The nucleotide residue A1408 in 16S rRNA of the 30S ribosomal subunit is a crucial ‘hot spot’ of aminoglycoside resistance. The universally conserved A1408 has a critical role in binding of aminoglycoside antibiotics to the A site of 16S rRNA. Structural studies have shown that the hydroxyl group on sugar ring I of 4,6- and 4,5-disubstituted 2-deoxystreptamines forms hydrogen bonds with the N1 and N6 atoms of A1408 (38–40
). The N1 methylation of A1408 by NpmA apparently disrupts the interaction of the 16S rRNA with the drug, as indicated in footprinting experiments (7
) thereby conferring resistance. The recent detection of the Kam family member NpmA in a clinical isolate (7
) indicates an imminent threat to the utility of aminoglycosides as antibacterial drugs and warrants attention. The structure of NpmA and its complex with the cofactors and substrate holds key to understanding its mode of action and potentially to develop specific inhibitors of the A1408 methylation activity that could reinstate the activity of aminoglycoside antibiotics.
Based on structural analyses we identified residues in NpmA potentially important for cofactor-binding and catalysis and carried out site-directed mutagenesis to obtain protein variants with alanine substitutions in the corresponding positions. As predicted, the ITC experiments revealed that NpmA variants with cofactor-binding residues substituted by Ala have completely lost or exhibited significantly reduced ability to bind AdoMet or AdoHcy (, Supplementary Figure S5
and ). We were, however, greatly surprised by the fact that the variant D55A retained the ability to carry out m1
A1408 methylation in vitro
, as well as to render the host-cell resistant to aminoglycosides, because substitutions of this conserved residue in other RNA methyltransferases, e.g. D55 in KamB (37
) or D156 in Sgm (41
) has lead to drastic reduction of these enzymes’ activity both in vivo
and in vitro
. We speculate that while the D55A variant of NpmA cannot form a binary complex with the cofactor, it may bind AdoMet and methylate the target when it is already engaged in interactions with the substrate. For some related methyltransferases, e.g. Dam enzyme from phage T4 phage, it has been demonstrated that the binding of the methyl group donor or the nucleic acid substrate reciprocally improve the affinity for each other in the ternary complex (42
). This suggests that NpmA may also exhibit significantly increased affinity for AdoMet when pre-bound to the 30S subunit. Other enzymes, however, e.g. Dam from E. coli
(which is very closely related to T4 Dam), exhibit similar affinity for AdoMet in the presence or in the absence of the substrate (43
). Apparently, large variations of parameters concerning the binding of the ligands and their mutual interactions are not uncommon among related methyltransferases. We speculate that in NpmA, binding of the 30S substrate increases the affinity for AdoMet to such extent that it can rescue the ligand binding ability of the D55A variant, while the similar effect has not been observed for KamB. Our results show that the relative importance of ‘the same’ residues in closely related proteins may vary. This study highlights the importance of comparative biochemical and functional analyses of proteins that belong to the same family and suggests that structural and functional characterization of just single members of each protein family (as in the structural genomics approach) may be insufficient to understand sequence-structure–function relationships in proteins. The finding that the side chain of D55 residue is dispensable for the in vivo
activity of NpmA, and hence that mutations altering that residue may appear in the nature, has to be taken into consideration in analyses aiming at structure-based design of inhibitors.
NpmA binding to the 30S subunit
NpmA was found to successfully methylate only fully assembled 30S subunits, while the entire 70S ribosome or naked 16S rRNA could not be methylated (7
). This reactivity towards 30S subunits is analogous to aminoglycoside resistance MTases from the Arm family, ArmA and Sgm, which methylate G1405 (44
). Here we present the analysis of interactions between a member of the Kam family and small ribosomal subunit as its substrate. Using DEPC and CMCT chemical probing and subsequent primer extension analysis we were able to identify helices 24 and 42 with associated loop as primary regions that interact with NpmA. In addition, two short sections of 16S rRNA 5′ from the target nucleotide residue A1408 showed increased non-specific reactivity to chemicals when isolated from 30S subunits incubated with the NpmA. This might be the result of a local structural change in the rRNA caused by NpmA binding, suggesting that NpmA-30S interaction could be associated with the top of helices 44 and 28 as well.
We have recently reported a similar footprinting and docking analysis for Sgm MTase from the Arm family that modifies G1405 (21
). While G1405 and A1408 are positioned very closely in rRNA, suggesting a similar mode of binding for Sgm and NpmA, we could not clearly define the region(s) of 16S rRNA that form distinctive interactions with Sgm. Instead, protected nucleotides were scattered over a wide area, together with a large number of nucleotides with the activity enhanced upon Sgm binding, indicating complex structural changes of a rather global character that enable the otherwise buried G1405 base to become available for methylation by Sgm. Unlike Sgm, NpmA binding to the 30S subunit does not lead to global structural rearrangements; instead we observe only local changes. Remarkably, the target base A1408 was not found to be protected upon NpmA binding, which suggests that it may be flipped-out and more solvent accessible in the complex with the enzyme than in the 30S subunit before modification. Its binding may be stabilized in the NpmA active site by residues W107 and W197, found here to be essential for the methylation to occur in vivo
. The conformation of W107 side chain is different in complexes with AdoMet and AdoHcy, which suggests that limited conformational changes upon binding may occur also on the protein side. In the crystal structures of NpmA, the side-chains of these Trp residues are too close to each other to enable binding of the target base, but a simple change to alternative rotamers allows the protein to form a binding pocket to accommodate the adenine moiety (data not shown). The necessity of minor conformational adjustments has to be taken into consideration in the future analyses aiming at structure-based design of inhibitors.
The results of footprinting experiments with Sgm and NpmA are in perfect agreement with the docking studies. In our previous work (21
) we found that the Sgm structure cannot be docked to its target G1405 without very severe steric clashes with the 30S subunit, which implies that significant conformational changes upon its binding must occur. On the other hand, NpmA can be easily docked to its target A1408, to form a relatively close-fitting complex with the 30S subunit. Therefore we postulate that Sgm and NpmA use very different mechanisms to access their target bases.
Regions of 16S rRNA that we found to be involved in interactions with NpmA come in close proximity and form functional sites in fully assembled 30S subunits, including the decoding site and site of association with 50S subunit, which clearly explains why NpmA cannot act on 70S ribosomes. In addition, regions identified as NpmA binding sites partly overlap with the KsgA-30S interaction sites identified by directed and solution hydroxyl radical footprinting that include helices 2, 11, 24, 27, 28, 44 and 45 and loop 790 (46
). KsgA is a housekeeping dimethyltransferase that modifies A1518 and A1519 (both to m6,6
A) in helix 45 of 16S rRNA. It acts as a regulator of ribosome biogenesis and binds to immature 30S subunits in a translationally inactive conformation (47
). NpmA is known to act on mature 30S subunits, but it has not been determined whether it can methylate any of the reconstitution intermediates (RIs) in the 30S subunit assembly. The first intermediate, RI, lacks tertiary ribosomal proteins S2, S3, S10, S14 and S21 in central and 3′-domain and is not competent to form a small ribosomal subunit. rRNA in RI undergoes an extensive conformational rearrangement to form RI*, which can then bind the remaining five ribosomal proteins and form 30S subunit (48
). Holmes and Culver have performed an extensive analysis of conformational changes in 16S rRNA during the assembly of small ribosomal subunit (49
) and mapped structural differences between the assembly intermediates RI and RI* (50
). The majority of conformational changes related to the central and 3′-domain of the 30S subunit occur during transition from RI to RI*. These changes include functional sites on 30S, where NpmA binds and recognizes its target, hence we may propose that the earliest stage that supports the NpmA action could be a nearly assembled RI* of the 30S subunit.
Relationship of NpmA to other MTases; insight from structural analyses
The availability of a high-resolution NpmA structure allowed us to establish its relationship to other MTase families. NpmA is a member of the RFM superfamily and exhibits a class I MTase fold with the exceptional topological rearrangement involving the N-terminal helix. The arrangement of the N-terminal helix in NpmA is similar to those found in the structure of tRNA:m7
G56 MTase TrmB, a representative of a protein family that appears to be the closest homolog of Kam-family MTases (Supplementary Figure S6
). It is very intriguing that an RNA:m1
A MTase (NpmA) appears to be more closely related to an RNA:m7
G MTase (TrmB) than to other m1
A MTases, for which structures have been determined, including tRNA:m1
A58 MTase TrmI (51
) and tRNA:m1
A22 MTase TrmK (52
). NpmA is also structurally distinct from Arm/RmtB/Sgm enzymes that form m7
G1405 in 16S rRNA (Supplementary Figure S6
). G1405 is very close to A1408, the site of methylation of NpmA. While NpmA/Kam and Arm/RmtB/Sgm methyltransferases belong to the same RFM superfamily and methylate bases that are very close to each other in the 30S subunit leading to a similar phenotypic effect, i.e. resistance to aminoglycoside antibiotics, their catalytic domains are not very closely related and presumed substrate-binding domains are completely different.
Based on structural superposition we generated a multiple sequence alignment involving all members of the Kam family (including NpmA studied in this work), as well as individual representatives of m1
A MTase families TrmI and TrmK, and m7
G MTases TrmB and Sgm (Supplementary Figure S4
). Motifs I and II involved in the binding of the common cofactor AdoMet exhibit residues that are universally conserved between these enzymes (in particular carboxylates involved in coordination of the methionine and ribose moieties). On the other hand, motifs IV, VI, and VIII that commonly harbor residues involved in binding of the target base and catalysis are completely different. In the active sites, traces of similarity are observed only between NpmA and TrmB enzymes: Y193 is homologous and most likely functionally equivalent to the base-binding residue W197 of NpmA. Both proteins also share a common Phe residue (F116 in TrmB and F105 in NpmA), which stacks face to edge with the above-mentioned base-binding aromatic residue and thereby stabilizes it. Other than that, however, the active sites of TrmB and NpmA are different, consistent with the activity on a different substrate: the N7 atom of guanosine versus the N1 atom of adenosine.
The absence of common residues in motifs responsible for catalysis suggest that m1
A MTases acting on different RNA targets have non-homologous active sites and most likely developed independently from different ancestors within the RFM superfamily. In particular, our analysis suggests that the Kam family evolved from the TrmB/Trm8 family of m7
G MTases. This is in agreement with the results of our phylogenetic analysis of the entire RFM superfamily, in which the Kam, TrmI and TrmK families of m1
A MTases do not form a single branch on the tree and most likely have evolved independently (J.M.B. unpublished data). This situation resembles the polyphyletic evolution of m7
G MTases, whose five families (Arm/Sgm, TrmB/Trm8, RsmG, Abd1/Ecm1 and Bud23) are predicted to be independent evolutionary inventions with the same fold but different active sites (21
). Likewise, 2′-O
-ribose MTases include several families of enzymes with different specificities (e.g. RrmJ-like, HEN1 and Trm13) that exhibit different active sites anchored to the common RFM fold (54
) or even to a different fold (55
). Recently, a family of RNA MTases of the SPOUT fold have been characterized, in which some members exhibit m1
G specificity (56
), while others form m1
A but not m1
G or exhibit dual specificity and generate m1
A or m1
). This variety of protein families and protein folds with different active sites able to carry out RNA:m1
A methylation suggests that this type of reaction is probably relatively easy for an enzyme to evolve (e.g. from an MTase of a different specificity). On the other hand, the uniqueness of the adenosine-binding site of MTases from the Kam family suggests that they may be good targets for the development of Kam-specific inhibitors. The structural analyses presented in this work provide a stepping stone for structure-based virtual screening to find candidates for such compounds.