The crystal structures of three bacterial and one archaeal TrmIs have previously been reported (Table 1): from Mycobacterium tuberculosis, a mesophilic bacterium (_MtTrmI, initially called Rv2118c) 22 25 , Thermus thermophilus, a thermophilic bacterium (_TtTrmI) 26 , Aquifex aeolicus (PDB code 2YVL, _AaTrmI), a hyperthermophilic bacterium and Pyroccocus abyssi, a hyperthermophilic archaeon (_PaTrmI) that lives in an environment of extreme pressure 27 . Moreover, a search in the Dali database reveals that the PDB code 1O54, a putative SAM-dependent O-MTase from the thermophilic bacterium Thermotoga maritima, is inaccurately annotated and corresponds to the m¹A58 MTase (_TmTrmI). Finally, the PDB code 2B25, annotated as a human putative 1-methyladenosine MTase, corresponds to the product of the TRM61 gene (_HsTrmI-61), the SAM-binding subunit that composes the hetero-tetramer of m¹A58 MTase in eukaryotes and shows extensive sequence similarity to the bacterial and archaeal enzymes 23 . These organisms, with structurally characterized TrmI proteins, display very different optimal growth temperatures (Table 1). These TrmI proteins show sequence identity ranging from 24.8% to 40.8%, the bacterial proteins from M. tuberculosis and T. thermophilus being the most similar and the two hyperthermophilic proteins (P. abyssi and A. aeolicus) the most dissimilar (Additional File 1, Table S1). Except otherwise stated, the residue numbering for T. maritima is used throughout the text.

The TrmI proteins show a tetrameric organization in the crystals as it was shown in solution 19 21 22 23 24 . The crystallographic asymmetric unit consists of one, two or four subunits of the protein (Figure 1, Table 1) and the tetramer is assembled from the monomers using either the crystallographic or non-crystallographic symmetry. The four subunits of the homo-tetrameric TrmI proteins are related by a four-fold symmetry. Two opposite sides of the tetramer show positively charged grooves, wide enough to accommodate an A-form RNA helix 26 , that likely bind the tRNA substrate (Figure 1A). Interestingly, the electrostatic surfaces are not similar in all TrmI proteins, _TmTrmI showing a less positive surface than the four other proteins and _MtTrmI and _PaTrmI having the most positive surface (Figure 1A). Based on the characteristics of the different monomer-monomer interfaces, the tetramer can be described as a dimer of two tightly assembled dimers (A/B and C/D), which interact back to back as shown in Figure 1B. The two protruding antiparallel β-strands at the C-terminus of each monomer contribute significantly to the tetrameric assembly (Figure 1C).

_MtTrmI, _TmTrmI and _PaTrmI (space group I222) contain one TrmI monomer in the asymmetric unit and the tetramer is generated using the crystallographic symmetry. In _AaTrmI and in the two crystal forms of _PaTrmI in complex with SAH, the crystallographic asymmetric unit contains a full tetramer and there are similar relationships between the monomers except that all axes are non-crystallographic. The four monomers display an rmsd between equivalent Cα atoms of less than 0.27 Å, 0.66 Å and 0.05 Å for _AaTrmI, _PaTrmI (space group P2₁2₁2₁) and _PaTrmI (space group P3₁), respectively. In _TtTrmI and _HsTrmI, the asymmetric unit contains the tight A/B dimer and the A/D dimer, respectively, in which the two monomers are related by the non-crystallographic 2-fold axis. The two monomers are almost identical with an rmsd between equivalent Cα atoms of 1.04 Å and 0.36 Å for _TtTrmI and _HsTrmI, respectively. The full tetramer of _TtTrmI is generated by proper crystallographic 2-fold symmetry. Although a homo-tetramer of _HsTrmI-61 can also be formed using the 2-fold crystallographic symmetry, it is not biologically relevant because _HsTrmI, in contrast to bacterial and archeal TrmIs, is a hetero-tetramer. The fact that the asymmetric unit of the _HsTrmI-61 crystal consists of the A/D dimer is consistent with the modeling of the full yeast TrmI structure 24 , and strongly suggests that in eukaryotic TrmIs, the A/B and A/C dimers are formed by two different subunits (TrmI-6 and TrmI-61). Therefore, in eukaryotes, the dimeric and tetrameric contacts are formed only between different subunits. The dimers and tetramers of all TrmIs are very similar, and the tetramers of all structures can be superimposed with rmsd of 1.65-3.2 Å (Additional File 1, Table S2A). Therefore, there is no rigid body rearrangement of the monomers between the TrmI proteins, which, in some cases 33 , was shown to be a factor contributing to thermal stability.

The monomer structures of TrmI proteins with known three-dimensional structure are closely similar, with rmsd between 1.34 and 2.70 Å and Q-scores between 0.41 and 0.59 (Additional File 1, Table S2B; Figure 1(D)). One monomer is formed by two domains: a catalytic C-terminal domain that binds the SAM/SAH cofactor with a Rossmann-like fold characteristic of SAM-dependent MTases, and a smaller N-terminal domain with a β-structure. A structure-based multiple sequence alignment and secondary structure assignment for the TrmI monomers are shown in Figure 2. The N-terminal domain (residues 1-67) contains one helix (α1) and six β-strands (βA to βF). The C-terminal domain (residues 68-263) contains seven α-helices and eight β-strands, of which strands β6, βG and βH form an antiparallel β-sheet involved in the tetramer formation. Compared to the other enzymes, _HsTrmI-61 has a 10-residue insertion in the turn between αB and β3. The similarity of the structures extends even higher when only the catalytic domains are superposed (Figure 1(D)), revealing a slightly different relative orientation of the N-terminal and catalytic domains in the TrmIs studied. Moreover, comparison of the four monomers in the structure of _PaTrmI in the P2₁2₁2₁ space group, in which SAH adopts two different conformations 27 , reveals also mobility of the N-terminal domains relative to the tetramer core formed by four catalytic domains. Indeed, the four monomers in the asymmetric unit display a pair-wise rmsd of less than 0.66 Å comparing 253 pairs of Cα atoms, whereas the superposition of the catalytic domains alone gives an rmsd of less than 0.36 Å. The higher B-factors of the N-terminal domain also indicate its mobility relative to the tetramer core (Figure 1 (E)). It is possible that the mobility of the N-terminal domain may be critical to the activity of the enzyme and that a hinged movement may occur upon tRNA binding, as observed for other RNA modifying enzymes 34 . However, although the crystallographic contacts of _PaTrmI in the three different space groups are different (Figure 3), the relative orientation of the catalytic and N-terminal domains remains unchanged, which suggests the existence of a preferred conformation of the protein in the absence of tRNA.

Usually, proteins from thermophiles contain more charged and hydrophobic amino acids residues at the expense of polar ones 1 40 . Additionally, analysis of mesophilic and thermophilic proteins has previously pointed out the tendency towards shorter (even absent) loop regions in thermophilic organisms, which correlates with their compactness 8 41 . The number of alanine residues and aromatic amino acids is not higher in thermostable TrmIs compared to the less-stable ones (Additional File 1, Table S3). A higher number of prolines increases the backbone rigidity and has been shown to contribute in some cases to protein thermostability 42 . Here, the content of proline residues (between 3.6 and 6.3%; Additional File 1, Table S3) did not reveal outstanding differences between the various TrmI proteins, indicating that reduction of the backbone flexibility is not a factor contributing importantly to the thermostability of TrmI proteins. Noticeably, _AaTrmI displays some loop shortenings (Figure 2) and the smallest surface area among the structurally characterized TrmIs (Table 2), two features that could contribute to the thermal stability of _AaTrmI.

Increased ion-pairing and hydrogen bonding interactions are factors employed by thermophiles to stabilize their proteins at extreme temperatures. The participation of these interactions in the stability of the monomer was analyzed (Additional File 1, Table S4). The two hyperthermophilic TrmI proteins have the highest number of intra-monomer salt bridges (18 and 20). However, these interactions are not more numerous in the case of the thermophilic TrmI proteins compared to the mesophilic ones. Surprisingly, there are only 6 intra-monomer salt bridges in _TtTrmI compared to 11 to 20 in the other TrmI proteins. The number of H-bonds per monomer (203 to 231) is similar in all TrmI proteins.

Although oligomerization has been identified as one of the ways to achieve thermostability of proteins in thermophilic organisms 43 , both mesophilic and thermophilic TrmI proteins are organized as tetramers. This quaternary structure therefore does not result from adaptation to high temperatures but is important for binding the tRNA substrates and for catalytic activity 19 21 22 23 24 26 .

Although the fine interactions between monomers can obviously be modulated by the flexibility of residues located at the structures interfaces, the analysis of the crystallographic coordinates is likely to unveil several features of the quaternary structure characteristic of the thermozymes. To investigate whether TrmI extremozymes are more tightly packed than their mesophilic homologs, the surface areas and the molecular protein volumes of the monomer, A/B dimer and tetramer for each homo-tetrameric protein were calculated (Table 2). All five enzymes have similar surface areas. The monomer protein volume and the number and volume of cavities do not decrease with higher stability of the protein. However, the difference between the volume of the tetramer and the volume of four monomeric units (Δ4 = c-4a, Table 2) is negative for all thermophilic and hyperthermohilic TrmIs, indicating that the protein density increases upon tetramerization. In contrast, this number is positive for the mesophilic _MtTrmI protein. Yet, the amount of volume contraction is not directly dependent of the optimal growth temperature of the thermophilic protein. Similarly, the contraction upon dimer formation (Δ2 = b-2a, Table 2) is negative for both thermophilic and hyperthermophilic proteins. This reflects a tight packing of the hyperthermophilic A/B dimers. The surface to volume ratio is another way to measure the compactness (Table 2). Interestingly, this factor decreases as the thermostability of the TrmI protein increases, except for _PaTrmI. This might reflect the particularity of this enzyme to possess intermolecular disulfide bridges (see below). Again with the exception of the P. abyssi enzyme, increased buried surface areas within the A/B dimer and within the tetramer (Table 3) correlate with increased thermostability of the TrmI proteins. Indeed, the mesophilic protein has a smaller buried surface area compared to the thermophilic and hyperthermophilic ones. These features related to the oligomerization of the proteins suggested some critical differences at the interfaces of the TrmI monomers and led us to analyze in detail the network of interactions at these interfaces.

The architecture of the TrmI tetramer (Figure 1(B) and 4(C4) and Table 3) shows that the formation of the A/B dimer involves a large buried surface area, which correlates with a high number of residues present at the interface between monomers. The A/C dimer presents a buried surface area that is reduced by a factor around 4 compared to the A/B one and that involves much less residues at the interface. Therefore, TrmI proteins can be described as a dimer of tightly-assembled dimers (A/B and C/D). The A/C dimer, in all available TrmI structures, presents very similar numbers of contacts with about ten hydrogen bonds between the A and C monomers (Table 3). Most residues involved at this interface belong to the C-terminal β-strands (β6, βG and β7, Figures 1(C) and 2). Two charged residues (Glu226 and Arg230, _TmTrmI numbering) are involved in two conserved bidentate ionic interactions to form contacts specific of the tetramer at the A/B and C/D dimer interfaces in all TrmIs except _PaTrmI (Figures 2 & 4). It was shown that suppressing this conserved ionic interaction by replacing both equivalent interacting residues in the TrmI-6 and TrmI-61 subunits of the S. cerevisiae enzyme by alanine did not prevent formation of the tetramer 23 . These mutations by themselves were not sufficient to destabilize the tetramer because other important interactions remained intact. In _PaTrmI, these ionic interactions are replaced essentially by four inter-monomer disulfide bridges (Figure 4(E)). Concerning the A/B interface of TrmIs, the comparison of the number of H-bonds does not indicate a trend that correlates with higher thermostability. Other interactions inside the tight A/B dimer are rather extensive and mainly ionic, particularly for _TmTrmI (Figure 4). For the A/B dimer, the number of ionic interactions at the interface between monomers A and B is clearly increased for TrmI proteins from thermophilic organisms (Table 3).

The dimeric (A/B and C/D) and tetrameric (A/D and B/C) interactions also involve, in all TrmIs, van der Waals and hydrophobic interactions (Table 4; Additional File 1, Figure S1B). Although thermophilic TrmIs do not contain more alanine and aromatic residues than TrmIs from mesophiles (Additional File 1, Table S3), a higher number of residues participates in van der Waals interactions. Interestingly, _PaTrmI exhibits unusually low hydrophobic Pv values 44 for both the dimeric and tetrameric interface, which indicates specific hydrophobic spots. In particular, Phe225 makes hydrophobic contacts with Leu223 and Val242. This interaction substitutes, together with the inter-monomer disulfide bridges (see below), to the conserved ionic salt bridges involved in tetramer stabilization in all other TrmI proteins. Indeed, Phe225 occupies the position equivalent to Arg230 that conservatively establishes a bidentate ionic interaction with Glu226 (Figure 2 and Additional File 1, Figure S1B). _AaTrmI also exhibits a low hydrophobic P-value for the A/B interface. Therefore, increased hydrophobic interactions at the dimeric interface in the hyperthermophilic TrmIs could account for the fact that the number of ionic interactions is not increased compared to the mesophilic and thermophilic proteins.

In summary, whereas thermophilic TrmIs use a higher number of ionic interactions to stabilize the A/B interface, hyperthermophilic TrmIs display increased hydrophobic interactions. This is in agreement with other studies that conclude that ionic interactions stabilizing crucial areas of structure, together with increased hydrophobic packing, are the most common means for stabilizing proteins at high temperatures, particularly oligomeric proteins. For example, comparison of tetrameric malate dehydrogenases from thermophilic and mesophilic bacteria indicated that higher thermostability comes first from the presence of polar residues that form additional hydrogen bonds within each subunit and then with the use of charged residues to form additional ionic interactions along the dimer-dimer interface, as well as additional aromatic contacts at the monomer-monomer interface in each dimer 38 . Moreover, comparative structural analysis of various citrate synthases also showed that higher growth temperatures correlate with reinforced electrostatic interactions in the subunit interface, as well as a reduced exposure of hydrophobic surface 39 . Interestingly, _TmTrmI, which has an optimum growth temperature of 80°C (the limit temperature to distinguish a thermophilic and a hyperthermophilic organism), has the highest number both of salt bridges and van der Waals contacts at the A/B dimer interface (Table 3). Therefore, _TmTrmI displays the highest buried surface areas for the A/B and A/C dimers and seems to employ both strategies used by thermophilic and hyperthermophilic TrmI proteins to achieve thermostability.

In addition to enhanced hydrophobic interactions at the interfaces, the archaeal _PaTrmI further increases its thermostability through the use of intersubunit disulfide bridges 21 27 . In _PaTrmI, the subunits are more tightly bound than in TrmIs from thermophilic bacteria, as shown by the value of 41 kcal/mole for the free energy difference between the dissociated and associated states (Table 3), despite the fact that the buried surface areas of the dimers and tetramer are less extensive than anticipated for a hyperthermophilic organism. This increased stability of the _PaTrmI tetramer compared to that of the other TrmI proteins results from the presence of four intermolecular disulfide bonds between Cys196 and Cys233 from different subunits (Figure 4E). Disulfide bonds are extremely rare in intracellular proteins from mesophilic organisms, due to the reductive nature of the cytoplasm 45 . In contrast, their presence in several intracellular thermophilic proteins has been shown to increase the stability of the proteins from these organisms at extreme temperatures 46 47 48 49 . The presence of inter-subunit disulfide bonds in _PaTrmI is consistent with the presence, in this organism, of a specific disulfide oxidoreductase protein, which is usually involved in the formation of intramolecular disulfide bonds within intracellular proteins from thermophilic organisms 50 . To determine whether the inter-subunit disulfide bridges are important for the stability and function of _PaTrmI, the single and double mutant proteins, in which Cys196 or/and Cys233 were replaced by serine, were produced and purified 21 . Whereas both single mutants migrated as a mixture of monomers and dimers on SDS-PAGE under non-reducing conditions, the double mutant migrated as a monomer. Gel filtration chromatography indicated that the single mutants formed high molecular weight aggregates and that the double mutant behaved predominantly as a dimer. Differential scanning calorimetry experiments indicated that the melting temperature of the double mutant is lowered by 16.5°C compared to that of the wild-type enzyme 27 . Finally, the double mutant was completely inactivated after preincubation at 85°C for 30 min. Altogether, these experiments indicated that the intersubunit disulfide bridges are essential for the thermostability of the tetramer of _PaTrmI.