human brd9 plasmid  (Sangon Biotech)

Journal: bioRxiv

Article Title: The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

doi: 10.1101/2023.02.14.528445

Figure Lengend Snippet: A) Coarse-grained (CG) and atomistic simulation setups of PTCH1 (light blue, PDB: 6RVD (CG)/6DMY (atomistic)) embedded in 3:1 POPC:cholesterol (white/purple) bilayers. Water is shown as transparent surface and Na + /Cl − ions are shown as blue/salmon spheres respectively. The inset shows the structure of cholesterol. B) Schematic diagram of the free energy changes associated with cholesterol movement between the PTCH1 sterol sensing domain (SSD) and the sterol binding domain (SBD) for the direct (ΔG 1 ) and indirect (ΔG 2 , ΔG 3 , ΔG 4 ) pathways. PTCH1-molA (yellow) and PTCH1-molB (light blue) are shown with either the ‘SHH-cholesterol’ (dark blue) or the ‘free cholesterol’ (purple) molecules positioned in the SBD and SSD (teal/ochre). The position of the extracellular (EC) and intracellular (IC) membrane leaflets are indicated in grey. C-E) CG potential of mean force (PMF) profiles for movement of ‘free cholesterol’ (purple) and ‘SHH-cholesterol’ (dark blue) between the SBD and the solvent (ΔG 4 ) ( C ) or the SSD and the bulk membrane (ΔG 2 ) ( E ) or for extraction of cholesterol from the membrane into the solvent (ΔG 3 ) ( D ). Bayesian bootstrapping (2000 rounds) was used to estimate profile errors (grey). Stars indicate the position of the ‘SHH-cholesterol’ (within PTCH1-molA) and ‘free cholesterol’ (within PTCH1-molB) densities in a revised cryo-EM structure (PDB: 6RVD ).

Article Snippet: Human Patched1 (hPtch1), comprising amino acids 75–1185 including a deletion of intracellular loop 3 (ICL3) (Δ630–717), was cloned into the pHR-CMV-TetO2 vector (Addgene plasmid #113893).

Techniques: Binding Assay, Membrane, Solvent, Extraction, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

doi: 10.1101/2023.02.14.528445

Figure Lengend Snippet: A) Schematic diagram of the free energy changes associated with cholesterol movement between the SSD and SBD of PTCH1 for the direct pathway, coloured identically to in . Decoupling of cholesterol from the SBD/SSD sites are indicated by red circles, with the difference between them (ΔΔG 1 ) shown as a red arrow to indicate alchemical transformation. A black arrow indicates shows movement of cholesterol between the ECD base and the SBD, used in PMF calculations to derive ΔG 1 a. Grey arrows correspond to currently uncertain regions of the transport pathway. B) The free energy values of decoupling cholesterol from the SSD (PTCH1-molA) and SBD (PTCH1-molA and PTCH1-molB) as obtained from absolute binding free energy (ABFE) calculations. C) Snapshots from PMF-1a indicating movement of cholesterol between the ECD base and SBD of PTCH1 (residues used in the steered MD are labelled in red). CG representation of PTCH1 backbone beads are shown in transparent light blue/yellow, ‘free cholesterol’ is coloured purple, ‘SHH-cholesterol’ is coloured dark blue and lipid phosphate beads are shown in grey. D) PMF profile for cholesterol movement through the ECD of PTCH1-molA/B (ΔG 1 a) (see C ). Bootstrapping errors (2000 rounds) are shown in grey. The position of cholesterol within the SBD pocket in the cryo-EM model (PDB: 6RVD ) is starred. Arrows in D indicate energetic peaks 1–3 within the ECD core conserved between PTCH1-molA and PTCH1-molB (see ). E) HH signalling strength is determined by measuring endogenous Gli1 mRNA abundances (normalized to the control Gapdh) in response to SHH ligands (200 nM, 20 hours) in Ptch1 −/− cells stably expressing the indicated variants. Statistical significance is determined by a Student’s t-test with a Welch’s correction. Exact P values for comparisons: Ptch1 −/− untreated vs. SHH = 0.085, WT untreated vs. SHH < 0.0001, D513Y untreated vs. SHH = 0.2712, and P155R/N802E untreated vs. SHH = 0.0205. not significant (ns), * P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Article Snippet: Human Patched1 (hPtch1), comprising amino acids 75–1185 including a deletion of intracellular loop 3 (ICL3) (Δ630–717), was cloned into the pHR-CMV-TetO2 vector (Addgene plasmid #113893).

Techniques: Transformation Assay, Binding Assay, Cryo-EM Sample Prep, Control, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

doi: 10.1101/2023.02.14.528445

Figure Lengend Snippet: Comparison of ΔG 1 values for ‘free cholesterol’ (purple) and ‘SHH-cholesterol’ (dark blue) movement between the PTCH1 SSD and SBD, derived from the indirect (PMF, ) and direct (ABFE, ) pathways. Step wise free energy changes are shown as connecting arrows and are labelled. ΔG 1 from the indirect pathway was calculated in quadrature since PMFs 2–4 were independent. In all cases cholesterol movement from the SSD to the SBD gives ΔG > 0 kJ mol −1 .

Article Snippet: Human Patched1 (hPtch1), comprising amino acids 75–1185 including a deletion of intracellular loop 3 (ICL3) (Δ630–717), was cloned into the pHR-CMV-TetO2 vector (Addgene plasmid #113893).

Techniques: Comparison, Derivative Assay

Journal: bioRxiv

Article Title: The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

doi: 10.1101/2023.02.14.528445

Figure Lengend Snippet: A) Snapshot from atomistic simulations of PTCH1 (light blue, PDB: 6DMY ) indicating the location of cation binding sites within the TMD. Na + ions are shown as blue spheres and lipid phosphates are grey. Inset: cation-like density surrounded by anionic tired residues at Site 1 within the cryo-EM structure , as viewed from the extracellular face. A cylinder (length 4 nm, radius 1.3 nm) centred on the midpoint of V520 and I1092 Cα atoms is shown in grey and was used to identify water and ions within the PTCH1 TMD ( B) . B) The z coordinates of water oxygen atoms (light blue), Na + (blue) and Cl − (salmon) ions localised within the PTCH1 TMD (see A , grey cylinder) over the length of 3 × 100 ns simulations initiated with Na + bound at the density observed in A . Snapshots of Site 1 ( C ) and Site 2 ( D ) showing coordination of bound Na + ions by surrounding residues (stick representation) or waters (w 1–6 ). E) Free energy perturbation (FEP) calculations for alchemical transformation of Na + into K + within the solvent or bound to Site 1. F) Schematic representation of the free energy cycle used to calculate the difference in Na + binding to Site 1 compared to K + (ΔΔG).

Article Snippet: Human Patched1 (hPtch1), comprising amino acids 75–1185 including a deletion of intracellular loop 3 (ICL3) (Δ630–717), was cloned into the pHR-CMV-TetO2 vector (Addgene plasmid #113893).

Techniques: Binding Assay, Cryo-EM Sample Prep, Transformation Assay, Solvent

Journal: bioRxiv

Article Title: The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

doi: 10.1101/2023.02.14.528445

Figure Lengend Snippet: Residue contacts with Na + bound at Site 1 across 3 × 100 ns simulations of PTCH1. Residues with < 1 % contact are excluded.

Article Snippet: Human Patched1 (hPtch1), comprising amino acids 75–1185 including a deletion of intracellular loop 3 (ICL3) (Δ630–717), was cloned into the pHR-CMV-TetO2 vector (Addgene plasmid #113893).

Techniques: Residue

Journal: bioRxiv

Article Title: The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

doi: 10.1101/2023.02.14.528445

Figure Lengend Snippet: A) Time averaged water density (blue isosurface) across a 100 ns simulation of PTCH1 (PDB: 6DMY ) initiated with Na + bound at Site 1. PTCH1 TMD is shown in ribbon representation and anionic triad residues are shown as spheres. Yellow arrows indicate paths for water entry into the TMD. B) Residues comprising the hydrophobic cap (red box in A ) shown in stick and surface representation, as viewed from the extracellular (EC) face. V510 and I1092 forming part of the conserved GXXXDD and GXXX(E/D) motifs on TM4 and TM10 are boxed in blue. Residues mutated in disease phenotypes are boxed in black. C) Mean number of waters per frame within the EC half of the PTCH1 TMD across the final 10 ns of 3 × 50 ns simulations of WT PTCH1 or PTCH1 mutants (see ). D) HH signalling strength is determined by measuring endogenous Gli1 mRNA abundances (normalized to the control Gapdh) in response to SHH ligands (200 nM, 20 hours) in Ptch1 −/− cells stably expressing the indicated variants. Statistical significance is determined by a Student’s t-test with a Welch’s correction. Exact P values for comparisons: Ptch1 −/− untreated vs. SHH = 0.085, WT untreated vs. SHH < 0.0001, D513Y untreated vs. SHH = 0.2712, V510F untreated vs. SHH = 0.0002, L517C/P1125C untreated vs. SHH = 0.0255, and L570C/V1081C untreated vs. SHH = 0.0014. not significant (ns), * P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. E) Comparison of cation binding sites within PTCH1 and DISP1 (PDB: 7RPH ) TMDs. Anionic triad residues are shown as sticks. F) Time averaged water density profiles across 100 ns simulations of DISP1 in apo or ‘3x Na + ’ bound conformations. G) Mean number of waters per frame within the intracellular (IC) half of the DISP1 TMD across the final 10 ns of 3 × 100 ns simulations of DISP1 in apo and ‘3x Na + ’ bound states, or in ‘2x Na + ’, ‘1x Na + ’ and ‘0x Na + ’ bound states generated by sequential ion removal from the end of the previous Na + bound state or PTCH1 in apo, ‘3x Na + ’ and ‘3x K + ’ bound states (see ). C and G report the mean and standard deviation between repeats.

Article Snippet: Human Patched1 (hPtch1), comprising amino acids 75–1185 including a deletion of intracellular loop 3 (ICL3) (Δ630–717), was cloned into the pHR-CMV-TetO2 vector (Addgene plasmid #113893).

Techniques: Control, Stable Transfection, Expressing, Comparison, Binding Assay, Generated, Standard Deviation

Journal: bioRxiv

Article Title: The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

doi: 10.1101/2023.02.14.528445

Figure Lengend Snippet: A) Proposed models for PTCH1 function (coloured as in ). Model-1: PTCH1 imports cholesterol extracted from SMO to a membrane sequestered pool or intracellular donor (energetically favourable). Model-2: Accessible cholesterol export by PTCH1 mediated by coupling to 1–3 Na + ions (blue, model-2a) or 2–3 K + ions (yellow, model-2b). Transition between ‘inward-open’ and ‘occluded’ states accompanied by breathing-like motions of intracellular helical segments (solid arrows) and alleviation of the hydrophobic cap (red). Model-3: PTCH1 re-partitions accessible cholesterol to an intramembrane sequestered pool (e.g. partnered with sphingomyelin) or intracellular acceptor. B) Free energy stored across the Na + (blue) and K + (yellow) membrane gradients as a function of membrane potential (ΔV) . C) Predicted number of Na + /K + coupling ions required per cholesterol exported by PTCH1 (defined as ‘cholesterol export free energy’/’free energy across cation potential’ from B ) vs membrane potential (ΔV). A cholesterol export free energy of +20 kJ mol −1 is indicated by a solid line, with export energies ranging between +10 to +40 kJ mol −1 indicated in transparent. Standard cellular ion concentrations ([Na + ] in : 12 mM, [Na + ] out : 145 mM, [K + ] in : 150 mM, [K + ] out : 4 mM) are assumed.

Article Snippet: Human Patched1 (hPtch1), comprising amino acids 75–1185 including a deletion of intracellular loop 3 (ICL3) (Δ630–717), was cloned into the pHR-CMV-TetO2 vector (Addgene plasmid #113893).

Techniques: Membrane