Structural Basis for Binding of RNA and Cofactor by a KsgA Methyltransferase

Abstract

Summary Among methyltransferases, KsgA and the reaction it catalyzes are conserved throughout evolution. However, the specifics of substrate recognition by the enzyme remain unknown. Here, we report structures of Aquifex aeolicus KsgA, in its ligand-free form, in complex with RNA and in complex with both RNA and S-adenosylhomocysteine (SAH, reaction product of cofactor S-adenosylmethionine), providing the first pieces of structural information on KsgA-RNA and KsgA-SAH interactions. Moreover, the structures show how conformational changes that occur upon RNA binding create the cofactor-binding site. There are nine conserved functional motifs (motifs I-VIII and X) in KsgA. Prior to RNA binding, motifs I and VIII are flexible, each exhibiting two distinct conformations. Upon RNA binding, the two motifs become stabilized in one of these conformations, which is compatible with the binding of SAH. Motif X, which is also stabilized upon RNA binding, is directly involved in the binding of SAH.

The Era GTPase recognizes the GAUCACCUCC sequence and binds helix 45 near the 3′ end of 16S rRNA

“…1A). Previously, we found that the stem loop opens up and dimerizes in the KsgA-h45 structure (22). In this study, we have obtained the structure of RNA301 in two conformations.…”

“…KsgA is a universally conserved RNA methyltransferase. In E. coli, it methylates two adjacent adenines (Ade) in the tetraloop of h45 (21,22) (Fig. 1A).…”

Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis

“…Based on the co-crystal structure of an RNA duplex-KsgA complex, a model of KsgA interacting with helix 45 was suggested that is inconsistent with the EM structure (24). The interactions involve the same positively charged surface of the protein, but the RNA binding mode differs significantly (supplemental Fig.…”

“…The N-terminal catalytic domain shares sequence and structural similarity to the adenine DNA methyltransferase (20). Furthermore, two structures of the Aquifex aeolicus KsgA in complex with an RNA fragments of helix 45 were solved (24,25). These structures show catalytically inactive complexes and are inconsistent with structural model based on footprinting data (19).…”

Nuclear Ribosome Biogenesis Mediated by the DIM1A rRNA Dimethylase Is Required for Organized Root Growth and Epidermal Patterning in Arabidopsis

“…The dim1A missense mutation alters a conserved Gly residue within the canonical SAM binding sequence, shown in bacterial KsgA to form part of the cofactor binding pocket (Tu et al, 2009) (Figure 2). Considering this, we hypothesized that the dim1A gene may be expressed normally and its product may process pre-rRNA normally, but it may have altered methylation ability due to the change in its predicted SAM binding sequence.…”