- Short Report
- Open Access
Sequence and structural evolution of the KsgA/Dim1 methyltransferase family
© Rife et al; licensee BioMed Central Ltd. 2008
- Received: 31 July 2008
- Accepted: 29 October 2008
- Published: 29 October 2008
One of the 60 or so genes conserved in all domains of life is the ksgA/dim1 orthologous group. Enzymes from this family perform the same post-transcriptional nucleotide modification in ribosome biogenesis, irrespective of organism. Despite this common function, divergence has enabled some family members to adopt new and sometimes radically different functions. For example, in S. cerevisiae Dim1 performs two distinct functions in ribosome biogenesis, while human mtTFB is not only an rRNA methyltransferase in the mitochondria but also a mitochondrial transcription factor. Thus, these proteins offer an unprecedented opportunity to study evolutionary aspects of structure/function relationships, especially with respect to our recently published work on the binding mode of a KsgA family member to its 30S subunit substrate. Here we compare and contrast KsgA orthologs from bacteria, eukaryotes, and mitochondria as well as the paralogous ErmC enzyme.
By using structure and sequence comparisons in concert with a unified ribosome binding model, we have identified regions of the orthologs that are likely related to gains of function beyond the common methyltransferase function. There are core regions common to the entire enzyme class that are associated with ribosome binding, an event required in rRNA methylation activity, and regions that are conserved in subgroups that are presumably related to non-methyltransferase functions.
The ancient protein KsgA/Dim1 has adapted to cellular roles beyond that of merely an rRNA methyltransferase. These results provide a structural foundation for analysis of multiple aspects of ribosome biogenesis and mitochondrial transcription.
- Ribosome Biogenesis
- Methyltransferase Activity
- Small Subunit rRNA
- Small Ribosomal Subunit
- Active Site Pocket
Ribosome biogenesis is a fundamental process in all cells, requiring the consumption of large quantities of cellular resources under the control of an extraordinary level of regulation. In comparing prokaryotic and eukaryotic ribosome biogenesis pathways, the conservation of the KsgA/Dim1 family is unique. The presence and function of this enzyme has been maintained in every evolutionary lineage, including eukaryotic organelles.
KsgA catalyzes the conversion of two adjacent adenosines in the small subunit rRNA (A1518 and A1519 of 16S rRNA, E. coli numbering) to N6,N6-dimethyladenosines . The KsgA family carries out this core methyltransferase function in all domains of life, including organelles, but has also added new roles as cellular organization became more complex. In eukaryotes, the KsgA ortholog Dim1 is essential for proper processing of the pre-18S small subunit rRNA , and Dim1 knockout is lethal. Pfc1, which is the KsgA ortholog found in chloroplasts of Arabidopsis thaliana, is important for chloroplast formation under chilling conditions .
A distinct eukaryotic ortholog, mtTFB, is transported into the mitochondria where, in addition to methylating the small subunit rRNA, it has adopted the ribosomally unrelated function of serving as a mitochondrial transcription factor [4, 5]. In some mitochondria, there are two separate mtTFB proteins, mtTFB1 and mtTFB2, which are proposed to have arisen from a gene duplication event . There is evidence that mtTFB1 has retained stronger methyltransferase activity, while mtTFB2 is more active as a transcription factor [5, 6]. The fungi have only a single mtTFB, suggesting either loss of one of the paralogs in this lineage, or that the duplication occurred later in evolution. In at least one case, S. cerevisiae, the single mtTFB protein (sc-mtTFB) serves as a transcription factor but has lost its methyltransferase activity entirely . sc-mtTFB lacks significant sequence homology to any of the KsgA/Dim1 enzymes; yeast mtTFBs are generally poorly conserved and difficult to identify via sequence homology .
Another important offshoot of the KsgA lineage is the Erm family of methyltransferases, which confer antibiotic resistance by methylating A2058 of the 23S rRNA . The present-day Erm family almost certainly resulted from one or more gene duplications of a KsgA gene and subsequent evolution to permit recognition of a distinct target base .
KsgA's remarkable degree of conservation, coupled with the adaptation of new cellular functions, give us a unique opportunity to look at structural/function evolution of a single protein lineage. Previous studies have established the structural similarity between some rRNA adenosine dimethyltransferases [11, 12]. Based on recent work in our groups we can now make predictions about how these proteins interact with their respective ribosomal targets. We also examine structural variations that may be important to the varied functions of KsgA orthologs and paralogs.
Divergent orthologues of bacterial KsgA, including Dim1 from the eukaryote S. cerevisiae and the archaeon Methanocaldococcus jannaschii as well as h-mtTFB1 and h-mtTFB2, can all complement for KsgA function in E. coli [5, 14, 15]. Therefore, it is expected that these functional orthologues all interact with substrate ribosome particles in a similar manner. We recently reported a mode of binding for KsgA onto 30S subunits . As was noted in this work, the binding site for KsgA on 30S involves protein interactions with 16S rRNA in helix 44 and in the 790 loop, but not in helix 45, the site of the methylated adenosines.
The KsgA enzymes share the Rossman-like structural fold common to many SAM-dependent methyltransferases [12, 19]. This fold consists of a seven-stranded beta sheet surrounded by a variable number of alpha helices, with well-defined binding pockets for SAM and the target nucleotide. In this way, the methyltransferase function has a clear structural basis. However, the structural basis for the other functions of the KsgA/Dim1 enzymes is unknown. Extensive sequence alignments of KsgA, Dim1 and mtTFB enzymes identify elements unique to each group. The addition of specific inserts relative to KsgA likely has permitted the eukaryotic Dim1 and mtTFB enzymes to gain functions while maintaining their roles as methyltransferases.
A similar analysis can be performed by comparing KsgA and sc-mtTFB. mtTFB proteins have three inserts relative to KsgA proteins ; all of these inserts lie in the N-terminal domain (Figures 3c and 3d). We have superimposed sc-mtTFB onto the KsgA/30S model to evaluate what roles these inserts play, if any, in ribosome binding (Figure 3b). The first of these inserts is comprised of residues 116–130 of sc-mtTFB, and takes the form of a random coil leading into an extended beta strand; the second insert, residues 155–162, clusters together with insert 1 . These two inserts, which are located on the opposite side of the protein from the 30S subunit, are generally conserved in all mtTFB proteins [see Additional file 2]; however, insert 1 is shorter in mtTFB1 than in mtTFB and mtTFB2, which have evolved as transcription factors perhaps at the expense of their full function as methyltransferases. Given that these two inserts do not appear as though they make direct interactions with the small ribosomal subunit, we suggest that they might be important to mtTFB's role as a transcription factor, perhaps comprising a single binding surface involved in transcription. The third insert, consisting of residues 209–230, is on the other side of the protein from the first two, and consists of two alpha-helices which form a long loop on the protein surface. Residues in this loop are important for binding of sc-mtTFB to the S. cerevisiae mitochondrial RNA polymerase, and this insert may also contribute to the protein's role as a transcription factor . The presence of insert 3 is conserved in mtTFB and mtTFB2 proteins, but not in mtTFB1 proteins [see Additional file 2]. Notably, mtTFB insert 3 is in a position to interact with the 30S subunit, either in a positive or a negative manner.
To our knowledge, the KsgA proteins are unique in both their level of conservation and their functional flexibility. KsgA was one of the first rRNA methyltransferases to be identified and has received episodic scrutiny over the last thirty years. Despite a collection of important observations, a thorough understanding of this protein family's role in ribosome biogenesis has been elusive. Certainly, the KsgA/Dim1 family has significance beyond the methyltransferase activity that was first described. Sequence and structural comparisons of these proteins, in concert with data about the binding of KsgA to the 30S subunit, implicate certain regions that might be important to the various functions of KsgA orthologs. Further experiments will help define individual characteristics of this important family of proteins.
This work was supported by the U.S. National Institutes of Health grants GM066900 to JPR and GM62432 to GMC.
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