TM0492-v-4 - Stanford University

Tm TrpRS_2g36

Lead Author: GW Han

PDB_id: 2g36

Acta F Section: Ligand in Xtal Structures 4/12/2020

SNTS_id: 354

Author List Approval by Core Leaders

Scott Lesley is _____OK _____not OK with Author List as submitted.

Adam Godzik is _____OK _____not OK with Author List as submitted.

Ashley Deacon is ____OK _____not OK with Author List as submitted.

Ian Wilson is _____OK _____not OK with Author List as submitted.

Target ID: Protein Accession: Local Accession:

Protein expression date: 2005-10-20

Structure deposited (PDBid): (2g36) on 2006-02-17

SNTS_id: 354

Acta F Section: Ligands in Crystal Structures that Aid in Functional Characterization

Notes/Observations:

1

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354

Crystal Structure of an Iron-Sulfur Cluster Containing Tryptophanyl-tRNA-synthetase from

Thermotoga maritima

Tentative Author List - Gye Won Han a,b

, Marc-André Elsliger a,b

, Xiang-Lei Yang b

, Yee Ting Esther

Chong b

, Min Guo b

, Christopher L. Rife a,c

(qc), QingPing Xu a,c

(data collection & processing),

Daniel McMullan d

, Sri Krishna Subramanian a,e,f

, Lukasz Jaroszewski a,e,f

(mr), Sanjay Agarwalla d

,

Tom Clayton d

(protein prep &crystallization), Dana Weeks a,e,f

(bic), Eric Hampton d

(dsc), Scott

Brittain d (mass spec), Mitch Miller a,c , Ashley Deacon a,c , Scott Lesley a,d , Adam Godzik a,e,f , Paul

Schimmel b and Ian Wilson a,b * a Joint Center for Structural Genomics (JCSG) b The Scripps Research Institute, La Jolla, California c

Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California d

Genomics Institute of the Novartis Research Foundation, San Diego, California e

Burnham Institute for Medical Research, La Jolla, California f

Center for Research in Biological Systems, University of California, San Diego,

La Jolla, California

Grant sponsor: National Institutes of Health, Protein Structure Initiative: Grant numbers: P50

GM62411, U54 GM074898.

*Fax number: 858-784-2980. Correspondence e-mail: wilson@scripps.edu

Keywords: TM0492, Tryptophan-tRNA ligase (TrpRS), Tryptophanyl tRNA synthetase class I, iron-sulfur cluster, Structural genomics

PDB Reference: Tryptophanyl-tRNA-synthetase from T. maritima , 2g36, 2g36.cif

2

Tm TrpRS_2g36


PDB_id: 2g36

Synopsis


SNTS_id: 354

For the first time, tryptophan tRNA-synthetase containing iron-sulfur cluster is reported.

The crystal structure of tryptophanyl-tRNA-synthetase from Thermotoga maritima reveals that iron-sulfur cluster is bound in the tRNA anti-codon binding region with four cysteine motif [C-x22-C-x6-Cx2-C]. This motif is conserved among anaerobic organisms from proteobacterial and archaeal groups.

A novel aminoacyl tRNA-synthetase containing an iron-sulfur cluster that efficiently charges tRNA with tryptophan is found in Thermotoga maritima. The crystal structure of Tm TrpRS [Tryptophanyl tRNA-synthetase (TrpRS; E.C 6.1.1.2)] reveals a [4Fe-4S] cluster bound to the C-terminal tRNA anti-codon binding (TAB) domain, and a L-tryptophan located in the active site. In the Thermotoga maritima genome, the [4Fe-4S] cluster binding motif [C-x22-C-x6-C-x2-C] is found only in the

TrpRS gene and not in any other Aminoacyl tRNA-synthetases (AARSs) genes. It has been known that MiaB tRNA modifying enzyme in Thermotoga maritima also contains iron-sulfur cluster which is essential for activity of the enzyme. This could be a hint that Tm TrpRS could have a role on recognition of modified tRNA.

3

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354

1. Introduction.

Aminoacyl tRNA-synthetases (AARSs) covalently append amino acids to their cognate tRNA’s.

This reaction proceeds in two steps. The first involves activation of the amino acid by ATP to form aminoacyl-adenylate which in the subsequent step reacts with its corresponding tRNA to form the aminoacyl-tRNA. Most organisms contain individual tRNA-synthetases specific for each of the twenty standard amino acids. Based on similarities in their sequences and structures, AARSs are grouped into two classes (class I and II) (Eriani et al.

, 1990)} each containing 10 members. Class I

AARSs contain Rossmann nucleotide fold, and two highly conserved sequence motifs “HIGH” and

“KMSKS” that are critical for function, while Class II AARSs containing anti-parallel β-sheet fold flanked by α-helices is characterized by three distinct sequence motif 1, 2 and 3.

Tryptophanyl tRNA-synthetase (TrpRS; E.C 6.1.1.2) which belongs to the class I AARS catalizes tryptophan activation by ATP and its subsequent aminoacylation to Trp-tRNA. The tRNA of all organisms contains a number of modified nucleotides (Winkler, 1998). The tRNA modification has been linked to regulatory mechanisms such as response to environmental stress where it serves to enhance fidelity of mRNA translation by preventing wobble on the 3’-side of the anti-codon as a consequence of reduced flexibility. One example of modified nucleoside is that 2thiocytidine in tRNA is found from E. Coli and Salmonella enterica serovar Typhimurium

(Jäger

et al.

, 2004). It has been shown that Thermotoga maritima MiaB tRNA-modifying enzyme ( Tm MiaB) involved in the posttranscriptional thiolation and methylation of tRNA, and contains an iron-sulfur cluster wih [C-x3-C-x2-C] binding motif (Pierrel et al.

, 2003).

Here we report, a novel Tm TrpRS that contains an [4Fe-4S] cluster bound to tRNA anticodon binding (TAB) domain, and a L-tryptophan located in the active site, from Thermotoga maritime.

The structure was determined using the semi-automated, high-throughput pipeline of the

4

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354

Joint Center for Structural Genomics (JCSG) (Lesley et al.

, 2002), as part of the National Institute of General Medical Sciences' Protein Structure Initiative (PSI).

2. Materials and Methods

2.1. Protein production and crystallization

Tm TrpRS (GenBank: AAD35577.1, GI:4981003; Swiss-Prot: Q9WYW2) was amplified by polymerase chain reaction (PCR) from Thermotoga maritima MSB8 genomic DNA using PfuTurbo

(Stratagene) and primer (forward primer: 5’-ctgtacttccagggc /TARGET_SEQUENCE/ -3’, reverse primer: 5’-aattaagtcgcgtta

/TARGET_SEQUENCE/ -3’, target sequence in upper case) corresponding to the predicted 5' and 3' ends. The PCR product was cloned into plasmid pMH2T7, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino terminus of the full-length protein. The cloning junctions were confirmed by DNA sequencing. Protein expression was performed in a modified Terrific Broth using the Escherichia coli methionine auxotrophic strain DL41. At the end of fermentation, lysozyme was added to the culture to a final concentration of 250 µg/mL, and the cells were harvested. After one freeze/thaw cycle, the cells were sonicated in Lysis Buffer [50 m M Tris pH 7.9, 50 m M NaCl, 10 m M imidazole, 1 m M Tris(2carboxyethyl)phosphine hydrochloride (TCEP)], and the lysate was clarified by centrifugation at

32,500 x g for 30 minutes. The soluble fraction was applied to nickel-chelating resin (GE

Healthcare) pre-equilibrated with Lysis Buffer, the resin was washed with Wash Buffer [50 m M

Tris pH 7.9, 300 m M NaCl, 40 m M imidazole, 10% (v/v) glycerol, 1 m M TCEP], and the protein was eluted with Elution Buffer [20 m M Tris pH 7.9, 300 m M imidazole, 10% (v/v) glycerol, 1 m M

TCEP]. The eluate was diluted ten-fold with Buffer Q [20 m M Tris pH 7.9, 5% (v/v) glycerol, 0.5 m M TCEP] containing 50 m M NaCl and loaded onto a RESOURCE Q column (GE Healthcare)

5

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354 pre-equilibrated with the same buffer. The protein was eluted with a linear gradient of 50 to 500 m M NaCl in Buffer Q, buffer exchanged with Crystallization Buffer [20 m M Tris pH 7.9, 150 m M

NaCl, 0.5 m M TCEP], and concentrated for crystallization assays to 18 mg/mL by centrifugal ultrafiltration (Millipore). Tm TrpRS was crystallized by mixing 200 nL protein with 200 nL crystallization solution and with a 50 μL reservoir volume using the nanodroplet vapor diffusion method 1 with standard JCSG crystallization protocols 2 . The crystallization reagent contained

12.5% (w/v) polyethylene glycol 3000, 0.25 M MgCl

2

, and 0.1 M cacodylate pH 6.5. A /shape of xtal/ crystal of approximate size /dimension/ μm x /dimension/ μ, x /dimension/ μm was harvested after 18 days at 277 K for data collection . Glycerol was added as a cryoprotectant to a final concentration of 10% (v/v). The data were indexed in orthorhombic space group C222

1

( Table 1 ).

The molecular weight and oligomeric state of Tm TrpRS were determined using a 1 cm x 30 cm

Superdex 200 column (GE Healthcare) in combination with static light scattering (Wyatt

Technology). The mobile phase consisted of 20 m M Tris pH 8.0, 150 m M NaCl, and 0.02% (w/v) sodium azide.

2.2. Data collection, structure solution and refinement

Native diffraction data were collected at the Advanced Light Source (ALS, Berkeley, USA).

The datasets were collected at 100K using an ADSC CCD detector. Data were integrated and reduced using XDS and then scaled with the program XSCALE (Kabsch, 1993). The structure was determined with the JCSG molecular replacement pipeline using TrpRS-R2 from Deinococcus radiodurans (PDB: 1yi8, sequence identity of 43%) as a search model. Model completion was performed with COOT (Emsley & Cowtan, 2004) and O (Jones et al.

, 1991) and TLS refinement was performed using REFMAC 5.2

(Winn et al.

, 2003) with three TLS groups (group 1: residues

6

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354

0-112, group 2: residues 120-196, group 3: residues 197-400) per chain. Residues 113-119 were disordered and not visible in the electron density maps.

2.3 Validation and deposition

The quality of the crystal structure was analyzed using the JCSG Quality Control server

( http://smb.slac.stanford.edu/jcsg/QC ). This server processes the coordinates and data through a variety of validation tools including AutoDepInputTool (Yang et al.

, 2004), MolProbity (Lovell et al.

, 2003), WHATIF 5.0

(Vriend, 1990), RESOLVE (Terwilliger, 2003), MOLEMAN2 (Kleywegt,

2000) as well as several in-house scripts, and summarizes the results. Protein quaternary structure analysis used the PQS server (Henrick & Thornton, 1998). The supplementary Figure 2 was adapted from an analysis using ClustalW (Larkin et al.

, 2007) and all others were prepared with

PyMOL (DeLano Scientific). Atomic coordinates and experimental structure factors for Tm TrpRS at 2.50 Å resolution have been deposited in the PDB ( http://www.PDB.org

) and are accessible under the code 2g36.

2.4. Pyrophosphate release assays

2.4.1. Aminoacylation Assay

Aminoacylation assay were performed with 50 m M HEPES (pH 7.5), 20 m M KCl, 10 m M

MgCl

2

, 4 m M ATP, 1



M [

3

H]-L-Trp, 19



M L-Trp, 2 m M DTT and 160



M bulk E. coli tRNA (or bulk yeast tRNA for the human enzyme). The aminoacylation reactions were initiated by the addition of 100 n M enzyme pre-incubated at either 37

˚

C or 60

˚

C. Samples were collected at various time points and quenched into a PVDF Multiscreen filter plate containing 100 m M EDTA,

300 m M sodium acetate (pH 3.0) and 0.5 mg/ml DNA as a carrier. After all the time points were

7

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354 collected, trichloroacetic acid was added to each well at a 10% final concentration to precipitate the tRNA. The plate then was vacuum dried, and washed 4 times with cold wash solution (5% trichloroacetic acid and 100 m M cold L-Trp for reducing background radioactivity from free [

3

H]-

L-Trp), and once with 95% ethanol before scintillation counting.

2.4.2. ATP-PPi exchange Assay

PPi exchange reactions were performed in 100 m M HEPES (pH 7.5), 20 m M KCl, 10 m M

MgCl

2

, 2 m M ATP, 2 m M sodium PPi, [

32

P]-sodium PPi, 2 m M L-Trp, and 5 m M



mercaptoethanol. Reactions were initiated by the addition of 1



M pre-incubated enzyme at either

37

˚

C or 60

˚

C. At each time point, samples were quenched into a PVDF Multiscreen filter plate containing 4% charcoal, 1 M HCl, 200 m M sodium PPi. The charcoal was collected and washed 4 times with 1 M HCl and 200 m M sodium PPi prior to scintillation counting.

2.5. Comparison with other tRNA-synthetases

The PSI-BLAST program was used to seach for homologs of Tm TrpRS protein in the

NCBI nonredundant (nr) protein sequence database. An iterative PSI-BLAST search was performed for 20 rounds of 3 iterations each, using as the initial query the Tryptophanyl-tRNA protein

Tm TrpRS sequence from Thermotoga maritima (gi|4981003). The resulting list of homolog sequences was then queried for the presence of pattern Cys-x6-Cys-x2-Cys from the [4Fe-4S] cluster binding motif. False-positive hits that contain the Cys pattern at a location other than the AB region were discarded, resulting in a total of 85 sequences that contained the cluster-binding motif.

In addition a PSI-BLAST filtered searched using the C-x6-C-x2-C pattern was performed for one iteration using the sequence of Tm TrpRS as a query. An additional 22 unique sequences that were

8

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354 not identified by the previous method were found. Moreover, to ensure we had exhaustively queried all proteins annotated as Tryptophan synthetases that contained the [4Fe-4S] cluster binding motif, a text search was performed where the nr database was mined for all annotations containing

“Tryptophan-tRNA synthetase”, and variants thereof. The resulting sequences were then searched for the aforementioned motif of interest. An additional 104 sequences were found using this last approach that were not identified in the previous methods. A taxonomy distribution of all sequences from the search methods are shown in Supplementary Figure 3 .

Further analysis was conducted to determine how many organisms existed that contained only one copy of a TrpRS, with the motif, and how many contained two copies of a TrpRS, where one contained the motif and the other does not. For example, Dienoccoccus radiodurans contains two TrpRS’s where only one contains the cysteine motif. Alignment of the resulting sequences is shown in Supplementary Figure 4.

Only one representative sequence from clustering at 50% is shown.

3. Results and Discussion

3.1. Overall structure description

The Tm TrpRS crystal structure, like other bacterial TrpRSs structures, contains two domains.

The N-terminal catalytic domain adopts a canonical Rossmann fold (RF; residues 1-181) with a central five-stranded parallel



-sheet (



1-

 strand order for the



-sheet is 32145), and a TAB domain adopts an all-helical fold (residues 187-328) at the C-terminus ( Fig. 1 ). The TAB domain is composed of five helices (H12-H16) that are packed as a bundle. A short hinge region (182-186) connects the RF and the TAB domains. The asymmetric unit contains one monomer of Tm TrpRS,

9

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354 which forms a crystallographic dimmer across the two-fold. The dimerization interface is centered around the connective polypeptide one (CP1) insertion of the RF.

As per the SCOP classification (Murzin et al.

, 1995), the catalytic domain of TrpRSs belong to the nucleotidylyl transferase superfamily. While all members of this superfamily retain the core elements of the Rossmann fold, substantial insertions to the catalytic domain, which confer novel functions, have been observed.

As expected, structure comparison by DALI (Holm & Sander, 1995) revealed extensive similarities to several class I AARSs with statistically significant Z-scores. Top DALI hits include

TrpRS-R2 from Deinococcus radiodurans ( Dr TrpRS-R2, (Buddha & Crane, 2005), PDB: 1yid,

Z=39.7), TrpRS from Bacillus stearothermophilus ( Bs TrpRS, PDB: 1d2r, Z=22.5), and human tyrosyl-tRNA synthetase ( Hs TyrRS; PDB: 1q11, Z=20.3), among many other class I AARSs. The structural similarity between Dr TrpRS-R2 and Tm TrpRS is particularly high, and a superimposition of the structure based on secondary structural elements from both the catalytic RF domain and the

C-terminal tRNA anti-codon binding (TAB) domain give an root mean squared difference (rmsd) of

1.7 Å ( Fig. 1 ).

The 2.5 Å resolution crystal structure of Tm TrpRS was determined by molecular replacement (MR) using Dr TrpRS-R2 ((Buddha & Crane, 2005), 1yid) as a search model

( Supplementary Figs 1-2 ). Remarkably, the structure of Tm TrpRS differs from other TrpRS by the appearance of an iron-sulfur cluster [4Fe-4S] bound at the C-terminal TAB domain in addition to a

L-tryptophan molecule bound to the active site.

3.2. [4Fe-4S] cluster binding site:

10

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354

The [4Fe-4S] cluster is chelated by the side-chains of residues Cys236, Cys259, Cys266, and Cys269 from the TAB domain arranged in a C-x22-C-x6-C-x2-C motif ( Figs 1 & 2 ). The presence of iron in the structure was confirmed by x-ray fluorescence scans. The presence of the

[4Fe-4S] cluster was based on electron density and geometry. Mass spectroscopy also corroborates the presence of the iron sulfur cluster. Although Tm TrpRS shares extensive sequence similarity at the TAB region with other TrpRSs, this motif is different from other TrpRS present in the PDB.

Homologs of Tm TrpRS that possess the [4Fe-4S] cluster binding motif are from anaerobic organisms from proteobacterial and archaeal groups ( Supplementary Fig. 3 ). However, no crystal structure of a TrpRS has yet been reported with a bound [4Fe-4S] cluster. Tm TrpRS is thus the first reported structure of a TrpRS that contains a [4Fe-4S] cluster.

Search of similar cysteine binding motif for an iron-sulfur cluster by SPASM (Kleywegt,

1999) provided the best hit with DNA glycosylase MutY (Guan et al.

, 1998) from Escherichia coli

( PDB: 1mun, rmsd = 0.88 Å). MutY belongs to the DNA repair enzyme superfamily and excises adenine from mispairs with 8-oxoguanine and guanine. The [4Fe-4S] cluster binding motif (C-x6-

C-x2-C-x5-C) with Cys192, Cys199, Cys202 and Cys208 in MutY is different from that of

Tm TrpRS.

Comparison of the Tm TrpRS structure to the recently determined human TrpRS-tRNA complex (PDB: 1r6t) (Yang et al.

, 2006) reveals that, although the sequence identity is very low

(19%), the structures are very similar with an root mean square difference (rmsd) of 1.9 Å for 238 superimposed C

α

atoms. A model of the Tm TrRS-tRNA complex based on the superimposed human complex structure indicates that Cyt34 of the tRNA molecule would interact with the iron of the

11

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354 cluster via Cys266 ( Figs 2a-c ). It would be possible that iorn cluster could play a functional role in selecting certain modified rRNAs. It has been shown that the MiaB containing iron-sulfur cluster in

Thermotoga Maritima is involved in the thiolation and methylation of tRNA of Adenine 37 which is close to the anticodon and iron-sulfur is essential for it. One interesting feature is that the helix



17

(D382-Q389) in the human TrpRS-tRNA complex is replaced with a loop in the Tm TrpRS structure

( Fig. 2c ). This helix in human TrpRS was suggested to be responsible for the anti-angiogenic activity associated with mini-TrpRS (Kise et al.

, 2004). Perhaps [4Fe-4S] cluster in Thermotoga maritima is providing a tether to hold this loop rigid .

The PSI-BLAST search for homologs of Tm TrpRS in the NCBI nonredundant protein sequence database shows that C-x(21-24)-C-x6-C-x2-C motif is mostly found in thermophiles or other extremophiles ( Supplementary Fig. 3 ). Interestingly this feature is conserved in some organisms that possess either only TrpRS gene or multiple genes encoding TrpRS. When it contains multiple TrpRS genes, only one copy contains the [4Fe-4S] cluster binding motif and the others

(doesn’t or don’t), which could indicate dual function. ( Supplementary Fig. 4 ).

3.3. ATP and Trp binding site :

TrpRS possesses ATP- and Trp-binding sites, which are located close to each other in the

RF domain. Although neither L-tryptophan nor ATP was added to the crystallization solution, the enzyme contains L-tryptophan molecules bound in both the subunits of the biologically relevant dimer ( Fig. 1 ). The dimer in solution was also confirmed by static light scattering. Typically, this enzyme, which is an obligate dimer, binds ATP and L-tryptophan in one subunit while the tRNA anti-codon region is recognized by the TAB domain from the other subunit. The orientation of L-

12

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354 tryptophan in Tm TrpRS is more similar to that seen in the human and Bs TrpRS than that of

Dr TrpRS-R2. TrpRS lacks an editing domain, consequently these enzymes are highly specific towards binding L-tryptophan. The L-tryptophan bound in the active site also supports it.

The ATP-binding site is located in a positively charged, solvent-exposed cleft present at the junction of the two domains ( Fig. 1 ). It is generated by residues 14 -17 (HIGH) and 193-197

(KMSKS) sequence motifs that are conserved across all members of the class I AARSs. In

Tm TrpRS, which represents an open conformation of the enzyme, the KMSKS motif is moved away from the ATP site and is not poised to bind ATP ( Fig. 1 ). Thus, as expected, no density for the ATP molecule was observed in the structure.

The TrpRS activity was confirmed for TM0492. In the presence of ATP, Tm TrpRS adenylated L-tryptophan and charged in vitro -transcribed E coli tRNA Trp with L-tryptophan demonstating that Tm TrpRS has thermophilic TrpRS activities ( Figs 3a-b).

Monitoring pyrophosphate (PPi) release in the presence of tryptophan and ATP indicated that it has increased charging activity at 60



C compared to 37



C. Furthermore, the PPi exchange activity was shown even when no Trp was added to the reaction (Fig. 3b). This result is consistent with the observation of the endogenously bound tryptophan molecule in the active site of the crystal structure.

In conclusion, for the first time, TrpRS containing [4Fe-4S] cluster is reported. The role of the iron-sulfur cluster in tRNA anti-codon recognition is still unclear. Furthermore, Tm TrpRS triple/quadruple mutants of cysteine residues are not expressed in soluble form. This observation suggests that the [4Fe-4S] cluster may play a role in structural fiedelity in the anticodon binding region.

13

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354

Furthermore, the cost for synthesizing [4Fe-4S] cluster suggests it plays a role in addition to lending stability. In biotin synthase, [4Fe-4S] cluster coordinates in radicals generations to catalyze the radical-mediated insertion of sulfur into dethiobiotin to form biotin (Berkovitch et al.

, 2004). As shown in Figure 2c, [4Fe-4S] cluster is in contact distance with t-RNA and could be crucial for the t-RNA recognition of Tm TrpRS. One hypothesis is that [4Fe-4S] cluster could be involved in the recognition of modified t-RNA.

Availability of more sequences and structures of the [4Fe-4S] cluster proteins might shed light on the evolutionary relationship of this enzyme. The information presented here, in combination with further biochemical and biophysical studies should yield valuable insights into the functional role of this enzyme.

4. Conclusions

We report the first crystal structure of the iron-sulfur cluster containing Tm TrpRS. Interestingly, this protein contains L-tryptophan in the active, and the iron-sulfur is located in the anti-codon binding region with four cysteine motif of C-x22-C-x6-C-x2-C. A model of the Tm TrpRS-tRNA complex based on the superimposed human complex structure indicates that Cyt34 of the tRNA molecule would interact with the iron of the cluster via Cys266. It would be possible that iorn cluster could play a functional role in selecting certain modified rRNAs .

The JCSG has developed The Open Protein Structure Annotation Network (TOPSAN), a wiki-based community project to create, share, and distribute information about protein structures determined at PSI centers. TOPSAN offers a combination of automatically generated, as well as comprehensive, expert-curated annotations, provided by JCSG personnel and members from the

14

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354 research community. Additional information about the protein described in this study is available at http://www.topsan.org/explore?PDBid=2g36 .

This work was done through the pipeline of the Joint Center for Structural Genomics

(JCSG). JCSG was supported by NIH Protein Structure Initiative grants P50-GM 62411 and U54

GM074898 from the National Institute of General Medical Sciences (www.nigms.nih.gov). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL) and the

Advanced Light Source (ALS).

The SSRL is a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural

Molecular Biology Program is supported by the Department of Energy, Office of Biological and

Environmental Research, and by the National Institutes of Health (National Center for Research

Resources, Biomedical Technology Program, and the National Institute of General Medical

Sciences) The ALS is supported by the Director, Office of Science, Office of Basic Energy

Sciences, Materials Sciences Division, of the U.S. Department of Energy under Contract No. DE-

AC03-76SF00098 at Lawrence Berkeley National Laboratory.

15

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354

Table 1 : Summary of crystal parameters, data collection, and refinement statistics for Tm TrpRS

(PDB id=2g36)

Space group

Unit cell parameters

Data Collection

Wavelength (Å)

Resolution range (Å)

Number of observations

C2221 a = 122.89 Å, b = 152.73 Å, c = 53.07 Å



0

1.0000

29.64 - 2.50

141072

Number of reflections

Completeness (%)

Mean I/



(I)

R merge

on I

Highest resolution shell (Å)

Model and refinement statistics

Resolution range (Å)

No. of reflections (total)

No. of reflections (test)

17636

99.3 (97.9) +

8.82 (2.1)+

0.08 (0.47)+

2.59 - 2.50

29.64 - 2.50 Data set used in refinement

Completeness (% total)

17623

894

99.3

Cutoff criteria

R cryst

R free

|F| > 0

0.19

0.26

Deviation from ideal geometry (rms):

Bond length

0.013 Å

Bond angle 1.60 o

Average isotropic B-value protein

Average isotropic B-value ligands

Average isotropic B-value water

43.9 Å 2

68.7 Å 2

41.3 Å 2

0.32 Å

ESU based on R value

Protein residues / atoms

Ligand / atoms

322 / 2,563

Solvent molecules

2 / 23

56

+

highest resolution shell

ESU = Estimated overall coordinate error (Tickle et al.

, 1998),(Collaborative Computational Project,

1994)

R merge

= Σ hkl

Σ i

R cryst

=



| |F

| I i

(hkl) - <I(hkl)> | / Σ hkl

Σ obs

|-|F calc

| | /



|F amplitudes, respectively.

 i

I i

(hkl) obs

| where F calc

and F obs

are the calculated and observed structure factor

R free

= as for R cryst

, but for 5.0% of the total reflections chosen at random and omitted from

§ refinement.

Typically, the number of unique reflections used in refinement is slightly lesser than the total number that were integrated and scaled. Reflections are excluded due to systematic absences, negative intensities and rounding errors in the resolution limits and cell parameters.

16

Tm TrpRS_2g36


PDB_id: 2g36

References


SNTS_id: 354

Berkovitch, F., Nicolet, Y., Wan, J. T., Jarrett, J. T. & Drennan, C. L. (2004). Science 303, 76-79.

Buddha, M. R. & Crane, B. R. (2005). J. Biol. Chem. 280, 31965-31973.

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17

Tm TrpRS_2g36


PDB_id: 2g36

Figure Legends


SNTS_id: 354

Figure 1 : Crystal structure of Tm TrpRS. (a) Most class I AARSs are functional as monomers with the exception of TrpRS, MetRS and TyrRSs, which are obligate homodimers. In Tm TrpRS the two relevant subunits pack against each other burying 2,365 Å

2 of hydrophobic surface. Analytical size exclusion chromatography in combination with static light scattering and crystallographic packing is consistent with a dimer as the biologically relevant form. (b) Tm TrpRS stays in a conformation similar to the “open” conformation of B. stearothermophilus TrpRS (BsTrpRS). In BsTrpRS, ligand binding induces “open” and “closed” conformation by hinge-like motion between the Rossmann fold domain and the anticodon recognition domain to close the ATP binding cleft. The 1



, 2Fo-Fc map of (c) iron-sulfur cluster shown in the cysteine motif [C-x22-C-x6-C-x2-C] with Cys236,

Cys259, Cys266 and Cys269 found in anticodon binding region of Tm TrpRS and (d) L-tryptophan found in the active site.

Figure 2 : Tm TrpRS-tRNA complex model. (a) Overall and (b) close up view of TrpRS-tRNA interactions at Cyt34 at the anticodon CCA region. The anticodon (CCA) were shown as stick model. Cyt34 of tRNA molecule interacts with the iron of the [4Fe-4S] cluster through Cys266. The tRNA model was taken from the human TrpRS-tRNA complex after human TrpRS-rRNA was superimposed on TrpRS Thermotoga maritima . TrpRS Thermotoga maritima is shown in grey-blue and light-green [4Fe-4S] cluster. (c) close up view of the loop conformation near the [4Fe-4S] cluster compared to the helix (Asp 382 – Asn 389) of the human TrpRS.

Figure 3 : Enzymatic activities of Tm TrpRS. (A) Aminoacylation activities assayed at 37

˚

C and 60

˚

C. Consistent with its thermophilic nature, Tm TrpRS has a more robust tRNA charging activity at

18

Tm TrpRS_2g36

Lead Author: GW Han Acta F Section: Ligand in Xtal Structures 4/12/2020

PDB_id: 2g36 SNTS_id: 354

60

˚

C compared to that at 37

˚

C. The assays were controlled by reactions lacking enzyme or tRNA at 60

˚

C. (B) ATP-PPi exchange activities assayed at 37

˚

C and 60

˚

C. The assays were controlled by reactions lacking enzyme or Trp at 60

˚

C. Consistent with the observation of an endogenously bound Trp molecule in the active site of its crystal structure, Tm TrpRS had PPi exchange activity even when no Trp was added to the reaction.

19

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354

(a) (b)

(c) (d)

Figure 1

20

(b)

(a)

Tm TrpRS_2g36


PDB_id: 2g36

Acta F Section: Ligand in Xtal Structures

21

4/12/2020

SNTS_id: 354

Tm TrpRS_2g36


PDB_id: 2g36

(c)


SNTS_id: 354

Figure 2

22

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354

Figure 3

23

Tm TrpRS_2g36


PDB_id: 2g36

(A)


SNTS_id: 354

Supplementary Figure 1 : (a) Structural superposition of Tm TrpRS (grey), Dr TrpRS-R2 (PDB:

1yid, green) and Dr TrpRS-R2 (PDB: 1yi8, blue). The structural alignment of Dr TrpRS-R2 with

Tm TrpRS can superimpose 319 C

α

atoms with an rmsd of 1.7 Å (sequence identity 45.0). (b)

Electrostatic surface of Tm TrpRS.

24

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354

Supplementary Figure 2 : A Structural Alignment of Tm TrpRS with known TrpRS structures. The sequence of DrTrpRS_I is included in the alignment. Four Cys motif [C-x22-C-x6-x2-C] for chelating [4Fe-4S] cluster are colored as red.

25

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354

Supplementary Figure 3 : Taxonomy distribution of TrpRS homologs containing the Cys-x6-Cysx2-Cys motif. Number of organisms at differing clustering criteria is in noted in brackets. For example, Euryarchaeota at 100%, there are 61 sequences. At 90%, only 24 clusters exists that contained Euryarchaeota. Archaea is shown in blue and Bacteria in grey. Red is the environmental samples that could not be classified under a particular taxonomy.

26

Tm TrpRS_2g36


PDB_id: 2g36


SNTS_id: 354

Supplementary Figure 4 : Sequence alignment of one or multiple annotated TrpRS in organisms.

Only a representative sample from clustering at 50% is shown. When the organisms are multiple

TrpRS annotated, it seems only one TrpRS gene contains [4Fe-4S] cluster binding motif.

27

TM0492-v-4 - Stanford University

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