RESUMO
In Arabidopsis thaliana, endogenous trans-acting and exogenous siRNA pathways are initiated by the interaction of DRB4 with trigger dsRNA. Further, DCL4:DRB4 complex cleaves the dsRNA into 21 bp siRNA. Understanding molecular determinants and mechanistic details of dsRNA recognition by DRB4 is vital for inducing long-term RNAi-mediated gene regulation in plants. Here, we present solution structures of individual and concatenated DRB4 dsRBDs and demonstrate modes of dsRNA binding by employing NMR, ITC and site-specific mutagenesis. While both dsRBDs adopt the canonical α-ß-ß-ß-α fold, key structural differences and ms-µs dynamics located at the RNA binding region were observed for dsRBD1. These features favor dsRBD1 to orient itself and make stronger tripartite contact with dsRNA, a feature missing in dsRBD2. Additionally, the inter-domain orientation induced by the linker restricts the mobility of dsRBD2, resulting in the steric hindrance of α1 helix in dsRBD2, and leads in further reduction of its dsRNA binding activity. Our study deciphers functional roles of DRB4 domains by showing that dsRBD1 drives the tasiRNA/siRNA pathway. Furthermore, we identify a potential role of the C-terminal region of DRB4 in protein:protein interaction as it possesses six PxxP motifs, binds to Zn2+ and contains a small structural domain.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA de Plantas/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação/genética , Calorimetria/métodos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA de Plantas/química , RNA de Plantas/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Zinco/química , Zinco/metabolismoRESUMO
In Drosophila melanogaster, Dcr-2:R2D2 heterodimer binds to the 21 nucleotide siRNA duplex to form the R2D2/Dcr-2 Initiator (RDI) complex, which is critical for the initiation of siRNA-induced silencing complex (RISC) assembly. During RDI complex formation, R2D2, a protein that contains three dsRNA binding domains (dsRBD), senses two aspects of the siRNA: thermodynamically more stable end (asymmetry sensing) and the 5'-phosphate (5'-P) recognition. Despite several detailed studies to date, the molecular determinants arising from R2D2 for performing these two tasks remain elusive. In this study, we have performed structural, biophysical, and biochemical characterization of R2D2 dsRBDs. We found that the solution NMR-derived structure of R2D2 dsRBD1 yielded a canonical α1-ß1-ß2-ß3-α2 fold, wherein two arginine salt bridges provide additional stability to the R2D2 dsRBD1. Furthermore, we show that R2D2 dsRBD1 interacts with thermodynamically asymmetric siRNA duplex independent of its 5'-phosphorylation state, whereas R2D2 dsRBD2 prefers to interact with 5'-P siRNA duplex. The mutation of key arginine residues, R53 and R101, in concatenated dsRBDs of R2D2 results in a significant loss of siRNA duplex recognition. Our study deciphers the active roles of R2D2 dsRBDs by showing that dsRBD1 initiates siRNA recognition, whereas dsRBD2 senses 5'-phosphate as an authentic mark on functional siRNA.
Assuntos
Arginina , Proteínas de Drosophila , Drosophila melanogaster , Interferência de RNA , RNA Interferente Pequeno , Animais , Drosophila melanogaster/metabolismo , Arginina/química , Arginina/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , RNA Helicases/metabolismo , RNA Helicases/química , RNA Helicases/genética , Domínios Proteicos , Proteínas de Ligação a RNARESUMO
In the model organism Drosophila melanogaster, one of the Dicer homologs, Dcr-2, initiates the RNA interference pathway by cleaving long double-stranded RNA into small interfering RNA (siRNA). The Dcr-2:R2D2 heterodimer subsequently binds to the 21-nucleotide siRNA to form the R2D2:Dcr-2 Initiator (RDI) complex, which is critical for initiating the assembly of the RNA-induced silencing complex containing guide siRNA strand. During RDI complex formation, R2D2 senses the stability of the 5' end of the siRNA and a 5'-phosphate group, although the underlying mechanism of siRNA asymmetry sensing and 5'-phosphate recognition by R2D2 is elusive. In this study, we present nearly complete chemical shift assignments of the backbone and the side chain of a construct that comprises the N-terminus dsRBD1 and linker of R2D2 (~ 10.3 kDa; henceforth: R2D2D1L). Our study would further aid in the structural and functional characterization of R2D2.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Interferência de RNA , Drosophila/genética , Drosophila/metabolismo , RNA de Cadeia Dupla/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Ligação a RNA/química , Ressonância Magnética Nuclear Biomolecular , Fosfatos/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are major signaling proteins in eukaryotic cells and are important drug targets. In spite of their role in GPCR function, the extramembranous regions of GPCRs are relatively less appreciated. The third intracellular loop (ICL3), which connects transmembrane helices V and VI, is important in this context since its crucial role in signaling has been documented for a number of GPCRs. Unfortunately, the structure of this loop is generally not visualized in x-ray crystallographic studies since this flexible loop is either stabilized using a monoclonal antibody or replaced with lysozyme. In this work, we expressed and purified the ICL3 region of the serotonin1A receptor and monitored its motional restriction and organization utilizing red edge excitation shift (REES) of its sole tryptophan and circular dichroism (CD) spectroscopy. Our results show that the tryptophan in ICL3 exhibits REES of 4â¯nm, implying that it is localized in a restricted microenvironment. These results are further supported by wavelength-selective changes in fluorescence anisotropy and lifetime. This constrained dynamics was relaxed upon denaturation of the peptide, thereby suggesting the involvement of the peptide secondary structure in the observed motional restriction, as evident from CD spectroscopy and apparent rotational correlation time. To the best of our knowledge, these results constitute one of the first measurements of motional constraint in the ICL3 region of GPCRs. Our results are relevant in the context of the reported intrinsically disordered nature of ICL3 and its role in providing functional diversity to GPCRs due to conformational plasticity.