1 INTRODUCTION
Protein-protein or protein-nucleic acid interactions drive a plethora of
biological processes. For the interaction to be biologically fruitful, a
protein must be capable of not only choosing its cognate partner from
the cellular soup but also be able to discriminate against non-cognate
partners present in its environment. A classic example is the case of
aminoacyl-tRNA synthetases (aaRSs), whose function is to aminoacylate or
charge its cognate tRNA and discriminate against all other non-cognate
tRNA (1). It is now established that each tRNA type, corresponding to a
particular amino acid, possesses unique nucleotides called identity
determinants that allow its recognition by cognate aaRS and
anti-determinants that discriminate against noncognate aaRSs (2). An
added layer of recognition/discrimination is encountered in glutamyl
tRNA-synthetases (GluRS) in some bacteria whose genomes lack
glutaminyl-tRNA synthetase (GlnRS) (3,4).
GluRS is a class-I aminoacyl-tRNA synthetase that catalyzes the
glutamylation of tRNAGlu (3). In absence of GlnRS,
GluRS in these bacteria are non-discriminatory (ND) and glutamylates
both tRNAGlu and tRNAGln.
Glutamylation of tRNAGln produces the mismatched
product Glu-tRNAGln (5,6). The misacylated
Glu-tRNAGln is then further edited to the correct
product Gln-tRNAGln by the enzyme
glutamyl-tRNAGln amidotransferase (gatCAB) through a
transamidation pathway (7). All ancient versions of bacterial GluRS were
tRNAGln-non-discriminatory GluRS (ND-GluRS). During
the course of evolution, bacteria acquired GlnRS of an eukaryotic origin
through horizontal gene transfer (4,8,9). The newly acquired GlnRS was
evolutionarily selected in some bacteria, where the native ND-GluRS that
charged both tRNAGlu and tRNAGlnevolved into a tRNAGln-discriminatory (D-GluRS) that
charged only tRNAGlu. In addition to ND-GluRS and
D-GluRS, a large number of proteobacterial species (for
example Helicobacter pylori (10), Acidithiobacillus
ferrooxidans (11)) possess two copies of GluRS (GluRS1 and GluRS2) with
distinct tRNAGlx-specificities (GluRS1: mostly
tRNAGlu-specific; GluRS2:
tRNAGln-specific), suggesting a gene duplication of
the primordial version of GluRS.
The structural features of Thermus thermophilus GluRS
(Tth -GluRS) have been extensively studied (12,13). GluRS is
composed of two structural domains. The N-terminal domain, also known as
the catalytic domain, contains the L-glu and ATP binding sites, along
with the class-I specific signature motifs HIGH and KMSKS in the ATP
binding site along with. This domain also encompasses a sparse binding
interface with the acceptor stem and the D-helix nucleotides of
tRNAGlu. The other domain (C-terminal) interacts with
the anticodon nucleotides of tRNAGlu and is aptly
known as the anticodon-binding domain.
Although the most important structural insights for bacterial GluRSs
came from Tth -GluRS, a non-proteobacterial GluRS, majority of
biochemical studies for the mechanistic understanding of GluRS:tRNA
interactions have been performed on GluRS/tRNAGlu in
the proteobacterium Escherichia coli (14–17). Mutational studies
performed on the E. coli GluRS (Eco -D-GluRS) and
tRNAGlu identified several ‘hot-spots’ (important
amino acids and nucleotides) for efficient glutamylation reaction (16).
For example, it has been shown that
tRNAGlu-specificity of GluRS in E. coli and
some other proteo-bacterial GluRSs arise due to subtle conformational
differences between tRNAGlu and
tRNAGln, originating at the D-helix (Figure 1A, Figure
S1) — augmented (presence of base-triple interaction 13:22:46 and the
absence of nucleotide 47) in tRNAGlu versus
non-augmented (absence of base-triple interaction 13:22:46 and the
presence of nucleotide 47) in tRNAGln (8,18).
Interestingly, this is not true in case of non-proteobacterium T.
thermophilus , which, despite possessing a D-GluRS, displays augmented
D-helix in both tRNAGlu and in
tRNAGln. A zinc ion present in the catalytic domain
of Eco -GluRS was shown to play a critical role glutamylation
reaction (19), although many bacterial GluRSs do not contain a bound
Zn2+, including Tth -GluRS, implying the
irregular occurrence of the zinc atom (20). In another study, when an
arginine residue (R266) in the tRNA-binding interface
of Eco -GluRS was mutated to leucine, glutamylation efficiency of
the protein was drastically reduced (more than 2500 fold) (16).
Interestingly, sequence analysis of bacterial GluRSs revealed that this
arginine residue is exclusively present only in proteobacterial GluRS.
In other words, hot-spot signatures for GluRS-tRNAGlxinteraction are not homogeneously conserved in all bacterial GluRSs (4),
indicating that factors responsible for the interactions are
phylum-specific and not universal. Therefore, to completely understand
sequence and structural signatures that drive specificity of
tRNAGlx-glutamylation reaction in bacteria, the
sequence and structural signatures must be analyzed in a phylum-specific
manner and the structural insights obtained from Tth -GluRS may
not be enough to understand the results of the functional studies
performed on Eco -GluRS (4).
From the perspective of GluRS evolution, the proteobacterial domain in
bacteria had experienced multiple sets of events (horizontal gene
transfer, gene duplication and perhaps domain fusion) while adapting a
tRNAGlu-specific aminoacylation pathway (4). In order
to achieve tRNAGlu-specificity, the evolving GluRS
must have undergone major adaptations, especially in residues lining its
tRNA-binding interface. To understand the rationale behind such
adaptations in proteobacterial GluRS, structural insights from crystal
structures of proteobacterial GluRS are needed. Further, since a number
of mutational studies related to tRNAGlx-specificity
have been performed on Eco -GluRS, it is important to analyzeEco -GluRS structure to elucidate clues behind
tRNAGlx-specificity. Here, we report the crystal
structure of Eco -GluRS. From structural and sequence analysis of
a large number of bacterial GluRS, both proteo- and non-proteobacterial,
we identify structural features present in proteobacterial GluRSs that
are required for tRNAGlu-specificity.
Our results highlight that the specific structural feature responsible
for the tRNAGlx-specificity of bacterial GluRSs is the
presence or the absence of an unique “towards tRNA” or “away from
tRNA” conformation adopted by a short loop connecting Helix 8 and Helix
9. The “towards tRNA” conformation is compatible with the augmented
D-helix but incompatible with the non-augmented D-helix, while the
“away from tRNA” conformation is compatible with both.