Improved Model of Proton Pump Crystal Structure Obtained by Interactive Molecular Dynamics Flexible Fitting Expands the Mechanistic Model for Proton Translocation in P-Type ATPases

Abstract

The plasma membrane H⁺-ATPase is a proton pump of the P-type ATPase family and essential in plants and fungi. It extrudes protons to regulate pH and maintains a strong proton-motive force that energizes e.g., secondary uptake of nutrients. The only crystal structure of a H⁺-ATPase (AHA2 from Arabidopsis thaliana) was reported in 2007. Here, we present an improved atomic model of AHA2, obtained by a combination of model rebuilding through interactive molecular dynamics flexible fitting (iMDFF) and structural refinement based on the original data, but using up-to-date refinement methods. More detailed map features prompted local corrections of the transmembrane domain, in particular rearrangement of transmembrane helices 7 and 8, and the cytoplasmic N- and P-domains, and the new model shows improved overall quality and reliability scores. The AHA2 structure shows similarity to the Ca²⁺-ATPase E1 state, and provides a valuable starting point model for structural and functional analysis of proton transport mechanism of P-type H⁺-ATPases. Specifically, Asp684 protonation associated with phosphorylation and occlusion of the E1P state may result from hydrogen bond interaction with Asn106. A subsequent deprotonation associated with extracellular release in the E2P state may result from an internal salt bridge formation to an Arg655 residue, which in the present E1 state is stabilized in a solvated pocket. A release mechanism based on an in-built counter-cation was also later proposed for Zn²⁺-ATPase, for which structures have been determined in Zn²⁺ released E2P-like states with the salt bridge interaction formed.

Keywords: Arabidopsis thaliana AHA2; P-type ATPases; crystallography; iMDFF; membrane transport; molecular dynamics; plasma membrane proton pump; proton gradient.

Figure 1

Overall resemblance of AHA2-AMPPCP to the SERCA-SLN complex. (A) Superposition of the revised structure of AHA2-AMPPCP (domains colored as indicated) and the SERCA-SLN complex (gray; pdb id: 4H1W) both representing E1 state. The structures are superimposed on Cα position of the TM or P domains only. Mg²⁺ and K⁺ ions are shown by green and purple-blue spheres, respectively. The spatial organization of the cytoplasmic domains is similar in both E1 state structures. Two loops connecting the A-domain with TM1 and TM3, respectively, align well in AHA2 and SERCA, although for the latter TM1 is longer and kinks into a short helix parallel to the membrane. The TM3 connector forms a longer helix in the A-domain of SERCA compared to AHA2. The N-domain is moved slightly closer toward the A-domain in the SERCA structure. The adenine base part of the AMPPCP molecule is coordinated by side chains of Asp372 and Asp375 and the backbone oxygen of Ser457. The major differences between the P-domains are visible in a region between Pro533^AHA2 to Asp559^AHA2, which covers one loop and two short helixes. (B) Side view of the aligned pumps (superimposed on TM2–TM10 domains, r.m.s.d 3.07 Å for 159 Cα atoms) showing a tilt of the cytoplasmic headpiece of AHA2 (in colors) relatively to SERCA (gray). The N- and P-domain headpiece of AHA2 is tilted by ~25° (measured between Cα of Ser436^AHA2 and Thr538^SERCA for the N-domain and between Cα of Pro550^AHA2 and Pro662^SERCA for the P-domain, using Cα of Lys625^AHA2 in TM5 in both cases as an apex). Asterisks mark helices from the P- and N-domain of AHA2. (C) Alignment of the transmembrane region. Top view from the plane marked by dashed line at segment A. Catalytically important residues of AHA2 and conserved Asp800 of SERCA are labeled. (D) Superposition of the N-domain of AHA2 and SERCA-SLN (r.m.s.d 0.97 Å for 123 atoms superimposed Cα atoms), both in E1 states and showing overlapping binding of β-γ phosphates of AMPPCP. The AMPCPP molecule represents the revised AHA2 model.

Figure 2

Improvement of the AHA2 model. (A) The initial AHA2 model (pdb id: 3B8C). (B) Final, revised model after re-refinement (pdb id: 5KSD). Spheres mark changes in the position of the Cα atoms between the two models (coloring according to the legend below). N and C-termini are indicated; DDM indicates bound n-Dodecyl β-D-maltoside. (C) Electron density map contoured at 1.0 r.m.s.d for the Asn106-Asp684 pair. (D) Electron density map contoured at 2.0 r.m.s.d. for the K⁺ ion binding site.

Figure 3

Overview of the P-type ATPases cycle. Schematic presentation of structural changes occurring during the catalytic cycle of P-type ATPases, represented by SERCA structures. Insets show changes in relative orientation of the residues involved in formation of SERCA ion binding throughout the transport cycle. White dashed lines mark the planes corresponding to the zoomed cross-sections. Cytoplasmic domains are colored as P-domain in blue, N-domain in red, A-domain in yellow. The transmembrane domain is shown in green. Ligands and ions are shown as spheres (K⁺ in purple-blue, Na⁺ in purple, Ca²⁺ in wheat, Mg²⁺ in green) and ball-and-sticks representation for nucleotides and metallofluorides. SERCA1a [structures 4H1W (Winther et al., 2013), 1T5T (Sørensen et al., 2004), 3B9B (Olesen et al., 2007), 2C8K (Jensen et al., 2006)]. The structures were aligned by their TM domains on TM7-TM10.

Figure 4

Binding of AMPPCP. (A) Binding of the phosphate groups of AMPPCP includes two Mg²⁺ ion, side chains of Thr511 and Thr331 and backbone atoms of Gly512 and Thr331. The adenine base can interact with side chains of Asp375 and Asp372, the latter of which also interacts with the Mg²⁺ ion that coordinates α-phosphate group. The ribose ring interacts with the backbone carbonyl of Ser457. (B) AHA2 binding of AMPPCP (green carbons) compared to ${\text{ADP}-\text{AlF}}_4^{-}$ (gray) binding to SERCA in the E1P-like state, pdb id: 1T5T (Sørensen et al., 2004). Mg²⁺—green spheres in AHA2, gray spheres in SERCA.

Figure 5

Comparison of AHA2-AMPPCP model with available SERCA structures. Orientation of a reference helix from the P-domain (blue) shows that the AHA2-E1 structure adopts an intermediate state of the transition between SERCA-E2 and SERCA-E1 states. Structures were aligned using helices TM5-TM9. TM5 marked in green. Corresponding helices from the N-domain are marked in red.

Figure 6

Sequence alignment of AHA2, SERCA, PMA1, ZntA. Secondary structure markings (below the aligned sequences) are based on AHA2 crystal structure with tubes for α-helix, arrows–β-strand, and lines for coil. The alignment was performed with MUSCLE (Edgar, 2004), using CLC Workbench (Genomics Workbench 7.7). and accession numbers: AHA2–P19456; PMA1–P05030; SERCA1a–P04191; ZntA–Q3YW59.

Figure 7

Overview of the AHA2-AMPPCP structure. (Left) AHA2 (yellow cartoon) modeled into a lipid bilayer. Ions and AMPPCP are shown by sphere representation. The circle zooms onto a negatively charged pocket formed between TM1 and TM2 in a close proximity of the Asn106-Asp684 pair. (Right) Overall surface electrostatic potential of AHA2 ± 5 K_bT/e_c indicated in blue (positive) and red (negative) mapped on a surface representation. Negative electrostatic charge comes from two Glu residues situated in helix 2. The unstructured loop connecting the A domain and TM1 carries positive charges (blue color), which may interact with lipid head-groups.

Figure 8

Comparison between AHA2 and Zn²⁺-ATPase. Despite different overall structure, the TM domains of AHA2 and the Shigella sonnei Zn²⁺-ATPase ZntA exhibit significant resemblance regarding the distribution of charged residues in the TM helices associated with transport. In both cases the TM helix 2 harbors a residue involved in closure of the ligand pathway from the cytoplasmic site. Phe210^ZntAserves as a gating residue, while overlapping Asn106^AHA2 is implicated in stabilization of the occluded, protonated Asp684. The Asp side chains placed in both cases in TM6 (Asp684^AHA2 and Asp714^ZntA) likely fulfill similar roles. Zinc binding in ZntA most likely involves Asp714 (and two conserved cysteines of TM4, not shown), while zinc release and the phosphorylation is stabilized by interactions with the conserved Lys693 of TM5. In AHA2 the equivalent Asp684 is essential for transport and most likely becomes deprotonated by interaction with Arg655 of TM5 along with E2P dephosphorylation.

Figure 9

Solvent cavity in the TM domain of AHA2. (A) Overview on the cavity location within the TM domain. The cavity likely encloses 8–10 bound water molecules in this conformation of the pump. (B) Zoom-in of the cavity viewed in the plane of the membrane. The cavity is formed by backbone atoms of Ile282, Gly283, and Ile285 along with Gly 284, Pro286 and side chains of conserved Tyr645, Tyr648, Tyr653, and Arg655 of TM5 and Asn683 of TM6. (C) Molecular Dynamics simulations indicate that a continuous string of water molecules (shown in green) can reach from the cytoplasm to the Asp684 residue, the proposed proton binding site, i.e., an open proton translocation pathway, which must close later in the functional cycle, most likely along with E1P formation. Two stable water molecules reached almost half way through the membrane into the TM domain where they can interact with Ile282, Thr653 and backbone oxygen of Ala649, and Cys247, next to the central solvent cavity.