Characterization of the Staphylococcus aureus rRNA methyltransferase encoded by orfX, the gene containing the staphylococcal chromosome Cassette mec (SCCmec) insertion site.

The gene orfX is conserved among all staphylococci, and its complete sequence is maintained upon insertion of the staphylococcal chromosome cassette mec (SCCmec) genomic island, containing the gene encoding resistance to ß-lactam antibiotics (mecA), into its C terminus. The function of OrfX has not been determined. We show that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth, and that insertion of SCCmec did not alter gene expression. We solved the crystal structure of OrfX at 1.7 Å and found that it belongs to the S-adenosyl-L-methionine (AdoMet)-dependent α/ß-knot superfamily of SPOUT methyltransferases (MTases), with a high structural homology to YbeA, the gene product of the Escherichia coli 70 S ribosomal MTase RlmH. MTase activity was confirmed by demonstrating the OrfX-dependent methylation of the Staphylococcus aureus 70 S ribosome. When OrfX was crystallized in the presence of its AdoMet substrate, we found that each monomer of the homodimeric structure bound AdoMet in its active site. Solution studies using isothermal titration calorimetry confirmed that each monomer bound AdoMet but with different binding affinities (K(d) = 52 ± 0.4 and 606 ± 2 µm). In addition, the structure shows that the AdoMet-binding pocket, formed by a deep trefoil knot, contains a bound phosphate molecule, which is the likely nucleotide methylation site. This study represents the first characterization of a staphylococcal ribosomal MTase and provides the first crystal structure of a member of the α/ß-knot superfamily of SPOUT MTases in the RlmH or COG1576 family with bound AdoMet.

Staphylococcus aureus and Escherichia coli have disparate dependences on KsgA for growth and ribosome biogenesis.

BACKGROUND: The KsgA methyltransferase has been conserved throughout evolution, methylating two adenosines in the small subunit rRNA in all three domains of life as well as in eukaryotic organelles that contain ribosomes. Understanding of KsgA's important role in ribosome biogenesis has been recently expanded in Escherichia coli; these studies help explain why KsgA is so highly conserved and also suggest KsgA's potential as an antimicrobial drug target. RESULTS: We have analyzed KsgA's contribution to ribosome biogenesis and cell growth in Staphylococcus aureus. We found that deletion of ksgA in S. aureus led to a cold-sensitive growth phenotype, although KsgA was not as critical for ribosome biogenesis as it was shown to be in E. coli. Additionally, the ksgA knockout strain showed an increased sensitivity to aminoglycoside antibiotics. Overexpression of a catalytically inactive KsgA mutant was deleterious in the knockout strain but not the wild-type strain; this negative phenotype disappeared at low temperature. CONCLUSIONS: This work extends the study of KsgA, allowing comparison of this aspect of ribosome biogenesis between a Gram-negative and a Gram-positive organism. Our results in S. aureus are in contrast to results previously described in E. coli, where the catalytically inactive protein showed a negative phenotype in the presence or absence of endogenous KsgA.

Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis.

The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.

Control of substrate specificity by a single active site residue of the KsgA methyltransferase.

The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and N(6)-methyladenosine as substrates to catalyze formation of the final product N(6),N(6)-dimethyladenosine. KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. We demonstrate that part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of S-adenosyl-L-methionine-dependent methyltransferases. Mutation of the leucine to a proline mimics the sequence found in DNA methyltransferases. The L114P mutant of KsgA shows diminished overall activity, and its ability to methylate the N(6)-methyladenosine intermediate to produce N(6),N(6)-dimethyladenosine is impaired; this is in contrast to a second active site mutation, N113A, which diminishes activity to a level comparable to L114P without affecting the methylation of N(6)-methyladenosine. We discuss the implications of this work for understanding the mechanism of KsgA's multiple catalytic steps.

The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway.

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the ribosome, has been traced to the ArmA/RmtA family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosyl-L-methionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA of the 30S ribosomal subunit. ArmA methylates mature 30S subunits but not 16S rRNA, 50S, or 70S ribosomal subunits or isolated Helix 44 of the 30S subunit. To more fully characterize this family of enzymes, we have investigated the substrate requirements of ArmA and to a lesser extent its ortholog RmtA. We determined the Mg+² dependence of ArmA activity toward the 30S ribosomal subunits and found that the enzyme recognizes both low Mg+² (translationally inactive) and high Mg+² (translationally active) forms of this substrate. We tested the effects of LiCl pretreatment of the 30S subunits, initiation factor 3 (IF3), and gentamicin/kasugamycin resistance methyltransferase (KsgA) on ArmA activity and determined whether in vivo derived pre-30S ribosomal subunits are ArmA methylation substrates. ArmA failed to methylate the 30S subunits generated from LiCl washes above 0.75 M, despite the apparent retention of ribosomal proteins and a fully mature 16S rRNA. From our experiments, we conclude that ArmA is most active toward the 30S ribosomal subunits that are at or very near full maturity, but that it can also recognize more than one form of the 30S subunit.

Binding of adenosine-based ligands to the MjDim1 rRNA methyltransferase: implications for reaction mechanism and drug design.

The KsgA/Dim1 family of proteins is intimately involved in ribosome biogenesis in all organisms. These enzymes share the common function of dimethylating two adenosine residues near the 3'-OH end of the small subunit rRNA; orthologs in the three kingdoms, along with eukaryotic organelles, have evolved additional functions in rRNA processing, ribosome assembly, and, surprisingly, transcription in mitochondria. The methyltransferase reaction is intriguingly elaborate. The enzymes can bind to naked small subunit rRNA but cannot methylate their target bases until a subset of ribosomal proteins have bound and the nascent subunit has reached a certain level of maturity. Once this threshold is reached, the enzyme must stabilize two adenosines into the active site at separate times and two methyl groups must be transferred to each adenosine, with concomitant exchanges of the product S-adenosyl-l-homocysteine and the methyl donor substrate S-adenosyl-l-methionine. A detailed molecular understanding of this mechanism is currently lacking. Structural analysis of the interactions between the enzyme and substrate will aid in this understanding. Here we present the structure of KsgA from Methanocaldococcus jannaschii in complex with several ligands, including the first structure of S-adenosyl-l-methionine bound to a KsgA/Dim1 enzyme in a catalytically productive way. We also discuss the inability thus far to determine a structure of a target adenosine bound in its active site.

Structural and functional divergence within the Dim1/KsgA family of rRNA methyltransferases.

The enzymes of the KsgA/Dim1 family are universally distributed throughout all phylogeny; however, structural and functional differences are known to exist. The well-characterized function of these enzymes is to dimethylate two adjacent adenosines of the small ribosomal subunit in the normal course of ribosome maturation, and the structures of KsgA from Escherichia coli and Dim1 from Homo sapiens and Plasmodium falciparum have been determined. To this point, no examples of archaeal structures have been reported. Here, we report the structure of Dim1 from the thermophilic archaeon Methanocaldococcus jannaschii. While it shares obvious similarities with the bacterial and eukaryotic orthologs, notable structural differences exist among the three members, particularly in the C-terminal domain. Previous work showed that eukaryotic and archaeal Dim1 were able to robustly complement for KsgA in E. coli. Here, we repeated similar experiments to test for complementarity of archaeal Dim1 and bacterial KsgA in Saccharomyces cerevisiae. However, neither the bacterial nor the archaeal ortholog could complement for the eukaryotic Dim1. This might be related to the secondary, non-methyltransferase function that Dim1 is known to play in eukaryotic ribosomal maturation. To further delineate regions of the eukaryotic Dim1 critical to its function, we created and tested KsgA/Dim1 chimeras. Of the chimeras, only one constructed with the N-terminal domain from eukaryotic Dim1 and the C-terminal domain from archaeal Dim1 was able to complement, suggesting that eukaryotic-specific Dim1 function resides in the N-terminal domain also, where few structural differences are observed between members of the KsgA/Dim1 family. Future work is required to identify those determinants directly responsible for Dim1 function in ribosome biogenesis. Finally, we have conclusively established that none of the methyl groups are critically important to growth in yeast under standard conditions at a variety of temperatures.

Scintillation proximity assay for measurement of RNA methylation.

Methylation of RNA by methyltransferases is a phylogenetically ubiquitous post-transcriptional modification that occurs most extensively in transfer RNA (tRNA) and ribosomal RNA (rRNA). Biochemical characterization of RNA methyltransferase enzymes and their methylated product RNA or RNA-protein complexes is usually done by measuring the incorporation of radiolabeled methyl groups into the product over time. This has traditionally required the separation of radiolabeled product from radiolabeled methyl donor through a filter binding assay. We have adapted and optimized a scintillation proximity assay (SPA) to replace the more costly, wasteful and cumbersome filter binding assay and demonstrate its utility in studies of three distinct methyltransferases, RmtA, KsgA and ErmC'. In vitro, RmtA and KsgA methylate different bases in 16S rRNA in 30S ribosomal particles, while ErmC' most efficiently methylates protein-depleted or protein-free 23S rRNA. This assay does not utilize engineered affinity tags that are often required in SPA, and is capable of detecting either radiolabeled RNA or RNA-protein complex. We show that this method is suitable for quantitating extent of RNA methylation or active RNA methyltransferase, and for testing RNA-methyltransferase inhibitors. This assay can be carried out with techniques routinely used in a typical biochemistry laboratory or could be easily adapted for a high throughput screening format.

Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA.

While the general blueprint of ribosome biogenesis is evolutionarily conserved, most details have diverged considerably. A striking exception to this divergence is the universally conserved KsgA/Dim1p enzyme family, which modifies two adjacent adenosines in the terminal helix of small subunit ribosomal RNA (rRNA). While localization of KsgA on 30S subunits [small ribosomal subunits (SSUs)] and genetic interaction data have suggested that KsgA acts as a ribosome biogenesis factor, mechanistic details and a rationale for its extreme conservation are still lacking. To begin to address these questions we have characterized the function of Escherichia coli KsgA in vivo using both a ksgA deletion strain and a methyltransferase-deficient form of this protein. Our data reveal cold sensitivity and altered ribosomal profiles are associated with a DeltaksgA genotype in E. coli. Our work also indicates that loss of KsgA alters 16S rRNA processing. These findings allow KsgAs role in SSU biogenesis to be integrated into the network of other identified factors. Moreover, a methyltransferase-inactive form of KsgA, which we show to be deleterious to cell growth, profoundly impairs ribosome biogenesis-prompting discussion of KsgA as a possible antimicrobial drug target. These unexpected data suggest that methylation is a second layer of function for KsgA and that its critical role is as a supervisor of biogenesis of SSUs in vivo. These new findings and this proposed regulatory role offer a mechanistic explanation for the extreme conservation of the KsgA/Dim1p enzyme family.

Breaking the cycle of translation.

In a recent issue of Molecular Cell, Trobro and Aqvist (2007) reported mechanistic insight into release factor-induced peptide hydrolysis. Now, in this issue, the Green research group establishes unexpected complexity in decoding translation stop codons (Youngman et al., 2007).

The adenosine dimethyltransferase KsgA recognizes a specific conformational state of the 30S ribosomal subunit.

The methyltransferase KsgA modifies two adjacent adenosines in 16S rRNA by adding two methyl groups to the N(6) position of each nucleotide. Unlike nearly all other rRNA modifications, these modifications and the responsible enzyme are highly conserved phylogenetically, suggesting that the modification system has an important role in ribosome biogenesis. It has been known for some time that KsgA recognizes a complex pre-30S substrate in vitro, but there is disagreement in the literature as to what that substrate can be. That disagreement is resolved in this report; KsgA is unable to methylate 30S subunits in the translationally active conformation, but rather can modify 30S when in an experimentally well established translationally inactive conformation. Recent 30S crystal structures provide some basis for explaining why it is impossible for KsgA to methylate 30S in the translationally active conformation. Previous work identified one set of ribosomal proteins important for efficient methylation by KsgA and another set refractory methylation. With the exception of S21 the recent crystal structures of 30S also instructs that the proteins important for KsgA activity all exert their influence indirectly. Unfortunately, S21, which is inhibitory to KsgA activity, has not had its position determined by X-ray crystallography. A reevaluation of published biophysical data on the location also suggests that the refractory nature of S21 is also indirect. Therefore, it appears that KsgA solely senses the conformation 16S rRNA when carrying out its enzymatic activity.

Crystal structure of KsgA, a universally conserved rRNA adenine dimethyltransferase in Escherichia coli.

The bacterial enzyme KsgA catalyzes the transfer of a total of four methyl groups from S-adenosyl-l-methionine (S-AdoMet) to two adjacent adenosine bases in 16S rRNA. This enzyme and the resulting modified adenosine bases appear to be conserved in all species of eubacteria, eukaryotes, and archaebacteria, and in eukaryotic organelles. Bacterial resistance to the aminoglycoside antibiotic kasugamycin involves inactivation of KsgA and resulting loss of the dimethylations, with modest consequences to the overall fitness of the organism. In contrast, the yeast ortholog, Dim1, is essential. In yeast, and presumably in other eukaryotes, the enzyme performs a vital role in pre-rRNA processing in addition to its methylating activity. Another ortholog has been discovered recently, h-mtTFB in human mitochondria, which has a second function; this enzyme is a nuclear-encoded mitochondrial transcription factor. The KsgA enzymes are homologous to another family of RNA methyltransferases, the Erm enzymes, which methylate a single adenosine base in 23S rRNA and confer resistance to the MLS-B group of antibiotics. Despite their sequence similarity, the two enzyme families have strikingly different levels of regulation that remain to be elucidated. We have crystallized KsgA from Escherichia coli and solved its structure to a resolution of 2.1A. The structure bears a strong similarity to the crystal structure of ErmC' from Bacillus stearothermophilus and a lesser similarity to sc-mtTFB, the Saccharomyces cerevisiae version of h-mtTFB. Comparison of the three crystal structures and further study of the KsgA protein will provide insight into this interesting group of enzymes.

Crystallization and preliminary X-ray diffraction analysis of KsgA, a universally conserved RNA adenine dimethyltransferase in Escherichia coli.

The bacterial enzyme KsgA catalyzes the transfer of a total of four methyl groups from S-adenosylmethionine (SAM) to two adjacent adenosines in 16S rRNA. These modified adenosines are universally conserved in all species of eubacteria, eukaryotes and archaebacteria studied. Recombinant KsgA from Escherichia coli was overexpressed as a His-tagged fusion protein and purified. The recombinant protein was crystallized using PEG 4000 as a precipitant. The crystals belong to space group C2 and diffract X-rays to a resolution of 1.9 A. The unit-cell parameters are a = 173.9, b = 38.4, c = 83.0 A, beta = 90.0 degrees. Structure determination using the molecular-replacement method is at the early stages of refinement.