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235 a.a.
Key reference
DOI no: 10.1016/j.str.2009.01.010 Structure 17:374-385 (2009) PubMed id: 19278652
Structural Basis for Binding of RNA and Cofactor by a KsgA Methyltransferase. C.Tu, J.E.Tropea, B.P.Austin, D.L.Court, D.S.Waugh, X.Ji. ABSTRACT
Among methyltransferases, KsgA and the reaction it catalyzes are conserved throughout evolution. However, the specifics of substrate recognition by the enzyme remain unknown. Here we report structures of Aquifex aeolicus KsgA, in its ligand-free form, in complex with RNA, and in complex with both RNA and S-adenosylhomocysteine (SAH, reaction product of cofactor S-adenosylmethionine), revealing critical structural information on KsgA-RNA and KsgA-SAH interactions. Moreover, the structures show how conformational changes that occur upon RNA binding create the cofactor-binding site. There are nine conserved functional motifs (motifs I-VIII and X) in KsgA. Prior to RNA binding, motifs I and VIII are flexible, each exhibiting two distinct conformations. Upon RNA binding, the two motifs become stabilized in one of these conformations, which is compatible with the binding of SAH. Motif X, which is also stabilized upon RNA binding, is directly involved in the binding of SAH.
Selected figure(s)
Figure 2.
Figure 2. Ligand-free Aa-KsgA Structures
(A) Stereo view showing the ribbon diagram of the KsgA1 structure. The α helices, β strands, and 3[10] helices are numbered and shown in cyan, magenta, and blue, respectively.
(B) Cα superposition of KsgA1 (yellow), KsgA2 (magenta), Ec-KsgA-chain A (cyan; PDB ID code 1QYR), and Ec-KsgA-chain B (green, PDB entry 1QYR).
(C) Two distinct conformations of motif I.
(D) Two distinct conformations of motif VIII.Figure 6.
Figure 6. The Putative Catalytic Center of KsgA
(A) Stereo view showing the superposition of tetraloop structures with the KsgA-RNA-SAH complex. SAH is shown as spheres (carbon in gray, nitrogen in blue, oxygen in red, and sulfur in yellow). KsgA is shown as a ribbon diagram in gray. RNAs are illustrated as tube-and-stick models with the positions of A1518 and A1519 indicated by spheres. RNA in KsgA-RNA-SAH is shown in orange, a structure of product RNA with dimethylated A1518 and A1519 (PDB ID code 2AW7) in cyan, and a model for substrate RNA with unmethylated adenosines (PDB ID code 1AFX) in magenta.
(B) Stereo view showing the superposition of the putative catalytic center of KsgA as observed in the ternary KsgA-RNA-SAH complex (green) with the catalytic center assembly of M·TaqI as observed in the M·TaqI-DNA-NEA structure (PDB ID code 1G38; yellow). The alignment was based on the cofactor SAH (stick model with carbon in green, nitrogen in blue, oxygen in red, and sulfur in yellow) and the cofactor analog NEA (stick model with carbon in yellow). The target adenine dA606 for M·TaqI is shown as a stick model in magenta, whereas the A1518 and A1519 for KsgA are shown as stick models in red. The contact between atom N6 of dA606 in the M·TaqI complex and atom S of SAH in the KsgA complex is indicated with a dashed line in black.
(C) A close-up stereo view showing the details of the adenine-binding site. The amino acid side chains in motif IV (N105, P106, P107, and Y108) and motif VIII (F196) in M·TaqI are shown as line models in yellow. The corresponding side chains in KsgA (N101, L102, P103, Y104, and F166) are shown in green. The adenine substrate is shown in magenta.The above figures are reprinted by permission from Cell Press: Structure (2009, 17, 374-385) copyright 2009. Figures were selected by the author.
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