PDBsum entry 2g5h

Ligase

PDB id: 2g5h

Contents

Protein chains

485 a.a.

398 a.a.

99 a.a.

Metals

_MG

Waters ×139

References listed in PDB file

Key reference

Title

Ammonia channel couples glutaminase with transamidase reactions in gatcab.

Authors

A.Nakamura, M.Yao, S.Chimnaronk, N.Sakai, I.Tanaka.

Ref.

Science, 2006, 312, 1954-1958. [DOI no: 10.1126/science.1127156]

PubMed id

16809541

Abstract

The formation of glutaminyl transfer RNA (Gln-tRNA(Gln)) differs among the three domains of life. Most bacteria employ an indirect pathway to produce Gln-tRNA(Gln) by a heterotrimeric glutamine amidotransferase CAB (GatCAB) that acts on the misacylated Glu-tRNA(Gln). Here, we describe a series of crystal structures of intact GatCAB from Staphylococcus aureus in the apo form and in the complexes with glutamine, asparagine, Mn2+, and adenosine triphosphate analog. Two identified catalytic centers for the glutaminase and transamidase reactions are markedly distant but connected by a hydrophilic ammonia channel 30 A in length. Further, we show that the first U-A base pair in the acceptor stem and the D loop of tRNA(Gln) serve as identity elements essential for discrimination by GatCAB and propose a complete model for the overall concerted reactions to synthesize Gln-tRNA(Gln).

Figure 1.
Fig. 1. Bacterial GatCAB complex fastens a molecular belt. (A) Front-view ribbon diagram of the overall structure of S. aureus GatCAB/glutamine complex at 2.3 Å resolution, depicted in three different colors for each subunit: blue, green, and magenta for GatA, GatB, and GatC, respectively. Glutamine in the active site of GatA is drawn as yellow stick representations, whereas the purple sphere is the magnesium ion found in the active site of GatB. ADP from the cocrystal structure with ADP-AlF[4]^- is shown together as pink sticks. This color code is used throughout all the figures. (B) Top view of annularly shaped GatC. (C) Amphipathic helices at the N terminus of GatC form a helical bundle with the hydrophobic core of GatA (hydrophobic residues are colored gray). (D) Detailed interactions between the internal loop region of GatC and GatAB complex. Conserved residues involved are labeled, with hydrogen bonds indicated ( 3.2 Å, dashed black line). Polar interactions are prominent on the C-terminal side, whereas the hydrophobic interactions are clustered on the opposite side. (E) The C terminus of GatC tightens GatAB complex, constructing an antiparallel ß sheet and a hydrophobic platform.

Figure 2.
Fig. 2. A 30 Å long ammonia channel connects the two remote active centers of GatCAB. (A) The active site of a glutaminase reaction in GatA is composed of the conserved Ser-cis-Ser-Lys catalytic scissors shown as magenta stick representations. Residues involved in the hydrogen-bonded network (dashed black lines) in the active site are labeled. A plausible hydrolytic water molecule is colored light blue and is on the opposite side of a supposed ammonia product (orange sphere). The Fo-Fc electron density map (contoured at 3 , green mesh) calculated without the glutamine and Ser178 clearly demonstrates the tetrahedral covalent intermediate of the glutamine with Ser178. (B) The environment of the ADP binding site shown together with the omit Fo-Fc electron density map (2 , blue). Residues contributing to ADP (ball-and-stick) recognition are represented as stick models with labels. Two water molecules (light blue spheres) are coordinated to a magnesium ion (purple) and to ß phosphate. (C) The putative ammonia channel was calculated using the program CAVER (26), with the structure of the water-omitted GatCAB/glutamine complex. Glu125B blocking the ammonia transport route is shown in a space-filling representation for clarity. The channel was filled with a row of solvent molecules (light blue spheres), which interact with the conserved polar residues (colored sticks) along the pathway. A bound glutamine in GatA is drawn as spheres indicating the start point of the channel. (D) Schematic representation of the ammonia channel. Residues defining the channel are colored corresponding to their properties: red, negative; blue, positive; black, nonpolar side chain; gray, main chain. Hydrolyzed ammonia is colored orange. Strictly conserved residues are underlined and hydrogen-bonding distances are indicated (Å). The presumed movement of the Glu125B gate to open the ammonia channel is indicated by a black arrow.

The above figures are reprinted by permission from the AAAs: Science (2006, 312, 1954-1958) copyright 2006.