Volume 13, Issue 6, Pages 829-841 (March 2004) Alanyl-tRNA Synthetase Crystal Structure and Design for Acceptor-Stem Recognition  Manal A. Swairjo, Francella J. Otero, Xiang-Lei Yang, Martha A. Lovato, Robert J. Skene, Duncan E. McRee, Lluis Ribas de Pouplana, Paul Schimmel  Molecular Cell  Volume 13, Issue 6, Pages 829-841 (March 2004) DOI: 10.1016/S1097-2765(04)00126-1

Figure 1 Functional Domains and Sequence of AlaRS (A) Functional modules of AlaRS as defined by E. coli and A. aeolicus residue numbers (top and bottom, respectively). Locations of some of the site-directed mutations previously placed in E. coli AlaRS are indicated. (B) Sequence alignment of the AlaRS catalytic fragment from A. aeolicus and E. coli. Identities and similarities are highlighted in red and white boxes, respectively. Secondary structure elements––as defined by the crystal structure––are shown and labeled above the sequence. Solvent accessibility (acc) is shown in blue, cyan, and white for accessible, intermediate, and buried residues, respectively. Motifs 1, 2, and 3 are underlined in pink, orange, and cyan, respectively. Residues in E. coli AlaRS important for tRNA-dependent aminoacylation function are indicated with asterisks. Molecular Cell 2004 13, 829-841DOI: (10.1016/S1097-2765(04)00126-1)

Figure 2 Aminoacylation of RNA Substrates by Aa-AlaRS453 (A) The sequences of A. aeolicus tRNAAla and synthetic RNA substrates tested for aminoacylation by Aa-AlaRS453. Base pairs in the acceptor-stem region of Ec-minihelixAla that differ from the A. aeolicus sequence are labeled with asterisks. (B) Aminoacylation of wild-type Aa-minihelixAla (closed circles) and G3:C70 Aa-minihelixAla (open circles) by Aa-AlaRS453 at 37°C (pH 7.5). Inset: the same experiment repeated at 55°C. Aminoacylation of Ec-minihelixAla was done at 37°C only (closed triangles). Molecular Cell 2004 13, 829-841DOI: (10.1016/S1097-2765(04)00126-1)

Figure 3 Crystal Structure of Aa-AlaRS453 (A) Stereo view of a representative region of the FOM-weighted experimental electron density map (resolution 2.3 Å, contour level 2.0 σ), calculated after solvent flattening, in the vicinity of Met220 in the central β sheet of the active site domain. The map is superimposed on the refined model. (B) Stereo view of a ribbon diagram of the overall crystal structure of Aa-AlaRS453 showing the active site, middle, and C domains in red, yellow, and green, respectively. Every 20th residue is labeled. The three class II signature motifs 1, 2, 3 are colored in pink, orange, and cyan, respectively. F352 at the kink site of helix α12 is indicated. (C and D) Top views of the middle helical domain and the C domain. Hydrophobic regions are colored in gray. (E) Topology of the catalytic fragment. Domains and motifs are colored and secondary structure elements are labeled as in (B). Molecular Cell 2004 13, 829-841DOI: (10.1016/S1097-2765(04)00126-1)

Figure 4 Structural Comparisions of AlaRS with Other Class II Synthetases Superpositions of Cα traces of Aa-AlaRS453 with (A) T. thermophilus GlyRS (Logan et al., 1995) (rmsd 2.7 Å over 171 Cα atoms), (B) T. thermophilus AspRS (Poterzsman et al., 1994) (rmsd 2.9 Å over 166 Cα atoms), and (C) T. thermophilus PheRS α subunit (Mosyak et al., 1995) (rmsd 3.3 Å over 166 Cα atoms) as examples of synthetases from subclasses IIa, IIb, and IIc, respectively. AlaRS colors are as in Figure 3, other synthetases (with their N and C termini labeled) are in magenta. Active site insertions not found in AlaRS are shown in gray. Molecular Cell 2004 13, 829-841DOI: (10.1016/S1097-2765(04)00126-1)

Figure 5 Interdomain Interactions and the Role of the Helical Hairpin Motif (A) The helical hairpin motif, shown in the context of the middle and C domains. The molecule is oriented and colored as in Figure 3. Interactions of D271 and R300 with residues in their environment are shown. Red spheres represent water molecules. (B) Schematic of the interfaces of the middle domain with the active site domain (left) and with the C domain (right), showing interactions between conserved residues. Both A. aeolicus and E. coli sequence numbers are listed. Residues in E. coli AlaRS investigated by alanine scan mutagenesis in previous work are boxed. Residues highlighted in red are those found important for in vivo and in vitro function. Molecular Cell 2004 13, 829-841DOI: (10.1016/S1097-2765(04)00126-1)

Figure 6 Docking Model of tRNA onto Aa-AlaRS453 (A) Overall view with the protein shown as a Cα trace representation with the van der Waals surface superposed on top and the three domains colored as in Figure 3. The tRNA is shown in gray. Both the complex (top) and the separated enzyme and tRNA (bottom) are shown. Functionally important sites on protein and tRNA––identified in previous mutagenesis and biochemical studies––are shown in CPK representation in purple and blue, respectively. (B) Potential C domain interaction with G3:U70 from the major groove side of the acceptor stem. The five helices of the domain and the 3:70 and 2:71 base pairs are labeled. Side chains on α14 are shown as sticks. The short loop in Aa-AlaRS453 which marks the site of the D. melanogaster mitochondrial AlaRS-specific insertion is colored in magenta. The dotted line hypothetically indicates the longer loop and predicted helical pair formed by the insertion in D. melanogaster mitochondrial AlaRS. The arrow indicates the direction of the potential C domain shift, from 3:70 to 2:71 position up the acceptor stem. Molecular Cell 2004 13, 829-841DOI: (10.1016/S1097-2765(04)00126-1)