The structural basis of ATP as an allosteric modulator

Abstract

Adenosine-5'-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems.

Figure 1. Phylogenetic tree of 13 allosteric proteins.

The phylogenetic tree was derived using ClustalX program. The tree structure consists of nodes (represented as circles) and branches (the connecting lines). The internal and external nodes are represented by green and orange circles, respectively. Phylogenetic distance is proportional to branch length. The allosteric proteins belonging to different protein families are depicted in the pie chart. Orange represents the percentage of the number of proteins with a sequence identity greater than 30% when compared to the reported allosteric protein in this protein family deposited in ASD.

Figure 2. Comparison of evolutionary conservation of allosteric and substrate ATP-binding sites.

(A) Distributions of conservation scores for residues in the allosteric and substrate ATP-binding sites as well as in the surface of proteins. (B) Distributions of average conservation scores for residues in the substrate and allosteric ATP-binding sites and the surface per protein. The statistical significant (P-value) was calculated by the Mann-Whitney U test.

Figure 3. Comparison of conformational preferences of allosteric and substrate ATP molecules.

The landscapes with the reaction coordinates of distance (defined by the distance from the P_γ atom to the centroid of adenine moiety) and angle (defined by the centroids of triphosphate, ribose and adenine moieties) were plotted on ATP conformations from the MD trajectory. The compact and extended structures of ATP correspond to the two major conformation regions. The bound ATP molecules in the allosteric and substrate datasets were mapped to the MD-generated ATP conformations.

Figure 4. The interactions between UMP kinase and allosteric and substrate ATP molecules.

(A) The energy contributions to the binding of allosteric and substrate ATP molecules to the UMP kinase, and the binding modes between the allosteric and substrate ATP molecules and the UMP kinase. The green dashes represent hydrogen bonds. The hydrogen atoms are not displayed for clarity. (B) The volume and shape of the allosteric and substrate ATP-binding sites of the UMP kinase.

Figure 5. The energy landscape of ATP binding to the allosteric ATP-binding site of the UMP kinase as a function of the progress of ATP binding.

(A) The six snapshots of the allosteric ATP binding pathway represent the following configurations: A, the unbound state; B, the “encounter complex”; C, an intermediate state featuring unfavorable electrostatic repulsions between Arg117^A,B and adenine; D, the relatively high-energy state; E; an intermediate state featuring favorable electrostatic interactions between the UMP kinase and ATP; F, the fully bound state. The ATP and its interacting residues are shown as sticks, whereas subunits A and B of the UMP kinase are shown in pale cyan and light blue, respectively. The green dashes represent hydrogen bonds. The hydrogen atoms are not displayed for clarity. The error bars represent standard deviations of binding energies for the 20 snapshots from 20 trajectories.

Figure 6. The energy landscape of ATP binding to the substrate ATP-binding site of the UMP kinase as a function of the progress of ATP binding.

(A) The six snapshots of the substrate ATP-binding pathway represent the following configurations: A, the unbound state; B, the “encounter complex”; C, an intermediate state in which ATP does not interact with Lys10, Ser12 and Lys161; D, the relatively high-energy state; E; an intermediate state in which ATP interacts with Lys10, Ser12, Arg57, and Lys161; F, the fully bound state. The 165-DGVFTSDP-172 motif of UMP kinase in the recognition of the adenine moiety of ATP is shown in magenta. The ATP and its interacting residues are shown as sticks, whereas subunit A of the UMP kinase is shown in pale cyan. The green dashes represent hydrogen bonds. The hydrogen atoms are not displayed for clarity. The error bars represent standard deviations of binding energies for the 20 snapshots from 20 trajectories.

Figure 7. Structural identification of allosteric triggers of an allosteric molecule.

The crystal structures of allosteric ATP unbound and bound proteins are shown in pale cyan and dark green, respectively. The relatively large conformational changes in the allosteric ATP unbound and bound proteins are shown in red and orange, respectively, coupled with the residues in light pink and light green, respectively. The adenine in ATP is an allosteric trigger in two cases: (A) P2X₄ ion channel (PDB: 4DW0 vs. 4DW1; the former is allosteric ATP unbound structure and the latter is allosteric ATP bound structure); and (B) Aspartate carbamoyltransferase (PDB: 6AT1 vs. 4AT1). The triphosphate part of ATP is an allosteric trigger in eight cases: (C) Cytosolic 5’-nucleotidase II (PDB: 2XCX vs. 2XCW); (D) ClpX (PDB: 3HTE vs. 3HWS); (E) Glycogen phosphorylase (PDB: 1FC0 vs. 1FA9); (F) Ribonucleotide reductase (PDB: 1R1R vs. 3R1R); (G) MutS (PDB: 1E3M vs 1W7A); (H) DnaA (PDB: 1L8Q vs. 2HCB); (I) Phosphofructokinase 1 (PDB: 2PFK vs. 1PFK); (J) Chaperonin GroEL (PDB: 1KP0 vs. 1KP8).