PDBsum entry 2e3c

Ligase

PDB id: 2e3c

Contents

Protein chain

260 a.a. *

Waters ×41

* Residue conservation analysis

PDB id:

2e3c

Name:

Ligase

Title:

Crystal structure of the catalytic domain of pyrrolysyl-tRNA synthetase

Structure:

Pyrrolysyl-tRNA synthetase. Chain: a. Fragment: c-terminal catalytic domain. Engineered: yes

Source:

Methanosarcina mazei. Organism_taxid: 2209. Strain: jcm9314. Gene: pyls. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.65Å R-factor: 0.200 R-free: 0.254

Authors:

T.Yanagisawa,R.Ishii,S.Yokoyama,Riken Structural Genomics/proteomics Initiative (Rsgi)

Key ref:

T.Yanagisawa et al. (2008). Crystallographic studies on multiple conformational states of active-site loops in pyrrolysyl-tRNA synthetase. J Mol Biol, 378, 634-652. PubMed id: 18387634 DOI: 10.1016/j.jmb.2008.02.045

Date:

22-Nov-06 Release date: 11-Dec-07

Protein chain

Q8PWY1  (PYLS_METMA) -  Pyrrolysine--tRNA ligase from Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)

Seq: 454 a.a.

Struc: 260 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.6.1.1.26 - pyrrolysine--tRNA(Pyl) ligase.
• Reaction: tRNA(Pyl) + L-pyrrolysine + ATP = L-pyrrolysyl-tRNA(Pyl) + AMP + diphosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/j.jmb.2008.02.045

J Mol Biol 378:634-652 (2008)

PubMed id: 18387634

Crystallographic studies on multiple conformational states of active-site loops in pyrrolysyl-tRNA synthetase.

T.Yanagisawa, R.Ishii, R.Fukunaga, T.Kobayashi, K.Sakamoto, S.Yokoyama.

ABSTRACT

Pyrrolysine, a lysine derivative with a bulky pyrroline ring, is the "22nd" genetically encoded amino acid. In the present study, the carboxy-terminal catalytic fragment of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) was analyzed by X-ray crystallography and site-directed mutagenesis. The catalytic fragment ligated tRNA(Pyl) with pyrrolysine nearly as efficiently as the full-length PylRS. We determined the crystal structures of the PylRS catalytic fragment in the substrate-free, ATP analogue (AMPPNP)-bound, and AMPPNP/pyrrolysine-bound forms, and compared them with the previously-reported PylRS structures. The ordering loop and the motif-2 loop undergo conformational changes from the "open" states to the "closed" states upon AMPPNP binding. On the other hand, the beta 7-beta 8 hairpin exhibits multiple conformational states, the open, intermediate (beta 7-open/beta 8-open and beta 7-closed/beta 8-open), and closed states, which are not induced upon substrate binding. The PylRS structures with a docked tRNA suggest that the active-site pocket can accommodate the CCA terminus of tRNA when the motif-2 loop is in the closed state and the beta 7-beta 8 hairpin is in the open or intermediate state. The entrance of the active-site pocket is nearly closed in the closed state of the beta 7-beta 8 hairpin, which may protect the pyrrolysyladenylate intermediate in the absence of tRNA(Pyl). Moreover, a structure-based mutational analysis revealed that hydrophobic residues in the amino acid-binding tunnel are important for accommodating the pyrrolysine side chain and that Asn346 is essential for anchoring the side-chain carbonyl and alpha-amino groups of pyrrolysine. In addition, a docking model of PylRS with tRNA was constructed based on the aspartyl-tRNA synthetase/tRNA structure, and was confirmed by a mutational analysis.

Selected figure(s)

Figure 5.
Fig. 5. Comparison of the β7-β8 hairpin conformations. (a) A stereo view of superpositioned C^α traces of the pyrrolysyladenylate-bound (black), the pyrrolysine/AMPPNP-bound (green), AMPPNP-bound (yellow), and apo forms (red). The locations of the C^α atoms for residues 377–387 are numbered, with the same coloring as in each trace. (b and c) Stereo views of the superposition of the pyrrolysyladenylate-bound (gray), pyrrolysine/AMPPNP-bound (green), AMPPNP-bound (yellow), and apo forms (vermilion). Val377, Gly378, and Asp379 (b), and Tyr384, Gly385, Asp386, and Thr387 (c) are represented by ball-and-stick models. d and e, A stereo view of the 2F[o] – F[c] electron density map (contoured at 1.1 σ) around the β7-β8 regions. (d) The AMPPNP-bound form. Direct hydrogen-bonding interactions of Val402 with Asp386, and of Asp379 with Arg356, and the water-mediated hydrogen-bonding interaction of Asp379 with Thr387 are shown by pink, green, and orange dotted lines, respectively. (e) The AMPPNP/pyrrolysine-bound form. Direct hydrogen-bonding interactions of Val402 with Asp386, and of Asp379 with Thr387 are shown by pink and orange dotted lines, respectively. Gly385, Asp386, and Thr387 in each PylRS(c270) form adopt the β8-open conformation, as shown in sky blue circles. On the other hand, Gly378 and Asp379 adopt the β7-open conformation in the AMPPNP-bound form and the β7-closed conformation in the pyrrolysine/AMPPNP-bound form, as shown in purple circles, respectively.

Figure 6.
Fig. 6. Pyrrolysine recognition by PylRS(c270). (a) The 2F[o] – F[c] omit map (contoured at 3 σ) of the active site of PylRS(c270) complexed with pyrrolysine and AMPPNP. The pyrrolysine molecule was omitted for the map calculation. (b) Ball-and-stick models of pyrrolysine and AMPPNP bound within the PylRS(c270) active site. PylRS(c270) is represented as a surface model, calculated by MSMS.^53 Hydrophobic residues in the catalytic cleft (Ala302, Leu305, Tyr306, Leu309, Val401, and Trp417) and Asn346, which hydrogen bonds with pyrrolysine, are colored yellow and green, respectively. (c) Active-site mutations of PylRS(c270) affect the aminoacylation activity for pyrrolysine. The aminoacylation assay conditions are described in Materials and Methods. A concentration of 9 μM of was used for the wild-type and mutant PylRS(c270) proteins. Starting from the left, each lane shows a reaction with the following: no enzyme; wild-type PylRS(c270); W417A; Y306A; N346A; C348A; S399A; V401A; L305A; control tRNA^Pyl. d and e, The active sites of the pyrrolysine/AMPPNP-bound (d) and pyrrolysyladenylate-bound forms of PylRS(c270) (e).^15 The β7-β8 hairpin is shown in yellow. The pyrrolysine, AMPPNP, pyrrolysyladenylate, Arg330, Asn346, and Tyr384 are shown as ball-and-stick models. Water molecules (Wat) are shown as pink balls. The class-II aaRS specific motifs, motif 1, motif 2, and motif 3 (residues 258–268, 329–345, and 418–431, respectively) are colored red, green, and blue, respectively. The three hydrogen-bonding interactions of the PylRS(c270) active site with pyrrolysine or pyrrolysyladenylate in both forms, and the two additional hydrogen-bonding interactions in the pyrrolysyladenylate-bound form are represented by black and red dotted lines, respectively. The interaction between the pyrrolysine α-carboxyl group and the α-phosphate atom in the pyrrolysine/AMPPNP-bound form is also shown (pale blue dotted line).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 378, 634-652) copyright 2008.

Figures were selected by an automated process.