Crystal structure and ligand binding of the MID domain of a eukaryotic Argonaute protein

Abstract

Argonaute (AGO) proteins are core components of RNA-induced silencing complexes and have essential roles in RNA-mediated gene silencing. They are characterized by a bilobal architecture, consisting of one lobe containing the amino-terminal and PAZ domains and another containing the MID and PIWI domains. Except for the PAZ domain, structural information on eukaryotic AGO domains is not yet available. In this study, we report the crystal structure of the MID domain of the eukaryotic AGO protein QDE-2 from Neurospora crassa. This domain adopts a Rossmann-like fold and recognizes the 5'-terminal nucleotide of a guide RNA in a manner similar to its prokaryotic counterparts. The 5'-nucleotide-binding site shares common residues with a second, adjacent ligand-binding site, suggesting a mechanism for the cooperative binding of ligands to the MID domain of eukaryotic AGOs.

Figure 1

Structure of the Neurospora crassa QDE-2 MID domain. (A) Ribbon representation of the MID domain showing the positions of two bound sulphate ions (I and II) as sticks (red: oxygen; yellow: sulphur). Residues corresponding to those proposed by Kiriakidou et al (2007) to sandwich the m⁷GpppN cap are shown as purple sticks. Secondary structure elements are labelled. (B) Ribbon representation of an Af MID domain in complex with the 5′-end of a guide RNA (sticks), generated from Protein Data Bank entry 2bgg (Parker et al, 2005). (C) Superposition of the Nc QDE-2 MID domain onto the Af MID domain, illustrating the conservation of the Rossmann-like fold. (D) Structure-based sequence alignment of the Nc QDE-2 MID domain with archaeal (Af, Pf) and eubacterial (Aa, Tt) homologues, as well as with eukaryotic MID domains involved in the miRNA pathway. Positions involved in coordinating the two sulphate ions are shown in green. Positions proposed by Kiriakidou et al (2007) to bind the m⁷GpppN cap are shown in purple. The position proposed by Djuranovic et al (2010) to effect allosteric regulation of miRNA binding is shown in pink. Invariant or highly conserved positions are marked by an asterisk. Aa, Aquifex aeolicus; Af, Archaeoglobus fulgidus; MID, middle; miRNA, micro RNA; Nc, Neurospora crassa; Pf, Pyrococcus furiosus; QDE-2, quelling deficient 2; Tt, Thermus thermophilus.

Figure 2

Conservation of the guide RNA 5′-nucleotide-binding pocket. (A) Electrostatic potential mapped onto the molecular surface of the Nc QDE-2 MID domain, with the two bound sulphates shown as yellow sticks. Potentials are contoured from −10 (red) to +10 kT/e (blue). The 5′-RNA nucleotide from the superposition with the Af MID domain in complex with a guide RNA (Fig 1B,C) is shown as grey sticks. (B) Close-up of the RNA 5′-end nucleotide-binding site. The superimposed 5′-uridine from the Af complex structure is shown as sticks, together with the two sulphates and important side chains from the Nc QDE-2 structure. The uridine base stacks on the aromatic Tyr 595 ring, whereas the 5′-phosphate superimposes with sulphate I. Red: oxygen; blue: nitrogen; yellow: sulphur; violet: phosphorus. Af, Archaeoglobus fulgidus; MID, middle; Nc, Neurospora crassa; QDE-2, quelling deficient 2.

Figure 3

Coupled coordination of the two sulphate ion ligands. (A) Difference density for the sulphate ions. Difference density ((∣F_o∣−∣F_c∣)e(iα_c)) is contoured at 3.0 sigma values over the mean, where ∣F_o∣ is the observed structure factor amplitudes and ∣F_c∣ and α_c are structure factor amplitudes and phases calculated from a model prior to including the sulphates. (B) Potential hydrogen bonds of the two sulphate ions (I and II) to side-chain and main-chain atoms of the Nc QDE-2 MID domain. Relevant side chains and the main chain of strand β4 are shown as sticks. Hydrogen bonds are shown as dotted lines. Asp 603 (pink) corresponds to the aspartate proposed by Djuranovic et al (2010) to effect allosteric regulation of miRNA binding. Red: oxygen; blue: nitrogen; yellow: sulphur. MID, middle; miRNA, micro RNA; Nc, Neurospora crassa; QDE-2, quelling deficient 2.