ABSTRACT
Box C/D RNA protein complexes (RNPs) direct site-specific 2'-O-methylation of RNA and ribosome assembly. The guide RNA in C/D RNP forms base pairs with complementary substrates and selects the modification site using a molecular ruler. Despite many studies of C/D RNP structure, the fundamental questions of how C/D RNAs assemble into RNPs and how they guide modification remain unresolved. Here we report the crystal structure of an entire catalytically active archaeal C/D RNP consisting of a bipartite C/D RNA associated with two substrates and two copies each of Nop5, L7Ae and fibrillarin at 3.15-Å resolution. The substrate pairs with the second through the eleventh nucleotide of the 12-nucleotide guide, and the resultant duplex is bracketed in a channel with flexible ends. The methyltransferase fibrillarin binds to an undistorted A-form structure of the guide-substrate duplex and specifically loads the target ribose into the active site. Because interaction with the RNA duplex alone does not determine the site specificity, fibrillarin is further positioned by non-specific and specific protein interactions. Compared with the structure of the inactive C/D RNP, extensive domain movements are induced by substrate loading. Our results reveal the organization of a monomeric C/D RNP and the mechanism underlying its site-specific methylation activity.
Subject(s)
Models, Molecular , RNA, Archaeal/chemistry , RNA, Archaeal/metabolism , Ribose/chemistry , Ribose/metabolism , Sulfolobus solfataricus/chemistry , Sulfolobus solfataricus/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Methylation , Protein Structure, TertiaryABSTRACT
Box C/D guide RNAs are abundant noncoding RNAs that primarily function to direct the 2'-O-methylation of specific nucleotides by base-pairing with substrate RNAs. In archaea, a bipartite C/D RNA assembles with L7Ae, Nop5, and the methyltransferase fibrillarin into a modification enzyme with unique substrate specificity. Here, we determined the crystal structure of an archaeal C/D RNA-protein complex (RNP) composed of all 3 core proteins and an engineered half-guide RNA at 4 A resolution, as well as 2 protein substructures at higher resolution. The RNP structure reveals that the C-terminal domains of Nop5 in the dimeric complex provide symmetric anchoring sites for 2 L7Ae-associated kink-turn motifs of the C/D RNA. A prominent protrusion in Nop5 seems to be important for guide RNA organization and function and for discriminating the structurally related U4 snRNA. Multiple conformations of the N-terminal domain of Nop5 and its associated fibrillarin in different structures indicate the inherent flexibility of the catalytic module, suggesting that a swinging motion of the catalytic module is part of the enzyme mechanism. We also built a model of a native C/D RNP with substrate and fibrillarin in an active conformation. Our results provide insight into the overall organization and mechanism of action of C/D RNA-guided RNA methyltransferases.
Subject(s)
Carrier Proteins/chemistry , tRNA Methyltransferases/chemistry , Amino Acid Motifs , Archaeal Proteins/chemistry , Base Sequence , Catalysis , Chromosomal Proteins, Non-Histone/chemistry , Crystallography, X-Ray/methods , Molecular Conformation , Molecular Sequence Data , Nucleic Acid Conformation , Protein Engineering/methods , Protein Structure, Secondary , Protein Structure, Tertiary , RNA/chemistry , RNA, Archaeal/chemistryABSTRACT
LePRK1 is a receptor-like kinase involved in successful fertilization in Lycopersicon esculentum (tomato). Importantly, the extracellular leucine-rich repeat (LRR) domain of LePRK1 mediates transmembrane signal transduction for pollen-tube growth and pollen germination. In this study, the N-terminal extracellular LRR domain of L. esculentum-derived LePRK1 was purified using an insect-cell secretion expression system and was crystallized by the vapour-diffusion method. The crystals diffracted X-rays to a resolution of 2.75 Å using synchrotron radiation. The crystals belonged to space group C2, with unit-cell parameters a = 136.53, b = 56.01, c = 62.93 Å, ß = 108.99° and two molecules per asymmetric unit.
Subject(s)
Crystallography, X-Ray/methods , Plant Proteins/chemistry , Solanum lycopersicum/enzymology , Amino Acid Sequence , Cloning, Molecular , Molecular Sequence Data , Plant Proteins/genetics , Plant Proteins/isolation & purification , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
The tomato (Lycopersicon esculentum) pollen-specific receptor kinase 2 (LePRK2) is a member of the large receptor-like kinase (RLK) family and is expressed specifically in mature pollen and pollen tubes in L. esculentum. Like other RLKs, LePRK2 contains a characteristic N-terminal leucine-rich repeat (LRR) extracellular domain, the primary function of which is in protein-protein interactions. The LePRK2 LRR is likely to bind candidate ligands from the external environment, leading to a signal transduction cascade required for successful pollination. LePRK2-LRR was purified using an insect-cell secretion expression system and was crystallized using the vapour-diffusion method. The crystals diffracted to a resolution of 2.50 Å and belonged to space group I4(1)22, with unit-cell parameters a = b = 93.94, c = 134.44 Å and one molecule per asymmetric unit.