction of the dsRBD with human tRNA Lys 3 revealed how this domain binds to tRNA. Indeed, this domain has a particular mode of tRNA recognition involving, in addition to the well-known canonical interactions between dsRBDs and dsRNAs (ribose and phosphate recognition), specific interactions with RNA, i.e., hydrogen bonds between some residues of dsRBD and RNA bases.…”

“…122 Indeed, hDus2 has a structural insertion within the TIM-barrel and an additional double-stranded RNA binding domain (dsRBD) that is appended to the polypeptide just after the HD, both playing a role in tRNA recognition (Figures 5 and 6). 103,122,123 The dsRBD is a double-stranded RNA (dsRNA) recognition module and is mainly found in proteins involved in mRNA transport, processing or editing. 124−127 Our structures of the hDus2 dsRBD in complex with a dsRNA (Figure 6C), as well as in-depth investigations by site-directed mutagenesis, nuclear magnetic resonance (NMR), and small-angle X-ray scattering (SAXS) of the interaction of the dsRBD with human tRNA Lys 3 revealed how this domain binds to tRNA.…”

The Dihydrouridine landscape from tRNA to mRNA: a perspective on synthesis, structural impact and function

“…Bou-Nader et al [ 130 ], Guimaraes and Golinelli-Pimpaneau [ 131 ], Molinaro et al [ 132 ], Whelan et al [ 133 ].…”