Mol. Cells 2020; 43(4): 350-359
Published online February 24, 2020
https://doi.org/10.14348/molcells.2020.2287
© The Korean Society for Molecular and Cellular Biology
Correspondence to : yunje@postech.ac.kr
Pathogenic aminoacyl-tRNA synthetases (ARSs) are attractive targets for anti-infective agents because their catalytic active sites are different from those of human ARSs. Mupirocin is a topical antibiotic that specifically inhibits bacterial isoleucy-ltRNA synthetase (IleRS), resulting in a block to protein synthesis. Previous studies on Thermus thermophilus IleRS indicated that mupirocin-resistance of eukaryotic IleRS is primarily due to differences in two amino acids, His581 and Leu583, in the active site. However, without a eukaryotic IleRS structure, the structural basis for mupirocin-resistance of eukaryotic IleRS remains elusive. Herein, we determined the crystal structure of Candida albicans IleRS complexed with Ile-AMP at 2.9 Å resolution. The largest difference between eukaryotic and prokaryotic IleRS enzymes is closure of the active site pocket by Phe55 in the HIGH loop; Arg410 in the CP core loop; and the second Lys in the KMSKR loop. The Ile-AMP product is lodged in a closed active site, which may restrict its release and thereby enhance catalytic efficiency. The compact active site also prevents the optimal positioning of the 9-hydroxynonanoic acid of mupirocin and plays a critical role in resistance of eukaryotic IleRS to anti-infective agents.
Keywords active site closure, aminoacyl-tRNA synthetases, anti-infective agents, crystal structure, mupirocin
Developing structure-based anti-infective drugs requires structural information on the active sites of validated target proteins (Kuntz, 1992). These targets must be conserved and essential for the survival of the target organisms, and the active sites must possess structural differences between eukaryotic and pathogenic molecules (Kwon et al., 2019; Yao and Fox, 2013). Aminoacyl-tRNA synthetases (ARSs) catalyze the addition of amino acids to their cognate tRNAs with high fidelity in the initial step of protein synthesis (Delarue, 1995). This canonical function of ARSs is essential and responsible for carrying accurate genetic information in every living organism.
ARSs are grouped into class 1 and class 2, depending on the core structure and oligomeric state (Ribas de Pouplana and Schimmel, 2001). Class 1 ARSs are further divided into three subclasses, 1a, 1b, and 1c, according to sequence homology. Isoleucyl-tRNA synthetase (IleRS) is a multi-domain enzyme with catalytic, editing (connective polypeptide 1 or CP1), and anticodon-binding domains. Together with leucyl-, valyl-, methionyl-, cysteinyl-, and arginyl-tRNA synthetase (LeuRS, ValRS, MetRS, CysRS, and ArgRS), IleRS belongs to class 1a ARSs, which are characterized by a Rossmann fold in the catalytic domain harboring conserved His-Ile-Gly-His (HIGH) and Lys-Met-Ser-Lys-Ser/Arg (KMSKS/R) motifs in the active site. The aminoacylation process requires a two step-reaction: activation of amino acids with ATP to form aminoacyl-AMP; and transfer of amino acids to their cognate tRNA (Antonellis and Green, 2008; Schimmel, 2018). IleRS possesses error-correction activity in the editing domain that enhances the accuracy of aminoacylation and maintains translation fidelity (Ling et al., 2009). In the double-sieve mechanism, larger amino acids are first filtered in the synthetic active site of the catalytic domain, and those of smaller size are removed in the editing active site of the editing domain (Fersht and Dingwall, 1979; Fukai et al., 2000). The misactivated substrate is believed to be hydrolyzed in the editing domain via shuttling of the 3’-acceptor stem of tRNA between the syntheticand editing active sites (Silvian et al., 1999).
Mupirocin (pseudomonic acid A) is a topical antibiotic used to treat infection by
Crystal structures of
In the present work, we determined the crystal structure of IleRS from the fungus
The gene encoding full-length fungal IleRS (residues 1-1088) was amplified by polymerase chain reaction (PCR) from genomic DNA of
For microscale thermophoresis (MST) experiments,
Crystals of
The structure of
MST assays were performed with a Monolith NT.115 instrument (NanoTemper Technologies, Germany) (Duhr and Braun, 2006; Seidel et al., 2013). Each titration curve con- tained 16 points prepared by serial dilutions of analytes and a constant concentration of the fluorescein-labeled ligand. To measure the binding affinity between mupirocin and purified human IleRS and
The atomic coordinate has been deposited at the Protein Data Bank, with an accession code 6LDK.
In both
To understand the basis for the mupirocin-resistance of eukaryotic IleRS, we attempted to determine the structure of eukaryotic IleRS. We initially crystallized full-length
We determined the crystal structure of the C-terminal truncated form of
The overall structure of
At present, the crystal structures of two bacterial IleRSs,
When the catalytic domains of IleRSs are superimposed, the editing domain of
The structures of the CP insertions and anticodon-binding domains are clearly different from those in bacterial IleRSs (Figs. 3C-3E). The CP cores of both
The crystal structure of
Phe55 encloses the backbone of Ile, further stabilizing ligand binding. The space between Trp576 and the terminal methyl of Ile is optimal, preventing unfavorable interactions with Leu or Val. The phosphate oxygens of Ile-AMP are surrounded by the HIGH loop and the CP core loop linking the β16 and β17 strands. The backbone amide of Phe55 and the side chain of His64 in the HIGH loop form H-bonds with the phosphate oxygens of Ile-AMP. Arg410 in the CP core loop interacts with the phosphate oxygen and covers the ribose group. The ribose ring of Ile-AMP is recognized by the β25 strand and the α19 helix from the second half of the catalytic domain. The O2’ atom of the ribose ring are stabilized by H-bonds with Asp571 (α19) and the backbone amide of Gly569 (β25), and the O3’ atom forms an H-bond with Glu568. The adenine base of Ile-AMP is surrounded by the three regions of the catalytic domain: His61 and His64 in the HIGH loop; the backbone of Gly600 to Val602 of theβ26 strand; and Met610 of the KMSKR loop that stabilizes the adenine ring via van der Waals interactions. Lys612 (the second Lys of the 609KMSKR motif) is directed toward to the HIGH loop, and engages in H-bonds with the main chain of Thr59 and the side chain of Thr57. These interactions restrict the KMSKR loop of
There are three notable differences in the synthetic active sites between
Among the class 1a ARSs, IleRS, LeuRS, and ValRS share particularly high levels of sequence identity and are therefore thought to have evolved from a common ancestor (Brown and Doolittle, 1995). The crystal structures of
The conformation of the KMSKS motif in
Although the catalytic domains are highly conserved among all bacterial and eukaryotic IleRSs, mupirocin selectively inhibits bacterial and archaeal IleRSs but not eukaryotic enzymes (Hughes and Mellows, 1980). The crystal structures of two bacterial IleRSs,
The structures of apoand mupirocin-bound
When we modeled mupirocin in
In summary, we determined the structure of fungal IleRS and analyzed structural differences between prokaryotic and eukaryotic IleRSs. We showed that three key features cause the active site to adopt a compact conformation and thereby restrict the release of the Ile-AMP reaction intermediate. Binding of tRNA at the catalytic and editing domains might rearrange the orientation of the editing domain, resulting in the opening the active site conformation to allow ligation of an aminoacyl group to the 3’ acceptor of tRNA (Supplementary Fig. S2). Tight binding of Ile-AMP at the compact active site would limit the release of Ile-ATP, and thus allowing eukaryotic IleRSs to use ATP energy more efficiently in aminoacylation and tRNA charging processes. In addition, the structural basis for mupirocin action presented herein provides new insights that could be used to develop improved anti-infective and anti-fungal drugs.
This work was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korea government (MEST, No. 2015R1A2A1A05001694, 2017M3A9F6029733, and NRF-2013M3A6A4044580), and BK21 program (Ministry of Education) to YC.
Data collection and refinement statistics
Data collection | |
Space group | P21 |
Cell dimensions | |
56.5, 137.6, 73.7 | |
α, β, γ (°) | 90, 106, 90 |
Resolution (Å) | 49.3-2.9 (2.95-2.9)a |
Measured reflections | 110,098 |
Unique reflections | 23,327 |
Completeness (%) | 97.8 (99.2) |
Average (I/σ) | 11.4 (1.3) |
0.15 (1.16) | |
CC*a | 0.997 (0.862) |
CC1/2a | 0.987 (0.591) |
Redundancy | 4.7 (4.9) |
Wilson B factor (Å2) | 52.3 |
Refinement statistics | |
Resolution (Å) | 49.35-2.9 |
17.4/23.9 | |
No. of atoms | |
Protein | 6692 |
Ile-AMP | 31 |
Water | 4 |
B-factors | |
Protein | 53.0 |
Ile-AMP | 47.3 |
Water | 44.4 |
R.m.s. deviations | |
Bond lengths (Å) | 0.010 |
Bond angles (°) | 1.137 |
Clash scorec | 11.0 |
Ramachandran plotc | |
Most favored (%) | 94 |
Allowed (%) | 5.7 |
Disallowed (%) | 0.3 |
Values in parentheses are for the highest shell.
aKarplus and Diederichs (2012).
bR = |Fobs–Fcalc|/Fobs, where Fobs = Fpi and Fcalc is the calculated protein structure factor from the atomic model (
cClash score and Ramachandran plot are calculated by Molprobity (Chen et al., 2010).
Mol. Cells 2020; 43(4): 350-359
Published online April 30, 2020 https://doi.org/10.14348/molcells.2020.2287
Copyright © The Korean Society for Molecular and Cellular Biology.
Scisung Chung1 , Sulhee Kim2
, Sung Ho Ryu1
, Kwang Yeon Hwang2
, and Yunje Cho1,*
1Department of Life Sciences, Pohang University of Science and Technology, Pohang 37673, Korea, 2Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea
Correspondence to:yunje@postech.ac.kr
Pathogenic aminoacyl-tRNA synthetases (ARSs) are attractive targets for anti-infective agents because their catalytic active sites are different from those of human ARSs. Mupirocin is a topical antibiotic that specifically inhibits bacterial isoleucy-ltRNA synthetase (IleRS), resulting in a block to protein synthesis. Previous studies on Thermus thermophilus IleRS indicated that mupirocin-resistance of eukaryotic IleRS is primarily due to differences in two amino acids, His581 and Leu583, in the active site. However, without a eukaryotic IleRS structure, the structural basis for mupirocin-resistance of eukaryotic IleRS remains elusive. Herein, we determined the crystal structure of Candida albicans IleRS complexed with Ile-AMP at 2.9 Å resolution. The largest difference between eukaryotic and prokaryotic IleRS enzymes is closure of the active site pocket by Phe55 in the HIGH loop; Arg410 in the CP core loop; and the second Lys in the KMSKR loop. The Ile-AMP product is lodged in a closed active site, which may restrict its release and thereby enhance catalytic efficiency. The compact active site also prevents the optimal positioning of the 9-hydroxynonanoic acid of mupirocin and plays a critical role in resistance of eukaryotic IleRS to anti-infective agents.
Keywords: active site closure, aminoacyl-tRNA synthetases, anti-infective agents, crystal structure, mupirocin
Developing structure-based anti-infective drugs requires structural information on the active sites of validated target proteins (Kuntz, 1992). These targets must be conserved and essential for the survival of the target organisms, and the active sites must possess structural differences between eukaryotic and pathogenic molecules (Kwon et al., 2019; Yao and Fox, 2013). Aminoacyl-tRNA synthetases (ARSs) catalyze the addition of amino acids to their cognate tRNAs with high fidelity in the initial step of protein synthesis (Delarue, 1995). This canonical function of ARSs is essential and responsible for carrying accurate genetic information in every living organism.
ARSs are grouped into class 1 and class 2, depending on the core structure and oligomeric state (Ribas de Pouplana and Schimmel, 2001). Class 1 ARSs are further divided into three subclasses, 1a, 1b, and 1c, according to sequence homology. Isoleucyl-tRNA synthetase (IleRS) is a multi-domain enzyme with catalytic, editing (connective polypeptide 1 or CP1), and anticodon-binding domains. Together with leucyl-, valyl-, methionyl-, cysteinyl-, and arginyl-tRNA synthetase (LeuRS, ValRS, MetRS, CysRS, and ArgRS), IleRS belongs to class 1a ARSs, which are characterized by a Rossmann fold in the catalytic domain harboring conserved His-Ile-Gly-His (HIGH) and Lys-Met-Ser-Lys-Ser/Arg (KMSKS/R) motifs in the active site. The aminoacylation process requires a two step-reaction: activation of amino acids with ATP to form aminoacyl-AMP; and transfer of amino acids to their cognate tRNA (Antonellis and Green, 2008; Schimmel, 2018). IleRS possesses error-correction activity in the editing domain that enhances the accuracy of aminoacylation and maintains translation fidelity (Ling et al., 2009). In the double-sieve mechanism, larger amino acids are first filtered in the synthetic active site of the catalytic domain, and those of smaller size are removed in the editing active site of the editing domain (Fersht and Dingwall, 1979; Fukai et al., 2000). The misactivated substrate is believed to be hydrolyzed in the editing domain via shuttling of the 3’-acceptor stem of tRNA between the syntheticand editing active sites (Silvian et al., 1999).
Mupirocin (pseudomonic acid A) is a topical antibiotic used to treat infection by
Crystal structures of
In the present work, we determined the crystal structure of IleRS from the fungus
The gene encoding full-length fungal IleRS (residues 1-1088) was amplified by polymerase chain reaction (PCR) from genomic DNA of
For microscale thermophoresis (MST) experiments,
Crystals of
The structure of
MST assays were performed with a Monolith NT.115 instrument (NanoTemper Technologies, Germany) (Duhr and Braun, 2006; Seidel et al., 2013). Each titration curve con- tained 16 points prepared by serial dilutions of analytes and a constant concentration of the fluorescein-labeled ligand. To measure the binding affinity between mupirocin and purified human IleRS and
The atomic coordinate has been deposited at the Protein Data Bank, with an accession code 6LDK.
In both
To understand the basis for the mupirocin-resistance of eukaryotic IleRS, we attempted to determine the structure of eukaryotic IleRS. We initially crystallized full-length
We determined the crystal structure of the C-terminal truncated form of
The overall structure of
At present, the crystal structures of two bacterial IleRSs,
When the catalytic domains of IleRSs are superimposed, the editing domain of
The structures of the CP insertions and anticodon-binding domains are clearly different from those in bacterial IleRSs (Figs. 3C-3E). The CP cores of both
The crystal structure of
Phe55 encloses the backbone of Ile, further stabilizing ligand binding. The space between Trp576 and the terminal methyl of Ile is optimal, preventing unfavorable interactions with Leu or Val. The phosphate oxygens of Ile-AMP are surrounded by the HIGH loop and the CP core loop linking the β16 and β17 strands. The backbone amide of Phe55 and the side chain of His64 in the HIGH loop form H-bonds with the phosphate oxygens of Ile-AMP. Arg410 in the CP core loop interacts with the phosphate oxygen and covers the ribose group. The ribose ring of Ile-AMP is recognized by the β25 strand and the α19 helix from the second half of the catalytic domain. The O2’ atom of the ribose ring are stabilized by H-bonds with Asp571 (α19) and the backbone amide of Gly569 (β25), and the O3’ atom forms an H-bond with Glu568. The adenine base of Ile-AMP is surrounded by the three regions of the catalytic domain: His61 and His64 in the HIGH loop; the backbone of Gly600 to Val602 of theβ26 strand; and Met610 of the KMSKR loop that stabilizes the adenine ring via van der Waals interactions. Lys612 (the second Lys of the 609KMSKR motif) is directed toward to the HIGH loop, and engages in H-bonds with the main chain of Thr59 and the side chain of Thr57. These interactions restrict the KMSKR loop of
There are three notable differences in the synthetic active sites between
Among the class 1a ARSs, IleRS, LeuRS, and ValRS share particularly high levels of sequence identity and are therefore thought to have evolved from a common ancestor (Brown and Doolittle, 1995). The crystal structures of
The conformation of the KMSKS motif in
Although the catalytic domains are highly conserved among all bacterial and eukaryotic IleRSs, mupirocin selectively inhibits bacterial and archaeal IleRSs but not eukaryotic enzymes (Hughes and Mellows, 1980). The crystal structures of two bacterial IleRSs,
The structures of apoand mupirocin-bound
When we modeled mupirocin in
In summary, we determined the structure of fungal IleRS and analyzed structural differences between prokaryotic and eukaryotic IleRSs. We showed that three key features cause the active site to adopt a compact conformation and thereby restrict the release of the Ile-AMP reaction intermediate. Binding of tRNA at the catalytic and editing domains might rearrange the orientation of the editing domain, resulting in the opening the active site conformation to allow ligation of an aminoacyl group to the 3’ acceptor of tRNA (Supplementary Fig. S2). Tight binding of Ile-AMP at the compact active site would limit the release of Ile-ATP, and thus allowing eukaryotic IleRSs to use ATP energy more efficiently in aminoacylation and tRNA charging processes. In addition, the structural basis for mupirocin action presented herein provides new insights that could be used to develop improved anti-infective and anti-fungal drugs.
This work was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korea government (MEST, No. 2015R1A2A1A05001694, 2017M3A9F6029733, and NRF-2013M3A6A4044580), and BK21 program (Ministry of Education) to YC.
. Data collection and refinement statistics.
Data collection | |
Space group | P21 |
Cell dimensions | |
56.5, 137.6, 73.7 | |
α, β, γ (°) | 90, 106, 90 |
Resolution (Å) | 49.3-2.9 (2.95-2.9)a |
Measured reflections | 110,098 |
Unique reflections | 23,327 |
Completeness (%) | 97.8 (99.2) |
Average (I/σ) | 11.4 (1.3) |
0.15 (1.16) | |
CC*a | 0.997 (0.862) |
CC1/2a | 0.987 (0.591) |
Redundancy | 4.7 (4.9) |
Wilson B factor (Å2) | 52.3 |
Refinement statistics | |
Resolution (Å) | 49.35-2.9 |
17.4/23.9 | |
No. of atoms | |
Protein | 6692 |
Ile-AMP | 31 |
Water | 4 |
B-factors | |
Protein | 53.0 |
Ile-AMP | 47.3 |
Water | 44.4 |
R.m.s. deviations | |
Bond lengths (Å) | 0.010 |
Bond angles (°) | 1.137 |
Clash scorec | 11.0 |
Ramachandran plotc | |
Most favored (%) | 94 |
Allowed (%) | 5.7 |
Disallowed (%) | 0.3 |
Values in parentheses are for the highest shell..
aKarplus and Diederichs (2012)..
bR = |Fobs–Fcalc|/Fobs, where Fobs = Fpi and Fcalc is the calculated protein structure factor from the atomic model (
cClash score and Ramachandran plot are calculated by Molprobity (Chen et al., 2010)..
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