Trinitrophenyl derivatives bind differently from parent adenine nucleotides to Ca2+-ATPase in the absence of Ca2+

Abstract

Trinitrophenyl derivatives of adenine nucleotides are widely used for probing ATP-binding sites. Here we describe crystal structures of Ca(2+)-ATPase, a representative P-type ATPase, in the absence of Ca(2+) with bound ATP, trinitrophenyl-ATP, -ADP, and -AMP at better than 2.4-Å resolution, stabilized with thapsigargin, a potent inhibitor. These crystal structures show that the binding mode of the trinitrophenyl derivatives is distinctly different from the parent adenine nucleotides. The adenine binding pocket in the nucleotide binding domain of Ca(2+)-ATPase is now occupied by the trinitrophenyl group, and the side chains of two arginines sandwich the adenine ring, accounting for the much higher affinities of the trinitrophenyl derivatives. Trinitrophenyl nucleotides exhibit a pronounced fluorescence in the E2P ground state but not in the other E2 states. Crystal structures of the E2P and E2 ∼ P analogues of Ca(2+)-ATPase with bound trinitrophenyl-AMP show that different arrangements of the three cytoplasmic domains alter the orientation and water accessibility of the trinitrophenyl group, explaining the origin of "superfluorescence." Thus, the crystal structures demonstrate that ATP and its derivatives are highly adaptable to a wide range of site topologies stabilized by a variety of interactions.

Fig. 1.

Binding mode of ATP (or AMPPCP) and TNP-ATP in Ca²⁺-ATPase. (A) ATP in the absence of Ca²⁺ but in the presence of thapsigargin [E2·ATP(TG)], pH 6.1, at 2.15-Å resolution; (B) AMPPCP in the presence of Ca²⁺ (E1·AMPPCP) at 2.5-Å resolution; (C) TNP-ATP in the absence of Ca²⁺ but in the presence of thapsigargin [E2·TNP-ATP(TG)] at 2.15-Å resolution. The violet net in C represents an |F_obs|-|F_calc| electron density map (contoured at 3σ), before introducing TNP-ATP into the atomic model. The N domain appears green and the P domain yellow. Small red spheres represent water molecules; cyan spheres, Ca²⁺; green spheres, Mg²⁺. Small cyan disks represent main chain amide. Broken lines in blue show hydrogen bonds and metal coordination. A stereo version of this figure is presented as Fig. S1.

Fig. 2.

Local distortion of the adenine binding site of Ca²⁺-ATPase in the absence of Ca²⁺. Superimposition of the atomic models for E2·ATP(TG) (atom color) and E1·AMPPCP (green for the N domain, pink for the A domain; AMPPCP, orange). Broken lines in blue show hydrogen bonds and coordinations of Mg²⁺.

Fig. 3.

Binding of TNP-AMP in various intermediates (or analogues) of Ca²⁺-ATPase in the absence of Ca²⁺. (A) E2(TG), (B) , an E2 ∼ P transition state analogue, and (C) , an E2P ground state analogue. The A, N, and P domains are colored pink, green, and yellow, respectively. Blue broken lines show hydrogen bonds. Small red spheres represent water molecules; small cyan disks, main chain amide. Carbon atoms in the TNP group are colored gray.

Fig. 4.

Surface representation of the binding pocket for TNP-AMP. Views of the binding pocket in E2(TG) (A), (B), and (C). Prepared with PyMol (40). A stereo version of this figure is presented as Fig. S6.