Methylation of biomolecules is a frequent biochemical reaction within the cell, and a plethora of highly specific methyltransferases (MTases) catalyse the transfer of a methyl group from S-adenosylmethionine (AdoMet) to various substrates. The posttranslational methylation of lysine residues, catalysed by numerous lysine (K)-specific protein MTases (KMTs), is a very common and important protein modification, which recently has been subject to intense studies, particularly in the case of histone proteins. The majority of KMTs belong to a class of MTases that share a defining ‘SET domain’, and these enzymes mostly target lysines in the flexible tails of histones. However, the so-called seven-β-strand (7BS) MTases, characterized by a twisted beta-sheet structure and certain conserved sequence motifs, represent the largest MTase class, and these enzymes methylate a wide range of substrates, including small metabolites, lipids, nucleic acids and proteins. Until recently, the histone-specific Dot1/DOT1L was the only identified eukaryotic 7BS KMT. However, a number of novel 7BS KMTs have now been discovered, and, in particular, several recently characterized human and yeast members of MTase family 16 (MTF16) have been found to methylate lysines in non-histone proteins. Here, we review the status and recent progress on the 7BS KMTs, and discuss these enzymes at the levels of sequence/structure, catalytic mechanism, substrate recognition and biological significance.

INTRODUCTION

Protein lysine methylation

Methylation is a frequent and important protein modification, which occurs primarily at lysine and arginine residues. It was discovered more than half a century ago that some proteins contain methylated lysine residues [1], and these methylations are introduced by lysine (K)-specific MTases (methyltransferases) (KMTs), which catalyse the transfer of a methyl group from the methyl donor S-adenosylmethionine (AdoMet) to the target lysine (Figures 1A and 1B). The ε-nitrogen of lysine can potentially accept up to three methyl groups, resulting in three possible methylation states: mono-, di- and trimethyllysine (Figure 1B). For several decades, the interest in lysine methylation remained rather limited [2]. However, since the turn of the millennium, research activity on protein lysine methylation has increased dramatically. This was initially spurred by the discovery of numerous MTases that target specific lysine residues in the flexible tails of the histone proteins, as well as the appreciation that histone methylations are important regulators of transcriptional activity and chromatin state [35]. Furthermore, it was discovered that so-called reader domains, which are often found as part of chromatin-associated proteins, can specifically recognize certain methylation states on the histones, thereby providing a means of transforming the rather subtle lysine methylations into biological output [6]. Moreover, a number of different lysine-specific demethylases that remove lysine methylations from histones have been discovered, thereby allowing dynamic regulation of histone methylations [7].

Protein methylation by lysine-specific methyltransferases (KMTs)

Figure 1
Protein methylation by lysine-specific methyltransferases (KMTs)

(A) Chemical structure of the methyl donor AdoMet and its demethylated counterpart AdoHcy. The transferred methyl group is shown in red. (B) KMT-mediated protein methylation. A lysine residue can receive up to three methyl groups, leading to three possible methylation states.

Figure 1
Protein methylation by lysine-specific methyltransferases (KMTs)

(A) Chemical structure of the methyl donor AdoMet and its demethylated counterpart AdoHcy. The transferred methyl group is shown in red. (B) KMT-mediated protein methylation. A lysine residue can receive up to three methyl groups, leading to three possible methylation states.

Despite the important discoveries and intense research interest in histone lysine methylation, the vast majority of lysine methylations are actually found on non-histone proteins. Recent development within protein mass spectrometry has allowed for high-throughput identification of lysine-methylated proteins [810], and currently, close to 2000 methyl modifications on lysine residues have been reported in the human proteome, distributed between ∼1200 different proteins (Phosphosite, 2015) [11]. Importantly, the MTases responsible for the majority of these methylations still await identification.

Seven-β-strand methyltransferases

Before any three-dimensional structure of an MTase was available, it was noted that many MTases shared several conserved sequence motifs, suggesting that they also adopt a common structure [12,13]. Indeed, the two first MTase structures to be determined, those of the HhaI DNA MTase and catechol-O-MTase, showed a nearly identical core fold, which was similar to the so-called Rossmann-fold found in certain NAD-binding proteins [14,15]. The core MTase fold consists of a twisted seven-stranded β-sheet of characteristic topology, where the first six β-strands are parallel, and the seventh has the opposite orientation (Figures 2A and 2B). Since the elucidation of the first MTase structures, many additional MTases have been shown to adopt a seven-β-strand (7BS) structure or a slight variation of this fold [16,17].

The 7BS MTase fold

Figure 2
The 7BS MTase fold

(A) Structure of a typical 7BS MTase. A cartoon representation of the core 7BS fold of the human KMT METTL21A (PDB: 4LEC; Chain A) is shown. α-helices and loops are depicted in cyan, β-strands in orange. The β-strands are numbered in the N-to-C direction. (B) Schematic representation of the canonical topology of the 7BS MTases. The conserved hallmark motifs ‘I’ ‘Post I’, ‘II’ and ‘III’ are indicated. (C) Location of the hallmark motifs relative to the cofactor AdoMet in the 7BS fold. Contacts with AdoMet are primarily made by residues in motifs I, and Post I. The structural figures were made using the PyMOL Molecular Graphics System, Version 1.3 (Schrödinger, LLC).

Figure 2
The 7BS MTase fold

(A) Structure of a typical 7BS MTase. A cartoon representation of the core 7BS fold of the human KMT METTL21A (PDB: 4LEC; Chain A) is shown. α-helices and loops are depicted in cyan, β-strands in orange. The β-strands are numbered in the N-to-C direction. (B) Schematic representation of the canonical topology of the 7BS MTases. The conserved hallmark motifs ‘I’ ‘Post I’, ‘II’ and ‘III’ are indicated. (C) Location of the hallmark motifs relative to the cofactor AdoMet in the 7BS fold. Contacts with AdoMet are primarily made by residues in motifs I, and Post I. The structural figures were made using the PyMOL Molecular Graphics System, Version 1.3 (Schrödinger, LLC).

A more systematic analysis of 7BS MTase sequences, and comparison with corresponding structures, demonstrated that these enzymes display sequence homology at four distinct motifs, denoted ‘I’, ‘Post I’, ‘II’ and ‘III’, localized at specific positions within the 7BS fold (Figures 2B and 2C), and conserved residues within motifs I and Post I play important roles in coordinating the methyl donor AdoMet (Figure 2C) [12,18]. These motifs are rather degenerate, and many 7BS MTases lack one or several of them. However, a combination of the motifs, as well as the spacing between them, has proven a powerful tool for identification of novel 7BS MTases through database searches, and the search algorithms have been further improved by incorporating structural information and secondary structure prediction [1921].

Studies on bioinformatics prediction of MTases have mainly focused on budding yeast (Saccharomyces cerevisiae) and humans. The most recent of these studies predicted a total of 208 MTases to be present in humans, of which 131 belong to the 7BS class, whereas 56 of the 81 identified yeast MTases were predicted as 7BS enzymes [21]. Interestingly, the function of many of the 7BS MTases remains unknown, particularly in humans. In 2011, a function had been assigned to only 36 of the 131 human 7BS MTases, and, although this number has increased, the majority of these enzymes still await functional characterization [21].

Lysine-specific methyltransferases

The Rubisco protein in plants is subject to lysine methylation, and the responsible MTase was one of the first KMTs to be identified [22,23]. Later, a related protein was discovered in Drosophila, Su(Var)3-9 [5,24]. This protein had caught interest because the corresponding gene had been retrieved in a screen for mutants that suppressed an epigenetic phenomenon called position effect variegation [24]. It was found that Su(Var)3-9 and two related Drosophila enzymes, Enhancer of Zeste [E(z)] and Trithorax, shared a conserved domain that showed sequence homology to Rubisco KMT, dubbed a SET domain (reflecting the first letter of the names of the three Drosophila enzymes) [5,24]. The SET domain enzymes, which represent the second largest MTase class both in humans and yeast, exclusively encompass KMTs, and the majority of hitherto identified KMTs belong to this class [25]. Most of the SET domain proteins target the N-terminal tails of histone proteins in a highly specific manner; for example, Drosophila Su(Var)3-9 catalyses methylation of Lys-9 in histone protein H3. However, some subgroups of SET-proteins have also been shown to methylate non-histone proteins. These include SMYD proteins, as well as G9a and SETD7, which are rather broad-specificity enzymes that target a wide range of substrates [2629]. About 5 years ago, only a single eukaryotic KMT belonging to the 7BS MTase class had been identified, namely the histone-modifying disruptor of telomere silencing 1 (Dot1) enzyme from budding yeast, as well as its mammalian counterpart DOT1L. However, a number of novel 7BS MTases have been discovered in recent years, particularly in humans and budding yeast.

Since several excellent reviews have been devoted specifically to SET domain proteins and to protein lysine methylation in general [25,3032], we found it purposeful to limit an in-depth article specifically to the 7BS KMTs. Although the SET domain KMTs and the 7BS KMTs catalyse the same chemical reaction, there are, apart from the obvious differences regarding sequence and structure, several interesting differences between these two types of enzymes, with respect to both substrate recognition and enzymatic mechanism, further justifying a review dedicated primarily to the 7BS KMTs.

7BS KMTs IN THE THREE KINGDOMS OF LIFE

Bacterial and archaeal 7BS KMTs

Although protein lysine methylation is far less abundant in bacteria than in eukaryotes, lysine methylation was first observed in bacteria, and the first KMT enzyme to be identified was the bacterial PrmA enzyme [33,34]. Also, it has become clear that lysine methylation is abundant in archaea, and the first archaeal KMTs were recently identified [35,36]. An overview of the 7BS KMTs currently identified in bacteria and archaea is given in Table 1. 

Table 1
Bacterial and archaeal 7BS KMTs

n.d.=not determined.

Enzyme Studied in Substrate Lys Distribution Remarks Refs. 
PrmA E. coli, T. thermophilus L11 3,39 All bacteria, plants Also targets N-terminus of L11 [33,39,40
EftM P. aeruginosa EF-Tu γ-proteobacteria, firmicutes  [45
PKMT1/2 R. prowazekii, R. typhi OmpB Several α-, β-, γ-, ε-proteobacteria (patchy)  [48,49
AtMETTL20 A. tumefaciens ETFβ 193,196 α-proteobacteria (mainly) Orthologue of human METTL20 [53
  RpL7/L12 86    
aKMT S. islandicus Several n.d. Crenarchaeota Sequence homology to human FAM173A/B [35,36
Enzyme Studied in Substrate Lys Distribution Remarks Refs. 
PrmA E. coli, T. thermophilus L11 3,39 All bacteria, plants Also targets N-terminus of L11 [33,39,40
EftM P. aeruginosa EF-Tu γ-proteobacteria, firmicutes  [45
PKMT1/2 R. prowazekii, R. typhi OmpB Several α-, β-, γ-, ε-proteobacteria (patchy)  [48,49
AtMETTL20 A. tumefaciens ETFβ 193,196 α-proteobacteria (mainly) Orthologue of human METTL20 [53
  RpL7/L12 86    
aKMT S. islandicus Several n.d. Crenarchaeota Sequence homology to human FAM173A/B [35,36

Multiple methylation of ribosomal protein L11 by PrmA

It was discovered as early as in 1974 that the ribosomal protein L11 from Escherichia coli contains methylated lysine residues [37]. Soon thereafter, in 1977, the prmA mutant, which lacked L11 methylation, was reported, suggesting that the encoded PrmA protein is the KMT responsible for L11 methylation [33]. This was supported by the cloning of the prmA gene, which revealed that a part of the PrmA protein sequence matched a DXGXGXGXL motif found in several AdoMet-dependent MTases (corresponding to what was later designated ‘Motif I’) [34], as well as by the observation that the recombinant PrmA protein bound AdoMet [38]. Further studies of E. coli L11 showed that this protein actually contains 9 post-translationally added methyl groups, distributed between two trimethylated lysine residues (Lys-3 and Lys-39), and the trimethylated, N-terminal alanine residue (Ala-1) [39].

Following these initial studies in E. coli, PrmA from the thermophilic bacterium Thermus thermophilus was subject to both biochemical and structural studies. Mass spectrometric data indicated that L11 from T. thermophilus contains the same methylations as the E. coli protein (trimethylation of Ala-1, Lys-3 and Lys-39), but also three additional methyl groups, possibly accounted for by trimethylation of Lys-16 (E. coli L11 has Met at this position) [40]. Interestingly, PrmA was found to be responsible for all the observed methylations on L11, indicating a broad specificity and high flexibility at the active site [40]. Indeed, crystal structures of PrmA in complex with substrate have demonstrated several different binding modes, i.e. one with Ala-1 in the active site, another with Lys-39 in the active site, and a third where no putative substrate is found in the active site, apparently a sampling intermediate representing the enzyme scanning the L11 surface until a suitable substrate side chain is placed into the active site [41,42].

PrmA orthologues appear to be present in all eubacteria, suggesting an important and fundamental function. However, null mutants of prmA in E. coli and T. thermophilus were viable, demonstrating that L11 methylation is not required for growth of the bacteria under normal conditions [38,40]. Additionally, putative PrmA orthologues are found in plants, but appear to be absent from other eukaryotes, as well as in archaea. Recently, a PrmA orthologue was characterized in Arabidopsis thaliana, and found to target L11 in chloroplasts and mitochondria [43].

EftM-mediated methylation of Lys-5 in Pseudomonas EF-Tu

An initial study indicated that surface-localized translation elongation factor EF-Tu from Pseudomonas aeruginosa was post-translationally modified, based on recognition by anti-phosphorylcholine (ChoP) antibodies [44]. The modification was somewhat surprisingly shown to be a trimethylation of Lys-5 in EF-Tu, and the responsible MTase was identified and denoted EftM (EF-Tu modifying enzyme) [45]. For many pathogenic bacteria, ChoP epitopes on the surface act as a mimic of platelet-activating factor (PAF), and the interaction of ChoP with the PAF receptor (PAFR) is a crucial step in the infectivity of these pathogens. The trimethyllysine moiety on EF-Tu is structurally similar to ChoP, and was also shown to mediate interaction of the Pseudomonas bacteria with PAFR on host cells. Accordingly, EftM-deficient P. aeruginosa showed a strongly reduced respiratory infectivity, further supporting the notion that EF-Tu methylation plays an important role in the infection process. In a recent study, the biochemical activity of recombinant EftM on Lys-5 in EF-Tu was demonstrated for the first time, and it was also shown by structural modelling that EftM is a 7BS MTase [46]. In agreement with a previous study showing that EF-Tu methylation is enhanced at 25°C relative to 37°C, recombinant EftM was shown to be a thermolabile enzyme that rapidly lost its activity when incubated at 37°C [46]. EftM appears very narrowly distributed in the bacterial kingdom, being restricted to certain species of γ-proteobacteria and firmicutes, with no putative orthologues in eukaryotes.

PKMT1/PKMT2-mediated multimethylation of OmpB in Rickettsia

The outer membrane protein B (OmpB) is an important and abundant surface antigen on typhus-causing Rickettsia subspecies such as Rickettsia prowazekii and Rickettsia typhi, and OmpB is subject to extensive methylation at several lysine residues [47]. Two related MTases, denoted PKMT1 (protein RP789 in R. prowazekii; RT0776 in R. typhi) and PMKT2 (RP027-028 in R. prowazekii; RT0101 in R. typhi) (PKMT=protein lysine methyltransferase), have been shown to catalyse lysine methylation of OmpB [48,49]. PKMT1 was shown to catalyse monomethylation at several lysine residues in recombinant OmpB in vitro, whereas PKMT2 was found to introduce trimethylation at a subset of these residues [48,49]. The pattern of methylation on OmpB introduced by PKMT1 and PKMT2 in vitro was very similar to that of OmpB isolated from bacteria, indicating that PKMT1 and PKMT2 are the enzymes responsible for OmpB methylation in vivo [48,49]. Whereas OmpB from virulent Rickettsia strains is trimethylated on multiple lysines, the non-virulent R. prowazekii Madrid E strain carries an inactivating mutation in the PKMT2 and is devoid of OmpB trimethylation, suggesting a possible link between OmpB methylation and Rickettsia virulence [48].

The modelled structures of PKMT1 and PKMT2 showed a β-sheet consisting of five strands, thus deviating from the canonical 7BS structure [49]. Such a compacted version of the 7BS fold has previously been observed for arginine-specific protein MTases [17,50]. Putative orthologues of PKMT1 and PKMT2 appear to be limited to proteobacteria, where they show a rather patchy distribution, but representatives can be found in the α-, β-, γ- and ε-subdivisions of the proteobacteria.

AtMETTL20-mediated methylation of Agrobacterium RpL7/L12 and ETFβ

The human 7BS MTase METTL20 was recently characterized independently by two groups, and found to methylate two adjacent lysines in the β-subunit of the electron transfer flavoprotein (ETFβ; for details, see later section) [51,52]. Strikingly, METTL20 homologues are also found in a few bacteria, especially in the Rhizobiales order of α-proteobacteria, where they seem to be ubiquitous [51]. Indeed, a very recent study demonstrated that the METTL20 homologue from Agrobacterium tumefaciens, (AtMETTL20), methylated ETFβ on Lys-193 and Lys-196, corresponding to the sites targeted by human METTL20 in human ETFβ [53]. AtMETTL20-mediated methylation of ETFβ was found to diminish electron transfer from various ETF-dependent dehydrogenases, similarly to what had previously been observed with the corresponding human proteins [53]. Interestingly, AtMETTL20 was also shown to be responsible for methylation of the ribosomal protein RpL7/L12 at Lys-86, a methyl modification observed in a wide range of bacteria, including several that lack a sequence homologue of METTL20 [53]. This indicates that different bacteria independently have evolved two types of KMTs targeting the same residue in RpL7/L12, thus underscoring the likely functional significance of this methyl modification.

Pervasive aKMT-mediated lysine methylation in Crenarchaeota

Among archaea, lysine methylation appears to be particularly extensive in thermophilic members of Crenarchaeota [54]. By interesting analogy to the methylation of eukaryotic histone proteins, the crenarchaeal, chromatin-associated proteins Cren7 and Sul7d have been shown to be methylated [55,56]. Analysis of lysine methylation sites in crenarchaeal proteins did not reveal any preferred sequence or structural context, suggesting the existence of a broad-specificity KMT [54].

To identify the crenarchaeal KMT responsible for lysine methylation of Cren7, Chu et al. [35] used a biochemical approach. An MTase activity capable of methylating recombinant E. coli-expressed Cren7 was detected in protein extracts from Sulfolobus islandicus, and further purification of this activity led to the identification of the corresponding enzyme, which was denoted aKMT. Recombinant aKMT displayed broad substrate specificity and was active on various recombinant Sulfolobus substrates in vitro, suggesting that this enzyme could be responsible for many, if not all, of the observed lysine methylations on Sulfolobus proteins. In an independent study, Niu et al. [36] identified the same crenarchaeal aKMT enzyme, based on its slight similarity to the yeast KMT Dot1, and also that study demonstrated in vitro activity of aKMT on several different recombinant substrates. Interestingly, the activity of aKMT on the chromatin protein Sul7d was strongly stimulated by the presence of DNA, suggesting that this methylation occurs in a chromatin context in vivo [36]. Although aKMT clearly displays robust KMT activity in vitro, the role of this enzyme in mediating the many lysine methylations observed in vivo remains to be established. Future studies will probably address this through analysing protein lysine methylation in aKMT deficient archaebacterial mutants.

Modelling of the aKMT structure, using Dot1 as a template, suggests that aKMT contains a truncated five-stranded version of the 7BS fold, similarly to the Pseudomonas PKMT1/2 enzymes described above [36]. Interestingly, homologues of aKMT are present in eukaryotes, primarily in animals [35]. In humans and other vertebrates, two sequence homologues of aKMT are found, namely the yet uncharacterized FAM173A and FAM173B proteins.

Eukaryotic KMTs from MTase family 16 (MTF16)

Protein sequence analysis has categorized 7BS MTases into different families [20,21]. Recently, several members of the so-called methyltransferase family 16 (MTF16; Pfam: PF10294) have been established as KMTs [57,58]. A hallmark of the MTF16 enzymes is a conserved DXXY motif (consensus [D/E]XX[Y/F]), localized immediately downstream of Motif II, which is very similar to the DPPY motif (consensus [D/N/S]PP[Y/F/W]) found at the corresponding position in many MTases that modify exocyclic N-atoms on bases in DNA and RNA, as well as in glutamine MTases [5961]. So far, substrates have been identified for 11 out of the 15 MTF16 enzymes found in humans and budding yeast, and all but two of these enzymes have been shown to exclusively target lysine residues in proteins. The exceptions are Hpm1 (histidine protein methyltransferase 1) and Efm7 (elongation factor methyltransferase 7) from budding yeast. Hpm1, encoded by the YIL110W gene, has been shown to be required for the methylation of a histidine residue in ribosomal protein Rpl3 [62,63]. Very recently, Efm7 (Ylr285w; NntI) was found to specifically trimethylate the amino group of the N-terminal residue (Gly-2) in eukaryotic translation elongation factor 1 alpha (eEF1A), as well as to dimethylate the neighbouring lysine residue (Lys-3) [64].

The gene names of the human 7BS KMTs originally corresponded to the historical names of the respective ORFs, such as C14orf138, C17orf95, FAM86A, FAM119B etc. Later, most of these gene names were altered to reflect a likely MTase function, e.g. METTL20, METTL21A (HGNC gene family ‘Methyltransferase like’). However, as several of the corresponding enzymes now have been established as highly specific KMTs, a consensus nomenclature for the enzymes has been adopted that reflects their substrate specificity, e.g. CaM-KMT, VCP-KMT, HSPA-KMT, eEF2-KMT, and several of these names have now been accepted as the formal gene names (CAMKMT, VCPKMT, EEF2KMT; gene names are capitalized, without hyphens and in italics). In the following, the human 7BS KMTs will be referred to by their (non-italicized) formal gene names. An overview of human 7BS KMTs, also including their alternative names, is given in Table 2. The majority of the 7BS KMTs identified in S. cerevisiae target translation elongation factors, and have been named correspondingly as ‘elongation factor methyltransferases’ (Efm2, Efm3 etc.). Table 3 gives an overview of the 7BS KMTs identified in S. cerevisiae.

Table 2
Human 7BS KMTs

*For CaM and H3, amino acid numbering refers to protein without initiator methionine, in accordance with the prevailing practice in the scientific literature. **Refers to the distribution of putative and established orthologues among the common eukaryotic model organisms Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), A. thaliana (At) and S. cerevisiae (Sc).

Gene name UniProt ID Aliases Substrate Lys Distribution** Remarks Refs. 
CAMKMT Q7Z624 CaM-KMT CaM 115* Ce, Dm, At  [69,75
  C2orf34      
  CLNMT      
VCPKMT Q9H867 VCP-KMT VCP 315 Ce, At  [57,58
  METTL21D      
  C14orf138      
EEF2KMT Q96G04 eEF2-KMT eEF2 525 Ce, Dm, At, Sc Orthologue of [92
  FAM86A    Sc Efm3  
METTL21A Q8WXB1 HSPA-KMT HSPA1 561 – Also targets other HSPAs [57,82
  FAM119A      
  HCA557B      
METTL22 Q9BUU2 KIN-KMT Kin17 135 Dm, At  [57
  C16orf68      
METTL20 Q8IXQ9 ETFB-KMT ETFβ 200,203 Ce Orthologue in [51,52
  C12orf72    bacteria  
EEF1AKMT1 Q8WVE0 eEF1A-KMT1 eEF1A1 79 Ce, Dm, At, Sc Orthologue of [64
  N6AMT2    Sc Efm5  
METTL10 Q5JPI9 eEF1A-KMT2 eEF1A1 318 Ce, Dm, At, Sc Orthologue of Sc Efm4 [104
  C10orf138      
DOT1L Q8TEK3 KIAA1814 Histone H3 79* Ce, Dm, Sc Orthologue of Sc Dot1 [106
  KMT4      
Gene name UniProt ID Aliases Substrate Lys Distribution** Remarks Refs. 
CAMKMT Q7Z624 CaM-KMT CaM 115* Ce, Dm, At  [69,75
  C2orf34      
  CLNMT      
VCPKMT Q9H867 VCP-KMT VCP 315 Ce, At  [57,58
  METTL21D      
  C14orf138      
EEF2KMT Q96G04 eEF2-KMT eEF2 525 Ce, Dm, At, Sc Orthologue of [92
  FAM86A    Sc Efm3  
METTL21A Q8WXB1 HSPA-KMT HSPA1 561 – Also targets other HSPAs [57,82
  FAM119A      
  HCA557B      
METTL22 Q9BUU2 KIN-KMT Kin17 135 Dm, At  [57
  C16orf68      
METTL20 Q8IXQ9 ETFB-KMT ETFβ 200,203 Ce Orthologue in [51,52
  C12orf72    bacteria  
EEF1AKMT1 Q8WVE0 eEF1A-KMT1 eEF1A1 79 Ce, Dm, At, Sc Orthologue of [64
  N6AMT2    Sc Efm5  
METTL10 Q5JPI9 eEF1A-KMT2 eEF1A1 318 Ce, Dm, At, Sc Orthologue of Sc Efm4 [104
  C10orf138      
DOT1L Q8TEK3 KIAA1814 Histone H3 79* Ce, Dm, Sc Orthologue of Sc Dot1 [106
  KMT4      
Table 3
Yeast 7BS KMTs

*For Rpl1ab and H3, amino acid numbering refers to protein without initiator methionine, in accordance with the prevailing practice in the scientific literature.

Name UniProt ID Systematic name Aliases Substrate Lys Remarks Refs. 
Efm2 P38347 YBR271W  eEF2 613  [96,97
Efm3 P47163 YJR129C  eEF2 509 Orthologue of Hs EEF2KMT [9294
Efm4 P40516 YIL064W See1 eEF1A 316 Orthologue of Hs METTL10 [96,101
Efm5 P53200 YGR001C Aml1 eEF1A 79 Orthologue of Hs EEF1AKMT1 [102
Efm6 P53970 YNL024C  eEF1A 390  [99
Efm7 Q05874 YLR285W Nnt1 eEF1A Also targets N-term. of eEF1A [64
Rkm5 Q12367 YLR137W  Rpl1ab 46*  [98
Dot1 Q04089 YDR440W Kmt4, Pch1 H3 79* Orthologue of Hs DOT1L [107109
Name UniProt ID Systematic name Aliases Substrate Lys Remarks Refs. 
Efm2 P38347 YBR271W  eEF2 613  [96,97
Efm3 P47163 YJR129C  eEF2 509 Orthologue of Hs EEF2KMT [9294
Efm4 P40516 YIL064W See1 eEF1A 316 Orthologue of Hs METTL10 [96,101
Efm5 P53200 YGR001C Aml1 eEF1A 79 Orthologue of Hs EEF1AKMT1 [102
Efm6 P53970 YNL024C  eEF1A 390  [99
Efm7 Q05874 YLR285W Nnt1 eEF1A Also targets N-term. of eEF1A [64
Rkm5 Q12367 YLR137W  Rpl1ab 46*  [98
Dot1 Q04089 YDR440W Kmt4, Pch1 H3 79* Orthologue of Hs DOT1L [107109

CAMKMT-mediated methylation of calmodulin at Lys-115

Calmodulin (CaM) is an important and abundant calcium-binding protein regulating the function of many enzymes in a calcium-dependent manner. It was first reported approximately four decades ago that rat CaM contains a single trimethyllysine residue [65]. This modification, found on the conserved residue Lys-115, has been identified in several different plants and animals [6568]. Furthermore, a corresponding MTase activity was isolated from rat brain extracts [69], and a number of studies emerged, addressing the enzymology of the MTase as well as the functional significance of CaM methylation. It was shown that methylation substantially decreased the ability of CaM to activate one of its targets, nicotinamide dinucleotide (NAD) kinase [70]. Mutational analysis of CaM demonstrated that both sequence features in the vicinity of the methylation site, as well as the overall CaM structure were important for recognition by the MTase [71,72]. Also, transgenic plants expressing K115R mutated CaM showed several phenotypes [73,74].

From the initial discovery of the CaM MTase (CAMKMT) as an enzymatic activity in cell extracts, it took three decades until the actual enzyme was identified. Magnani et al. identified CAMKMT from activity-enriched mammalian extracts using tandem mass spectrometry, showing that it corresponded to the previously uncharacterized protein C2orf34, which has homologues in a wide range of multicellular eukaryotes, including plants, insects and nematodes [75]. It was also shown that recombinant CAMKMT from several different organisms all displayed the same activity, namely trimethylation of CaM at Lys-115.

The functional significance of CAMKMT was addressed in a recent study of the plant A. thaliana [76]. It was demonstrated that knockout or over-expression of CAMKMT caused increased or decreased root length respectively, indicating that CaM methylation has important functional consequences. Moreover, an analysis of the binding of methylated compared with unmethylated CaM to protein arrays, revealed several proteins that bound more avidly to either the methylated or unmethylated form, indicating that CaM methylation status strongly influences its ability to interact with other proteins.

A recent study reported that mice with a homozygous deletion of the Camkmt gene (Camkmt−/−), showed impaired development of muscle and somatosensory function, as well as reduced functionality of the brain and mitochondria in adult mice [77]. The so-called 2P21 syndrome in humans, which involves the deletion of four genes including CAMKMT at position p21 on chromosome 2, shows similar manifestations, suggesting that this syndrome is caused partly by defects in CaM methylation [78].

VCPKMT-mediated methylation of human VCP at Lys-315

Two independent studies identified the previously uncharacterized human MTase METTL21D (also known as C14orf138) as a KMT specifically targeting a single Lys residue, Lys-315, in the essential molecular chaperone VCP (valosin containing protein), and the enzyme was renamed VCPKMT [57,58]. Kernstock et al. [58] initially observed weak MTase activity of this enzyme on histone proteins and lysine homopolymers, but further experiments revealed that VCP, which had been identified as a binding partner in a yeast two-hybrid screen, was a superior substrate. VCPKMT was found to be a non-processive enzyme, capable of generating all three methylation states on its target site Lys-315, and this residue was predominantly found in the trimethylated state in VCP from mammalian cells and tissues [58]. Disruption of the VCPKMT gene in human cell lines abolished methylation, demonstrating that VCPKMT is the sole enzyme responsible for VCP methylation in mammalian cells [58]. Similarly, Cloutier et al. [57] identified VCP as a partner of VCPKMT, using tandem affinity purification (TAP), and also demonstrated VCPKMT-mediated methylation of VCP on Lys-315. VCP contains two ATPase domains, and whereas the methylation site is localized within the first of these, denoted D1, the second (C-proximal) domain, D2, is responsible for the bulk of ATPase activity. In the study by Cloutier et al., a VCP deletion mutant lacking the D2 domain was used to study the D1 domain in isolation, and, interestingly, it was found that VCPKMT-mediated methylation decreased the ATPase activity of the D1 domain. Kernstock et al. [58] observed similar ATPase activities from methylated and unmethylated VCP, isolated from VCPKMT-proficient and VCPKMT-deficient cells respectively, indicating that methylation does not substantially affect the major, D2-associated ATPase activity of VCP.

Interestingly, one study reported VCPKMT as a novel metastasis promoting protein, as it was found to be over-expressed in various tumour samples, and since its expression stimulated the migration and invasion of tumour cells in vitro and in animal models [79]. Also, VCPKMT deficient cells showed a reduced capacity for migration and invasion in vitro [58]. However, the molecular mechanism by which VCPKMT promotes tumour metastasis remains elusive. Moreover, a Vcpkmt−/− mouse has been generated, and, despite showing a complete loss of methylation of VCP at Lys-315, these mice were without any overt phenotype, demonstrating that VCPKMT is dispensable for mouse development [80].

METTL21A-mediated methylation of a conserved lysine in human HSPA proteins

Seventy kilodalton heat shock proteins (Hsp70) constitute a family of molecular chaperones, and the most studied human members are the heat and stress inducible HSPA1 (Hsp70), the constitutively expressed HSPA8 (Hsc70) and the ER-resident HSPA5 (BiP). In 1982, it was reported that the likely orthologues of HSPA1, HSPA5 and HSPA8 in chicken cells all contain a trimethyllysine modification [81], but the identity of the responsible MTase remained elusive for over three decades. Recently, two independent studies identified human METTL21A as the enzyme catalysing methylation of HSPA1 at Lys-561, as well as at the corresponding sites in HSPA8 (Lys-561) and HSPA5 (Lys-585) [57,82]. Cloutier et al. demonstrated METTL21A-catalysed methylation of HSPA (human 70 kDa heat-shock protein family member) proteins in vitro, whereas Jakobsson et al. showed enzyme-mediated trimethylation of HSPA1, HSPA5 and HSPA8 using a combination of in vitro and in vivo methods [57,82]. The latter study also showed that trimethylation of Lys-561 in HSPA8 reduced its chaperone activity towards the much-studied and Parkinson disease associated client protein α-synuclein [82]. Furthermore, it was recently shown that mutating Lys-561 to Arg in HSPA1 reduces its susceptibility to CHIP-mediated ubiquitination [83]. As CHIP primarily mediates Lys-63-linked polyubiquitination, which targets the substrate for proteasomal degradation, these results suggest that Lys-561 trimethylation, which is mutually exclusive with ubiquitination, may increase the half-life of HSPA1.

HSPA1 protein levels are often increased in cancers [84,85] and, consequently, Lys-561 methylation on HSPA1 has been assessed in tumour-derived samples. First, a paper by Cho et al. [86] suggested that dimethylated Lys-561 in HSPA1 was specifically enriched in cancers, but some of the main conclusions of that paper were recently challenged [87], and another, subsequent study showed that the trimethylated species of HSPA1 is the predominant form in human gynaecological tumours [88].

METTL22-mediated methylation of human KIN17 at Lys-135

The human KIN17 (official gene name: KIN) protein has been implicated in various processes involving nucleic acids, including DNA repair and replication, as well as mRNA processing (reviewed in [89]). Moreover, KIN17 expression is linked with tumour progression via cell proliferation [90]. However, the molecular function of KIN17 remains elusive. Cloutier et al. [57] identified KIN17 as a binding partner of the uncharacterized MTase METTL22. They further found that KIN17 was trimethylated on Lys-135, and that METTL22 was capable of catalysing the corresponding methylation reaction in vitro, indicating that METTL22 is also responsible for KIN17 methylation in vivo [57]. The methylation of Lys-135 has been suggested to modulate the association of KIN17 with chromatin, since over-expression of METTL22 diminishes the chromatin association of wild-type KIN17, whereas no such effect was observed with the K135R mutant [91].

Dual lysine methylation of human ETFβ by METTL20

Two independent studies by Rhein et al. [52] and Malecki et al. [51] identified human METTL20 as the first mitochondrial KMT. METTL20 specifically targets the beta subunit (ETFβ) of electron transfer flavoprotein (ETF) [51,52], which acts as a mobile carrier of electrons from several FAD-containing dehydrogenases to ETF:quinone oxidoreductase. The study by Rhein et al. [52] found ETFβ as a binding partner of METTL20, and identified two methylated lysine residues in ETFβ, namely Lys-200 and Lys-203, whereas the study by Malecki et al. [51] used a biochemical purification scheme to identify ETFβ as substrate of METTL20, and provided direct biochemical evidence that recombinant METTL20 methylates Lys-200 and Lys-203 in vitro. Both studies provided data indicating that METTL20 is required for methylation of Lys-200 and Lys-203 in ETFβ also in vivo, thus firmly establishing METTL20 as the enzyme responsible for ETFβ methylation.

The two METTL20-targeted Lys residues of ETFβ are located in the proximity of the so-called ‘recognition loop’ that is responsible for the interaction between ETF and the medium chain acyl-CoA dehydrogenase (MCAD) [51], and that is believed to be involved in the interaction also with other ETF-dependent dehydrogenases. Accordingly, it was shown that METTL20 decreases the ability of ETF to extract electrons from MCAD and glutaryl-CoA dehydrogenase in vitro [51]. Moreover, METTL20-dependent methylation of ETFβ was found to influence the O2 consumption rate in permeabilized mitochondria supplied with palmitoyl-L-carnitine [52], thus suggesting that METTL20-mediated ETFβ methylation may play a regulatory role in mitochondrial metabolism.

METTL20 shows a scattered and interesting organismal distribution. Among eukaryotes, putative METTL20 orthologues are present only in certain animals, primarily in chordates. As described above, functional METTL20 homologues are also found in some bacteria [53]. Thus, METTL20 appears to represent the first KMT found to catalyse the same reaction, i.e. methylation of ETFβ, both in prokaryotes and humans.

Methylation of eEF2 by human EEF2KMT and yeast Efm3

Eukaryotic translation elongation factor 2 (eEF2) is a highly conserved GTP-dependent translation factor required for protein synthesis in eukaryotes. Interestingly, eEF2 was identified by TAP as an interaction partner of the previously uncharacterized MTF16 member FAM86A [92]. Activity assays on hypomethylated HEK293 cell extracts showed that this MTase trimethylates a specific lysine residue on eEF2, Lys-525, and FAM86A was renamed EEF2KMT [92]. Lys-525 in eEF2 was found to exist largely in the trimethylated state in vivo, which suggests a constitutive optimizing function for the modification. However, eEF2 displayed considerably lower methylation levels in the rat brain, suggesting that eEF2 methylation may be subject to regulation in certain mammalian organs or tissues [92].

In addition, three studies independently identified the yeast protein Yjr129c as the enzyme responsible for trimethylating the analogous Lys-509 of the yeast eEF2 protein [9294]. This enzyme displays modest sequence similarity to human EEF2KMT and was named elongation factor methyltransferase 3, Efm3, in keeping with previous nomenclature [9294]. In various structures of eEF2 in complex with the ribosome, the eEF2 lysine in question appears to be located in close proximity to a ribosomal protein of the small subunit, RPS23, referred to as the ‘ribosomal accuracy centre’ [95]. Accordingly, efm3Δ yeast displayed a slight increase in −1 frameshifting as measured through luciferase reporter assays, and were hypersensitive to several inhibitors of protein translation, suggesting an important role of Efm3-mediated methylation, but its precise function in translation is still unclear [92,93].

Efm2-mediated methylation of yeast eEF2 at Lys-613

Studying lysine methylation in S. cerevisiae, Couttas et al. [96], observed reduced methylation of elongation factors eEF2 and eEF3 in a ybr271wΔ yeast strain, using Western blotting with pan-methyllysine antibodies. This indicated that the corresponding Ybr271w protein is a KMT targeting translation elongation factors, and, accordingly, the enzyme has been renamed Efm2. Further work has demonstrated that Efm2 is indeed required for methylation of Lys-613 in eEF2, whereas the possible role for Efm2 in eEF3 methylation yet remains to be firmly established [97]. Furthermore, efm2Δ yeast displayed hypersensitivity towards certain protein synthesis inhibitors, as well as increased stop codon read-through [93]. This indicates that Efm2-mediated methylation of translation factors is indeed important for optimal function of the protein synthesis machinery.

Rkm5-mediated methylation of yeast ribosomal protein Rpl1ab on Lys-46

Ribosomal proteins are frequently methylated, and, to identify the corresponding MTases, Webb et al. used top-down mass spectrometry to compare the molecular weight of ribosomal proteins from wild-type and MTase-deficient yeast strains [98]. Ribosomal protein Rpl1ab from the ylr137wΔ strain showed a 14 Da reduction in molecular mass, and, correspondingly, it was demonstrated that the Ylr137w protein catalysed the monomethylation of Rpl1ab on Lys-46 [98]. Consequently, the Ylr137w protein was, in keeping with previous nomenclature, renamed Rkm5 [ribosomal lysine (K) methyltransferase 5]. Sequence analysis has revealed that Rkm5 belongs to a distinct group of related 7BS MTases in yeast, Group J, representing the yeast members of MTF16 [20]. Putative Rkm5 orthologues are found exclusively in fungi, although the methylation site and surrounding residues in Rpl1ab appear to be conserved throughout the eukaryotic kingdom. In agreement with the apparent lack of Rpl1ab methylation in many eukaryotes, Rkm5-deficient yeast cells were without phenotypes regarding growth and sensitivity towards protein synthesis-inhibiting antibiotics.

Efm6-mediated methylation of yeast eEF1A on Lys-390

During the initial characterization of the related human VCPKMT and METTL21A (HSPAKMT) enzymes, it was noted that the Ynl024c protein is their closest sequence homologue in S. cerevisiae [58,82]. However, yeast orthologues of VCP and Hsp70 were found to be unmethylated on the relevant lysines, suggesting that Ynl024c had an enzymatic activity distinct from that of VCPKMT and METTL21A. Indeed, it was shown that the Ynl024c protein was responsible for methylation of Lys-390 in eEF1A, and in agreement with previous nomenclature the protein was renamed Efm6 [99]. Interestingly, Lys-390 showed a very low methylation level, being predominantly in the unmethylated state, with only a small fraction (∼25%) being monomethylated. However, ectopic over-expression of Ynl024c caused a dramatic increase in methylation, with trimethylation as the predominant state [99]. This may suggest that Lys-390 methylation may be particularly important under certain conditions, e.g. stress, where it may serve an adaptive or regulatory function.

Other eukaryotic 7BS KMTs

Methylation of eEF1A by human METTL10 and yeast Efm4

Eukaryotic translation elongation factor eEF1A, which is the counterpart of bacterial EF-Tu, contains several methylated lysine residues, and yeast eEF1A is methylated on Lys-30, Lys-79, Lys-316 and Lys-390 [100]. When analysing the molecular weight of eEF1A in yeast strains carrying inactivating insertions in putative MTase genes, Lipson et al. [101] found a mass loss of 28 Da for eEF1A from a yil064wΔ strain, possibly accounted for by the loss of two methyl groups. Also, the recombinant Yil064w protein was found to label a protein with molecular weight corresponding to eEF1A specifically in extracts from the yil064wΔ strain. From this it was concluded that Yil064w is an eEF1A-specific MTase, and the enzyme, which was originally named See1 (Secretion and Early Endocytosis 1), has now been redubbed Efm4 [102,103]. Lys-316 was suggested to be the target of Efm4, as it is the only dimethylated lysine residue in yeast eEF1A, and this was later confirmed by mass spectrometry [96]. Both Efm4 and its target site in eEF1A are highly conserved in eukaryotes, and it has indeed recently been demonstrated that the putative human Efm4 orthologue, METTL10, is responsible for trimethylation of the corresponding residue, Lys-318, in human eEF1A1 [104]. Since another human eEF1A-specific KMT, N6AMT2, was recently suggested to be renamed EEF1AKMT1 (see below), we propose that METTL10 is redubbed EEF1AKMT2.

Methylation of eEF1A by human EEF1AKMT1 and yeast Efm5

When screening yeast 7BS MTase deletion strains for lack of eEF1A methylation, it was found that eEF1A from a ygr001cΔ strain was devoid of trimethyllysine [102]. As Lys-79 is the only trimethylated residue in eEF1A, it was suggested as the target of this MTase, which was indeed confirmed by protein mass spectrometry. Consequently, the Ygr001c protein was redubbed Efm5. Interestingly, the Efm5 protein had previously been categorized as a putative RNA-specific MTase, as it contains a DPPY motif (after β-strand 4); a motif primarily found in RNA methyltransferases targeting the N6 position of adenine. Efm5 appears to be present in all eukaryotes, and its human orthologue, denoted N6AMT2 (N6-adenine methyltransferase), was recently shown also to methylate Lys-79 eEF1A in vitro, and hence this enzyme was renamed EEF1AKMT1 [64]. Another interesting feature of Efm5 and its orthologues is the absence of a canonical Motif 1.

Methylation of histone H3 at Lys-79 by human DOT1L and yeast Dot1

Dot1 was the first eukaryotic 7BS KMT to be discovered, and the Dot1 gene was initially identified in a genetic screen in S. cerevisiae, as one of several genes that, when over-expressed, caused activation of genes localized in heterochromatic regions at telomeres (Dot=disruptor of telomere silencing) [105]. Protein sequence searches revealed that Dot1 showed homology to 7BS MTases, in particular to protein arginine MTases, and it was therefore suggested that Dot1 may be involved in methylation of histone proteins. Indeed, it was demonstrated independently by several groups that yeast Dot1, as well as its human homologue DOT1L, specifically targets histone protein H3 [106109]. In contrast with histone-specific KMTs of the SET domain family, which typically target the flexible, unstructured tails found at the N- and C-termini of histone proteins, Dot1/DOT1L methylates Lys-79, which is found in the globular portion of H3. Correspondingly, recombinant Dot1/DOT1L does not methylate short H3-derived, K79-containing peptides nor recombinant H3 in vitro, but is only active on its substrate in the context of the nucleosome [109].

Since its initial discovery, numerous studies have addressed the biological and functional significance of Dot1/DOT1L-mediated methylation of Lys-79 in H3. Dot1/DOT1L-mediated histone methylation has been demonstrated to play an important part in a number of cellular processes, including telomere silencing, DNA repair and cell cycle regulation [108,110,111]. Moreover, the mammalian DOT1L protein has been linked to several important physiological processes, such as cardiac function and erythropoiesis, and also plays a crucial role in the development of certain leukaemias [112114].

Uncharacterized putative eukaryotic 7BS KMTs

A number of novel 7BS KMTs have been reported during recent years, and clearly, more will be added to the list. All six human MTF16 members characterized thus far (described above) have turned out to be KMTs, indicating that there will probably be KMTs also among the four remaining ones, METTL18, METTL21B, METTL21C and METTL23. However, one of these, METTL18, is less likely to be a KMT, as its putative yeast orthologue, Hpm1, was found to methylate a histidine residue in the ribosomal protein Rpl3. Moreover, given the close similarity of the uncharacterized human FAM173A and FAM173B proteins to the crenarchaeal aKMT, it is likely that also these enzymes will turn out to be KMTs.

7BS KMTs–GENERAL DISCUSSION

Diversity in sequence and structure

In terms of their primary amino acid sequence, the 7BS KMTs represent a rather diverse set of enzymes. To illustrate this, we generated a structure-guided sequence alignment of the putative and established human 7BS KMTs, also including some non-KMT 7BS enzymes, and an unrooted phylogenetic tree was made from the alignment (Figure 3A). The tree clearly shows that several of the 7BS KMTs are only distantly related to each other, and in many cases equally related to non-KMT enzymes. Overall, the less related human 7BS KMTs do not share sequence homology beyond the rather degenerate motifs ‘I’, ‘II’, ‘III’ and ‘Post I’, which are the hallmarks of the entire 7BS MTase family. Even at these motifs the sequence homology is low, and the 7BS KMTs again appear no more similar to each other than to the non-KMTs (Figure 3B). This underscores that, in terms of primary amino acid sequence, KMTs encompass a highly diverse set of enzymes within the 7BS family.

Sequence diversity of human 7BS KMTs

Figure 3
Sequence diversity of human 7BS KMTs

(A) Unrooted phylogenetic tree of human KMTs and selected non-KMT 7BS MTases. A structure-guided sequence alignment was generated using PROMALS3D [121]. From the resulting alignment, an unrooted phylogenetic tree was generated and rendered using the MRBAYES and TREEDYN programs respectively, embedded within the Phylogeny.fr package [122124]. Blue colour and underlined print indicates established KMTs, whereas blue colour and regular print indicates putative KMTs. Red, orange and green colours indicate protein (non-lysine), RNA and small-molecule MTases respectively. NTMT1, N-terminal Xaa-Pro-Lys N-methyltransferase 1; GNMT, glycine N-methyltransferase; COMT, catechol O-methyltransferase; NSUN5, 28S rRNA (cytosine-C(5))-methyltransferase, METTL1, tRNA (guanine-N(7))-methyltransferase. Note that, due to the low similarity of the sequences used, the actual topology of the tree is somewhat arbitrary; the main purpose of the figure is to illustrate that KMTs represent a diverse set of enzymes within the human 7BS MTase family, and that the MTF16 enzymes form a rather distinct group. (B) Motif alignment of 7BS KMTs and non-KMTs. An alignment of motifs I, Post I and II from various human KMTs and non-KMTs is shown. Colour coding as in (A).

Figure 3
Sequence diversity of human 7BS KMTs

(A) Unrooted phylogenetic tree of human KMTs and selected non-KMT 7BS MTases. A structure-guided sequence alignment was generated using PROMALS3D [121]. From the resulting alignment, an unrooted phylogenetic tree was generated and rendered using the MRBAYES and TREEDYN programs respectively, embedded within the Phylogeny.fr package [122124]. Blue colour and underlined print indicates established KMTs, whereas blue colour and regular print indicates putative KMTs. Red, orange and green colours indicate protein (non-lysine), RNA and small-molecule MTases respectively. NTMT1, N-terminal Xaa-Pro-Lys N-methyltransferase 1; GNMT, glycine N-methyltransferase; COMT, catechol O-methyltransferase; NSUN5, 28S rRNA (cytosine-C(5))-methyltransferase, METTL1, tRNA (guanine-N(7))-methyltransferase. Note that, due to the low similarity of the sequences used, the actual topology of the tree is somewhat arbitrary; the main purpose of the figure is to illustrate that KMTs represent a diverse set of enzymes within the human 7BS MTase family, and that the MTF16 enzymes form a rather distinct group. (B) Motif alignment of 7BS KMTs and non-KMTs. An alignment of motifs I, Post I and II from various human KMTs and non-KMTs is shown. Colour coding as in (A).

7BS KMTs are diverse also regarding their structure. They all share the core 7BS fold, but the additional N-terminal structural elements required to make up the catalytically active enzyme vary greatly. Several MTF16 enzymes, such as METTL21A (HSPA-KMT), typically only contain an addition of two β-strands preceding the 7BS core, whereas PrmA contains a three-stranded β-sheet sandwiched between two α-helices, and Dot1/DOT1L a bundle of four α-helices (Figure 4). Also, the amino acid residues implicated in recognition of the polypeptide substrate and in catalysis, are different (see also below). This is maybe not so surprising, as these three enzymes show important differences with respect to both substrate recognition and specificity. Catalytic activity of Dot1 does not only require interaction with the protein substrate, H3, but also with DNA, which is wound around H3 in the nucleosome [109]. Unlike most other 7BS KMTs, PrmA does not target a single lysine residue in its substrate (L11), but it methylates multiple lysine residues, as well as the N-terminal alanine.

Structural diversity of 7BS KMTs

Figure 4
Structural diversity of 7BS KMTs

Three-dimensional structures of human METTL21A (PDB: 4LEC; Chain A), T. thermophilus PrmA (PDB: 1UFK), as well as the catalytic domain of human DOT1L (PDB: 1NW3) are shown. α-helices and β-strands of the core 7BS fold are depicted in cyan and orange respectively, whereas structural elements N-terminal to the core 7BS fold are depicted in purple.

Figure 4
Structural diversity of 7BS KMTs

Three-dimensional structures of human METTL21A (PDB: 4LEC; Chain A), T. thermophilus PrmA (PDB: 1UFK), as well as the catalytic domain of human DOT1L (PDB: 1NW3) are shown. α-helices and β-strands of the core 7BS fold are depicted in cyan and orange respectively, whereas structural elements N-terminal to the core 7BS fold are depicted in purple.

The considerable diversity of the 7BS KMTs clearly suggests that these enzymes, despite catalysing the same chemical reaction and sharing a common structural scaffold, do not originate from a common ancestral enzyme. Rather, 7BS KMT probably evolved independently at different instances throughout evolution, suggesting that the 7BS scaffold readily evolves specificity towards lysine residues in protein substrates. Thus, extensive parallel evolution appears to have generated several different types of KMTs within the 7BS class of enzymes. This represents an interesting analogy to the evolution of KMTs both among SET-domain enzymes and 7BS enzymes, two unrelated MTase classes; also a likely result of convergent evolution.

Substrate recognition

The SET domain enzymes typically recognize their substrates through the interaction with a linear unstructured peptide sequence, constituted by the target lysine and a few surrounding residues, and such enzymes are therefore usually active on their target lysine residues in the context of a short synthetic peptide. In contrast, the 7BS MTases appear to recognize their substrates as a three-dimensionally folded structure. For example, a folded domain (∼80 aa) of VCP is required for interaction with VCPKMT, and the residues flanking the methylation site Lys-315 appear to be of less importance, as these can be individually or collectively mutated without preventing methylation [58]. Finally, several 7BS KMT substrates were initially identified through their ability to interact tightly with the MTase in their folded state, further supporting the notion that these enzymes are highly specific and recognize fully folded proteins.

The bacterial PrmA enzyme is the only 7BS KMT for which the three-dimensional structure of the enzyme in complex with its substrate has been elucidated. The structure of PrmA in complex with its target Lys-39 in the ribosomal L11 protein indicates that one residue, Asn-191, is of particular importance, being localized both in the vicinity of the methyl group of AdoMet and the targeted ε-nitrogen of Lys-39 (Figure 5A) [42]. Importantly, Asn-191 and surrounding residues are also highly conserved among putative PrmA orthologues from different bacteria, forming an A-N-I/L motif, localized immediately after Motif II (Figure 5B). Interestingly, other 7BS KMTs also contain highly conserved motifs at this position; the MTF16 enzymes contain the previously noted D/E-X-X-Y/F motif, whereas orthologues of DOT1L/Dot1, METTL10/Efm4 and N6MT2/Efm5 carry highly conserved N-N-F-X-F, D-K-G-T and D-P-P-Y/F motifs respectively (Figure 5B). These motifs, previously referred to as ‘Post II’, are, when comparing putative KMT orthologues from different organisms, much more conserved than the preceding Motif II, suggesting an important role in substrate recognition and/or catalysis. Indeed, structural comparison of DOT1L, VCPKMT and PrmA reveals that the conserved Post II motifs of DOT1L and VCPKMT include an Asn or Asp residue respectively, that is superimposable (using AdoMet as an anchor) with the key catalytic residue Asn-191 in PrmA (Figures 5C and 5D). In the case of DOT1L/Dot1, this residue (Asn-241/Asn-479) has been proposed to play an important role in positioning the targeted amino group of the substrate Lys residue in a favourable orientation for methyl transfer [115,116], and one may speculate that the conserved Asp/Asn residues, found at corresponding positions in all the 7BS KMTs, may play similar roles.

7BS groups are defined by a highly conserved ‘Post II’ motif

Figure 5
7BS groups are defined by a highly conserved ‘Post II’ motif

(A) Orientation of Asn-191 in PrmA relative to the substrate Lys-39 in L11 and AdoMet. (B) Sequence alignments encompassing motifs ‘II’ and ‘Post II’ for various 7BS KMTs, i.e. selected human MTF16 enzymes (upper panel), as well as putative orthologues (from various organisms) of PrmA, DOT1L, METTL10, N6AMT2 (lower panels). Green dots indicate the conserved, catalytically important Asp/Asn residue of motif ‘Post II’. Ec=E. coli; Tt=T. thermophilus; Cb=Clostridium botulinum; Ft=Francisella tularensis; Xc=Xanthomonas campestris; Hs=Homo sapiens; Dm=D. melanogaster; Ce=C. elegans; Sc=S. cerevisiae; At=A. thaliana. (C) Similar positioning of the catalytically important Asp/Asn residue of motif ‘Post II’ in different 7BS KMTs. The conserved Asp/Asn residue of motif ‘Post II’ (illustrated by green dot in B) is shown on the structures of PrmA (PDB: 2NXE; Chain A; Asn-191, upper panel), DOT1L (PDB: 1NW3; Asn-241, middle panel) and VCPKMT (PDB: 4LG1; Chain B; Asp-144, lower panel). (D) Orientation of the putatively catalytically important Asp/Asn residues of motif ‘Post II’ relative to AdoMet. The three structures shown in (C) were superimposed, and only the catalytic Asp/Asn residue and AdoMet are shown.

Figure 5
7BS groups are defined by a highly conserved ‘Post II’ motif

(A) Orientation of Asn-191 in PrmA relative to the substrate Lys-39 in L11 and AdoMet. (B) Sequence alignments encompassing motifs ‘II’ and ‘Post II’ for various 7BS KMTs, i.e. selected human MTF16 enzymes (upper panel), as well as putative orthologues (from various organisms) of PrmA, DOT1L, METTL10, N6AMT2 (lower panels). Green dots indicate the conserved, catalytically important Asp/Asn residue of motif ‘Post II’. Ec=E. coli; Tt=T. thermophilus; Cb=Clostridium botulinum; Ft=Francisella tularensis; Xc=Xanthomonas campestris; Hs=Homo sapiens; Dm=D. melanogaster; Ce=C. elegans; Sc=S. cerevisiae; At=A. thaliana. (C) Similar positioning of the catalytically important Asp/Asn residue of motif ‘Post II’ in different 7BS KMTs. The conserved Asp/Asn residue of motif ‘Post II’ (illustrated by green dot in B) is shown on the structures of PrmA (PDB: 2NXE; Chain A; Asn-191, upper panel), DOT1L (PDB: 1NW3; Asn-241, middle panel) and VCPKMT (PDB: 4LG1; Chain B; Asp-144, lower panel). (D) Orientation of the putatively catalytically important Asp/Asn residues of motif ‘Post II’ relative to AdoMet. The three structures shown in (C) were superimposed, and only the catalytic Asp/Asn residue and AdoMet are shown.

Enzyme processivity

A KMT can catalyse the addition of up to three methyl groups on to a lysine residue, and some KMTs are processive enzymes, meaning that they are capable of adding more than one methyl group to the substrate per binding event. In contrast, other KMTs have a distributive mode of action, and must dissociate from the substrate between each round of methylation, typically to allow exchange of S-adenosylhomocysteine (AdoHcy) for AdoMet. Whereas many SET domain KMTs are processive, the 7BS KMTs studied so far display a distributive mode of action. In the case of Dot1, it has been shown that all three different methylation states are generated in vivo and in vitro, and that they are functionally redundant [117]. VCP-KMT also appears to be a distributive enzyme, as a mixture of all three methylation states on VCP is obtained at low enzyme concentrations in vitro [58]. Also for METTL21A, METTL20 and EEF2KMT, a non-processive mode of action is suggested, since their respective substrates HSPA1, ETFβ and eEF2 have been observed to acquire a mixture of methylation states in vivo [51,82,92].

Obviously, for each round of methylation the KMT substrate changes, due to the acquisition of an additional methyl group. In some cases, this will substantially alter the enzyme's affinity for the substrate or the positioning of the substrate in the active site, thereby influencing the potential for further methylation. Thus, a 7BS KMT with a distributive mode of action may not necessarily be able to perform trimethylation. For example, this is likely the case for Efm4, which dimethylates its substrate, Lys-316 in yeast eEF1A [96,101].

Biochemical and biological significance of 7BS KMT-mediated methylation

Many SET domain enzymes can target multiple non-histone proteins, which makes it challenging to pinpoint the biologically relevant target, as well as to specifically demonstrate a functional/biochemical consequence of methylation of that particular substrate in vivo. In the case of the 7BS KMTs, the situation appears somewhat simpler as many of these enzymes seem to target a single protein (or a group of highly related proteins). Still, it has proven challenging to unravel functional consequences of methylation of the 7BS KMT targets, and in most cases the biological/biochemical relevance remains elusive. The reported cases of a biological/biochemical effect of methylation have been described in detail above, but there are no indications that these modifications serve a common function in all the substrates. Also, the substrates of the 7BS KMTs are highly diverse, with regard to both function and subcellular localization. Several of the MTF16 enzymes, such as VCPKMT, METTL21A and METTL20, show a rather scattered distribution among eukaryotes, whereas their substrates, including the targeted lysine residues, are typically highly conserved and omnipresent in the eukaryotic kingdom. This implies that these lysines are unmethylated in a wide range of organisms, and that the corresponding proteins are functional in the absence of methylation. Thus, one may speculate that such methylation may have a marginal effect, not readily observed at the levels of protein biochemistry or organism viability, but still making a difference during the selective process of evolution. In agreement with this notion, two recent studies demonstrated that homozygous lack-of-function mutations in the gene encoding METTL23, a putative MTF16 KMT, only caused mild phenotypes in humans, i.e. a syndrome associated with learning disability and mild intellectual impairment [118,119]. Similarly, a recent study that investigated the presence of homozygous loss of function mutations in the Icelandic population showed that a number of Icelanders actually lack the METTL20 function, indicating that this enzyme is clearly not essential in humans [120]. Finally, and as mentioned above, VCPKMT knockout mice were viable and without any overt phenotype [80]. In conclusion, although these lysine methylations are clearly important on an evolutionary time scale, as the corresponding enzymes have been maintained throughout evolution, their functional significance appears to be more challenging to demonstrate at the molecular and cellular levels.

CONCLUSIONS AND FUTURE PERSPECTIVES

A number of previously uncharacterized 7BS MTases have been established as KMTs during recent years, particularly from yeast and humans. The current knowledge regarding the substrate specificity of human and yeast 7BS KMTs has been summarized in Figure 6. These enzymes appear highly specific, and typically target non-histone proteins involved in protein synthesis or folding.

Human and yeast 7BS KMTs and their targets

Figure 6
Human and yeast 7BS KMTs and their targets

An unrooted phylogenetic tree of established and putative 7BS KMTs from H. sapiens (blue) and S. cerevisiae (red) is shown. In order to provide more space for enzyme names, some of the branches in the tree have been extended by dotted lines. Substrates are indicated by rounded rectangles, and the numbers indicate the position of the targeted Lys residue(s) in the substrate. In the cases where yeast and human KMTs display identical activities, i.e. the methylation of equivalent residues in orthologous substrates, this is indicated by dual (red/blue) colouring of the substrates.*Efm7 also methylates the N-terminal amino group of eEF1A.

Figure 6
Human and yeast 7BS KMTs and their targets

An unrooted phylogenetic tree of established and putative 7BS KMTs from H. sapiens (blue) and S. cerevisiae (red) is shown. In order to provide more space for enzyme names, some of the branches in the tree have been extended by dotted lines. Substrates are indicated by rounded rectangles, and the numbers indicate the position of the targeted Lys residue(s) in the substrate. In the cases where yeast and human KMTs display identical activities, i.e. the methylation of equivalent residues in orthologous substrates, this is indicated by dual (red/blue) colouring of the substrates.*Efm7 also methylates the N-terminal amino group of eEF1A.

Despite substantial recent progress, several important challenges remain. Very little is still known regarding the functional importance of the methylated lysine residues introduced by the 7BS KMTs, although it appears that the effects in many cases may be subtle. Moreover, it will be interesting to learn whether these methylations, similarly to histone lysine methylations, are recognized by specific reader domains, or whether they are subject to reversal by demethylases. Finally, future investigations will certainly address whether defects in such enzymes are important for the development of human diseases, such as cancer or neurological disorders.

We thank Dr Michael McDonough for advice on visualization of protein structures.

FUNDING

This work was supported by the Norwegian Cancer Society [grant number 107744-PR-2007-0132]; and the Research Council of Norway [grant number FRIMEDBIO-240009].

Abbreviations

     
  • AdoHcy

    S-adenosylhomocysteine

  •  
  • AdoMet

    S-adenosylmethionine

  •  
  • 7BS

    seven-β-strand

  •  
  • CaM

    calmodulin

  •  
  • ChoP

    phosphorylcholine

  •  
  • Dot1

    disruptor of telomere silencing 1

  •  
  • eEF1A

    eukaryotic translation elongation factor 1A

  •  
  • eEF2

    eukaryotic translation elongation factor 2

  •  
  • Efm

    elongation factor methyltransferase

  •  
  • EftM

    EF-Tu modifying enzyme

  •  
  • ETF

    electron transfer flavoprotein

  •  
  • Hpm1

    histidine protein methyltransferase 1

  •  
  • HSPA

    human 70 kDa heat-shock protein family member

  •  
  • KMT

    lysine-specific methyltransferase

  •  
  • METTL

    methyltransferase like

  •  
  • MTase

    methyltransferase

  •  
  • MTF16

    methyltransferase family 16

  •  
  • OmpB

    outer membrane protein B

  •  
  • PAF

    platelet-activating factor

  •  
  • PAFR

    platelet-activating factor receptor

  •  
  • PKMT1 and PKMT2

    protein lysine methyltransferase 1 and 2

  •  
  • Rkm5

    ribosomal lysine (K) methyltransferase 5

  •  
  • TAP

    tandem affinity purification

  •  
  • VCP

    valosin containing protein

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