Fig 1.
Chemical structures of the ATP analogs used in this study.
The structures of ATP, 7-deazaATP, 2'-deoxyATP and AMPPCP with key atom positions indicated. Boxes denote the region—nucleobase, sugar, triphosphate—containing the modification for that analog.
Table 1.
Data collection and refinement statistics.
Fig 2.
GRP78ATPase domain structure and canonical ATP/ADP binding site.
(A). Overview of the GRP78ATPase subdomain structure: Ia (cyan), Ib (yellow), IIa (purple), and IIb (green). (B). Close-up of ATP binding site indicates key interacting residues. (C). Overlay of the ATP (cyan) and ADP-Pi-Mg++ (green) binding sites highlights the difference between the ATP γ-phosphate and Pi. (D). Fourier difference maps (Fo-Fc) of ATP and ADP-Pi-Mg++ contoured to 2.5 σ. Dashed lines indicate potential hydrogen bonds (green) and metal coordination (black). Non-bonding spheres indicate water (blue) and magnesium (green). For clarity, the oxygen atoms of ADP are shown in magenta.
Table 2.
GRP78 ATPase nucleotide affinities (Kd) determined by SPR.
Fig 3.
(A). The binding site of AMPPCP-Mg++-bound (yellow) GRP78ATPase illustrates the key residues interacting with the phosphate groups. Asp224, which interacts with the other Mg++-coordinated waters, has been removed for clarity. Dashed lines indicate potential hydrogen bonds (green) and metal coordination (black). Non-bonding spheres indicate water (blue) and magnesium (green; from AMPPCP-Mg++). (B). Fourier difference map (Fo-Fc) of AMPPCP-Mg++ contoured to 3.0 σ; the arrow denotes the carbon bridging the β- and γ-phosphates. (C). Superposition of α-, β- and γ-phosphate groups in ATP (gray) and AMPPCP-Mg++ (orange and red). Residues correspond to the AMPPCP-Mg++-bound structure at the active site of GRP78ATPase. (D). Superposition of α-, β- and γ-phosphate/Pi groups in ADP-Pi (gray) and AMPPCP-Mg++ (orange and red) at the active site of GRP78ATPase. Residues correspond to the AMPPCP-Mg++-bound structure.
Fig 4.
(A) 7-deazaATP bound to GRP78ATPase illustrates that the 7-position of the adenine ring faces an opening (dash wedge) bordered by Ser300 and Arg367. (B). Overlay of ATP ligand (cyan) and 7-deazaATP-bound GRP78ATPase demonstrates the minimal effect of a 7-position modification. (C). Fourier difference map (Fo-Fc) of 7-deazaATP contoured to 3.0 σ. The arrow denotes the carbon at the 7-position of the adenine ring.
Fig 5.
(A). Surface representation of GRP78ATPase shows the outward binding mode of 2'-deoxyATP (magenta) and the inward binding mode of ATP (cyan). (B). Close-up of the 2'-deoxyATP binding site with superimposed ATP (cyan) shows key residues in grey. (C). Alternate view of 2'-deoxyATP binding site highlights interactions with Arg60, Arg367 and Asn389. (D). Fourier difference map (Fo-Fc) of 2'-deoxyATP contoured to 3.0 σ. Green dashed lines indicate potential hydrogen bonds.