Membrane surface recognition by the ASAP1 PH domain and consequences for interactions with the small GTPase Arf1

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Science Advances  30 Sep 2020:
Vol. 6, no. 40, eabd1882
DOI: 10.1126/sciadv.abd1882
  • Fig. 1 Sequence and structure of the ASAP1–PH domain.

    (A) Residues truncated in ΔN14ASAP1–PH are provided in the dotted box. Structured residues in the crystal structure (PDB: 5C79) are shown in plain font ([334-437]–ASAP1, referred to as 5C79). (B) Ribbon representation of the structure ASAP1–PH in PDB: 5C79. For visual guidance, N and C termini are labeled along with all β strands, as well as loops linking the β strands. Approximate sites of PI(4,5)P2 of interaction are labeled CA (for canonical sites) and NCA (for noncanonical site). Isoleucine side chains are shown as stick models.

  • Fig. 2 ASAP1–PH:PIP2 binding interface and ASAP1–PH:PS interactions identified through CSP and MD analyses.

    CSPs between free and bound PH domain are plotted against residue number. (A) ASAP1–PH was titrated by diC4-PI(4,5)P2. (B) ASAP1–PH was titrated by PI(4,5)P2-containing NDs. Lipid composition of the NDs was 16:0-18:1 PC:18:1-18:1 PI(4,5)P2 (95:5). A carbon-detected experiment was used to measure diC4-PI(4,5)P2–induced CSPs. (C) Normalized PI(4,5)P2:ASAP1–PH interaction counts plotted against residue number. Data were averaged over three MD trajectories with a cutoff of 3.5 Å to detect nonhydrogen proximities. (D) (Left axis, ●) Free energy of binding of ASAP1–PH to 16:0-18:1 PC NDs containing 1.25 mol % of 18:1-18:1 PI(4,5)P2 and increasing mole fraction of 18:1-18:1 PS. Dissociation constants were calculated by following 1H-13C chemical shift changes of I371, A381, and L383 methyls as a function of ND concentration. (Right axis, ○) The percentage of GTP bound to myr-Arf1 hydrolyzed in 3 min by PZA is plotted against the mole fraction of PS in large unilamellar vesicles (LUVs) containing PI(4,5)P2 (1 mol %) and increasing concentration of PS. (E) Plot of 1HN-15N CSPs of ASAP1–PH bound to 16:0-18:1 PC NDs containing 1.25 mol % of 18:1-18:1 PI(4,5)P2 (x axis) and 1.25 mol % 18:1-18:1 PI(4,5)P2 and 15 mol % of 18:1-18:1 PS (y axis). 1HN-15N CSPs were collected at 0.1, 0.2, and 0.6 of bound PH domain (as measured by the CSP of I371, A381, and L383 methyls by 1H-13C HMQC). The data presented are for the fully bound ASAP1–PH domain. (F) Density distributions of PI(4,5)P2 (left) and PS (right) headgroups bound to ASAP1–PH, calculated using the last 100 ns of one of the all-atom simulation. A diffused density for PS highlights the nonspecific interaction of PS with the PH domain. Peaks in the density distribution correspond to the CA and NCA.

  • Fig. 3 Membrane and solvent PRE data.

    Bilayer proximities of I, V, L, T, and A side chains were measured to PC:PI(4,5)P2 (95:5) NDs doped with an average of two (A) 5-doxyl spin-labeled PC molecules or (B) 10-doxyl spin-labeled PC molecules per leaflet. The resulting PREs were plotted as ΔR2 = R2paramagR2diamag, the difference between methyl proton relaxation rates with and without doxyl-substituted dipalmitoylphosphatidylcholine (DPPC). The uncertainties indicated are those of the fits of the exponential decays and their differences. (C) Solvent PRE expressed as the change of relaxation rate normalized to the probe concentration (mM−1·s−1) as a function of the residue numbers for bound (dashed) or free (solid) ASAP1–PH domain. n = 2, *P < 0.0001, t test. (D) Membrane PRE data mapped on the crystal structure of the ASAP1–PH domain; I/L/V/A/T residues are represented with spheres corresponding to ΔR2 > 30 s−1 (red), 15 s−1 < ΔR2 < 30 s−1 (orange), and ΔR2 < 15 s−1 (green). (E) Solvent PRE data mapped on the crystal structure of the ASAP1–PH domain. Sites protected by more than 50% are marked on the crystal structure by red spheres. An orange sphere is used to label A374 (~30% protection). Deprotection is marked by green spheres. Spectral superimposition prevented the measurement of reliable data for residues marked by ice blue spheres (T375, T408, T444, and L449).

  • Fig. 4 Docking geometry of the ASAP1–PH domain on stBLMs [16:0-18:1 PC:18:1-18:1 PI(4,5)P2 (95:5)] by NR.

    (A) CVO profiles of a lipid bilayer with the surface-bound ASAP1 PH domain (ΔN14ASAP1) parameterized as a Hermite spline. The protein distribution is indicated with its median (black dashed line) and 68.2% confidence region. (B) Data evaluation by rigid-body modeling with the crystal structure (PDB: 5C79) and comparison with the free-form model [same data as in (A)]. Inset: Comparison of protein CVO profiles for ΔN14ASAP1–PH (black line) and ASAP1–PH (dashed line with confidence region) from free-form fitting. (C) Orientation analysis of ΔN14ASAP1–PH at the membrane in terms of two Euler angles (β,γ), as indicated in the inset. Protein orientations are realized by first rotating the reference orientation of the high-resolution structure (see Materials and Methods) by an angle β about the x. Second, the structure is rotated by an angle γ about z′. (D) Visualization of the PH crystal structure on the stBLM in a configuration (orientation and insertion depth) on the membrane at the center of the 68.2% probability region shown in (C). Phospholipids and substrate are only schematically displayed—for example, atomistic corrugation of the interface has been neglected—but lipids are shown at the same scale as the protein. The CVO profile at the left shows the same data as in (B).

  • Fig. 5 MD simulations reveal PH domain approach to the membrane surface and docking geometry.

    (A) Ensemble-averaged Cα profile of 5C79 calculated during the last 50 ns of HMMM simulations in PI(4,5)P2-containing membranes for all nine independent simulations. Red dots correspond to the average z position of each residue, and blue bars represent SD over the averaged time, where z is the distance from the bilayer phosphate plane. (B) Comparison of the 68% probability distribution of ΔN14ASAP1–PH to average orientations obtained from the HMMM MD simulation trajectories.

  • Fig. 6 PI(4,5)P2 triggered conformational switch and interface with L8KArf1•GTP identified through CSP.

    (A) 1H-13C CSPs observed when ASAP1–PH is bound to NDs containing 18:1-18:1 PI(4,5)P2 (filled bars) and when bound to diC4-PI(4,5)P2 (open bars). n = 3, *P < 0.0001, **P < 0.05, t test. (B) Methyl residues that show significant CSP differences and are not located in the CA or NCA sites are plotted in orange on the crystal structure. Residues in the CA or NCA sites are plotted in dark blue. (C) Difference between the 1H-13C CSPs observed for PH binding to NDs in the absence and in the presence of L8KArf1•GTP at a ratio ASAP1–PH:Arf1 1:1. In addition to CSPs on the α helix, small CSPs were observed for residues around the PIP2 binding sites, likely indicating tighter binding of the PH domain in the presence of Arf1 or altered PI(4,5)P2 interactions. n = 2, **P < 0.05, t test. (D) Methyl residues exhibiting significant CSPs upon L8KArf1•GTP binding are plotted in light blue on the crystal structure.

Supplementary Materials

  • Supplementary Materials

    Membrane surface recognition by the ASAP1 PH domain and consequences for interactions with the small GTPase Arf1

    Olivier Soubias, Shashank Pant, Frank Heinrich, Yue Zhang, Neeladri Sekhar Roy, Jess Li, Xiaoying Jian, Marielle E. Yohe, Paul A. Randazzo, Mathias Lösche, Emad Tajkhorshid, R. Andrew Byrd

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