August 1, 2021 - Free Activators

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August 1, 2021 - Free Activators

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With τi, Eqs. (2) and (3) define the perpendicular (\({{{{{{\mathbf{F}}}}}}}_{{{{{{\mathbf{i}}}}}}}^{{{\perp }}}\)) and parallel (\({{{{{\bf{F}}}}}}_{{{{{{\bf{i}}}}}}}^{{\parallel }}\)) components of the total force, respectively.

$${{{{{\mathbf{F}}}}}}_{{{{{\bf{i}}}}}}^{\perp }=-{\nabla }{{{{{\rm{V}}}}}}(Pi)+({\nabla }{{{{{\rm{V}}}}}}({P}_{i}){\cdot }{{{{{\boldsymbol{\tau }}}}}}_{{{{{\bf{i}}}}}}){\cdot }{{{{{\boldsymbol{\tau }}}}}}_{{{{{\bf{i}}}}}}$$

(2)

$${{{{{{\mathbf{F}}}}}}}_{{{{{{\mathbf{i}}}}}}}^{\parallel }=\left({{{{{{\mathbf{F}}}}}}}^{{{{{{\mathbf{s}}}}}}}{{\cdot}}{{{{{{\boldsymbol{\tau }}}}}}}_{{{{{{\mathbf{i}}}}}}}\right){{\cdot }}{{{{{{\boldsymbol{\tau }}}}}}}_{{{{{{\mathbf{i}}}}}}}$$

(3)

$${{{{{{\mathbf{F}}}}}}}^{{{{{{\mathbf{total}}}}}}}={{{{{{\mathbf{F}}}}}}}_{{{{{{\mathbf{i}}}}}}}^{\perp }{+{{{{{\mathbf{F}}}}}}}_{{{{{{\mathbf{i}}}}}}}^{\parallel }$$

(4)

where ∇V(Pi) represents the gradient of the energy regarding the atomic coordinates in replica i, namely the potential force originating from the force fields. Fs is the force provided by the elastic bands. As a result, the force field parameters only contribute to the perpendicular part of the total force, and the spring force is only responsible for the parallel part [Eq. (4)]89,90.

After the preparation, the inactive (PDB 4YAY) and active (PDB 6DO1) structures were set as the initial and end states, respectively. The Amber ff14SB force field was adapted for atom interactions91. First, we conducted 4000 steepest descent and 6000 conjugate gradient energy minimization cycles to our two systems. Then, 20 replicas were created between the inactive and active AT1 receptors with elastic bands connecting each other. The translational and rotational differences between the replicas were excluded by moving the systems to the coordinate of the center of mass and applying an optimal rotation matrix to the replicas to minimize the RMSD between the systems. In the initial heating process, the systems were gradually heated from 0 to 300 K with a spring force of 10 kcal mol−1 Å−2, a timestep of 0.5 fs, and a Langevin collision frequency of 1000 ps−1. During the next equilibration, simulated annealing, and cooling runs, a spring force of 50 kcal mol−1 Å−2 was used. The equilibration runs of the replicas were carried out at 300 K with a timestep of 1 fs. In the simulated annealing runs, the systems were first heated to 500 K, then gradually cooled and equilibrated alternatively at 0 K with a timestep of 0.5 fs. Finally, the replicas were completely cooled at 0 K for 2 ns with a timestep of 1 fs. The detailed workflow has been confirmed in the previous studies27,92.

MD simulation settings

The NEB process provided 22 replicas, but some had highly similar conformations. Thus, we calculated the RMSD between adjacent structures and accordingly selected the most different 15 replicas, including the starting and end structures, as the initial structures for the MD simulations.

All structures were oriented in the Orientations of Proteins in Membranes server93. Next, they were inserted into a POPC membrane using the CHARMM36 additive force field in the CHARMM-GUI server94. TIP3P water molecules with a length of 10 Å were added to the top and bottom of the system. The counterions K+ or Cl and an additional 0.15 mol L−1 KCl were also solvated in the systems. The components of bilayers have been commonly used in other simulations38,41. With the help of the input generator of CHARMM-GUI, we obtained the Amber format coordinate and topology files.

The systems were first minimized with restraint of 500 kcal mol−1 Å−2 on the AT1 receptor and lipids, while water and counterions 2021 - Free Activators minimized in 8000 steepest descent cycles, followed by 7000 conjugate gradient cycles. Second, all atoms encountered 1.5 × 104 cycles of steepest descent and 1.5 × 104 cycles of conjugate gradient minimization. Next, with 10 kcal mol−1 Å−2 position restraint on proteins and lipids, the systems were gradually heated from 0 to 300 K in 300 ps and equilibrated for 700 ps under canonical ensemble conditions. Then, the 15 systems underwent 10 rounds of 2 μs MD simulations, with an integration step of 2.0 fs. Finally, we collected 150 independent repeat trajectories with random initial velocities. The total simulation timescale was 300 μs. During simulations, the Particle mesh Ewald method was applied to calculate the long-range electrostatic interactions, and a cutoff of 10 Å was used for 2021 - Free Activators electrostatic and van der Waals interactions. The SHAKE algorithm was employed for covalent bonds containing hydrogen. A temperature of 300 K was controlled by Langevin Thermostat, while the collision frequency was 1.0 ps−1. The snapshots were written out every 200 ps.

Markov State Model construction

According to the activation parameters, an MSM was built using the PyEMMA protocol (http://www.emma-project.org/latest/)95. Through the implied timescale validation (Supplementary Note 5), we confirmed that the AT1 receptor systems were Markovian and reliable with a 200 microstate model with a lag time of 8 ns and a maximum k-means iteration number of 200. Then, the microstates were clustered into three macrostates via the PCCA+ algorithm, which was confirmed by a Chapman–Kolmogorov test. Using TPT, we measured the transition probability matrix of the MSMs and computed the mean first passage time for each activation and inactivation process96. Based on the mdtraj package, we extracted the structures close to the microstate cluster centers of each macrostate into the trajectories for the corresponding macrostates. Then, using three trajectories, we selected the representative conformation of each macrostate according to the similarity score Sij. As shown in Eq. (5), the conformation with the highest Sij among the trajectories was regarded as the most representative conformation of the macrostate. The dij is the RMSD between the conformations i and j, while dscale is the standard deviation of d.

$${S}_{{ij}}={e}^{-{d}_{{ij}}/{d}_{{{{{{\rm{scale}}}}}}}}$$

(5)

Measurement of receptor expression by ELISA

HEK293 cells were transiently transfected with Flag-tagged WT AT1 receptor or mutants, Flag-tagged V2R receptor, or Flag-tagged AT1R conformational sensor or sensor-based mutants in 24-well plates. After incubation at 37 °C for 48 h, the cells were fixed with DPBS containing 4% (w/v) formaldehyde for 5 min at room temperature. For whole-cell ELISA, the cells were incubated in blocking solution (5% BSA in DPBS) containing 0.2% Triton X-100 for 1 h at room temperature. For cell surface ELISA, the cells were incubated in blocking solution without Triton X-100. The cells were washed three times with DPBS and incubated overnight with an anti-Flag primary antibody (Sigma Aldrich, Cat# F1804, 1:1000) at 4 °C, followed by incubation with a horseradish peroxidase-conjugated secondary anti-rabbit antibody (Thermo Fisher, Cat# A-21235, 1:5000) for 1 h at room temperature. After washing, tetramethyl benzidine solution was added and the reaction was stopped by adding an equal volume of 0.25 M HCl solution. The solution was incubated for 5 min. The optical density of each well was measured at 450 nm using the TECAN luminescence counter (Infinite M200 Pro NanoQuant). The optical density was plotted against the transfecting amounts of respective plasmids to determine the relative expression levels of each receptor or mutants. The levels of the AT1 receptor mutants were normalized to that of the WT receptor.

Site-directed mutagenesis

All AT1 receptor mutants used in the present study were generated by site-directed mutagenesis. The successful introduction of the mutations in the polymerase chain reaction products was verified by DNA sequencing. Corresponding primer sequences were shown in Supplementary Data 2.

BRET measurement

β-arrestin 2 recruitment

HEK293 cells were transiently cotransfected with β-arrestin 2-Rluc and YFP-tagged WT or mutated AT1 receptor. After 24 h, the cells were seeded on 96-well microplates and incubated for 24 h at 37 °C, after which were washed twice with HBSS and stimulated with AngII at different concentrations. Luciferase substrate coelenterazine-h (Promega) was added at the concentration of 5 μM before light emissions were recorded using a Mithras LB940 microplate reader (Berthold Technologies). The BRET signal was determined by calculating the ratio of luminescence at 530/485 nm.

G protein activation

Gq (Gq-RLuc8, Gβ3, Gγ9-GFP10), Gi (Gi1-RLuc8, Gβ3, Gγ9-GFP10), and G12(G12-RLuc8, Gβ3, Gγ9-GFP10) BRET probes were from the TRUPATH kit, which was a gift from Bryan Roth (Addgene kit #1000000163)97. HEK293 cells were transiently co-transfected with WT or mutated AT1 receptor along with specific G protein BRET probes according to the experimental setting. After 24 h, the cells were seeded on 96-well microplates and incubated for an additional 24 h. For the constitutive activity measurement, cells transfected with varying amounts of WT or mutated AT1 receptor, or V2R were washed twice with HBSS buffer and the BRET signal was directly recorded after the addition 2021 - Free Activators 5 μM luciferase substrate coelenterazine 400a using a Mithras LB940 microplate reader. For the AngII-stimulated G protein activation, the cells were washed twice with HBSS and stimulated with AngII at different concentrations. BRET signal was subsequently measured after the addition of the luciferase substrate and was calculated as the ratio of light emission at 510/400 nm.

The data obtained in the G protein activation assay and β-arrestin recruitment assay were normalized as a percentage of the Emax of WT AT1 Avira Antivirus Pro 2018 Crack + Serial Key Free Download (reference receptor) in each pathway. The normalized data were analyzed using the “Operational Model” in GraphPad Prism to determine the transduction coefficient log(τ/KA), in which τ is the transducer ratio and KA is an agonist-receptor dissociation constant98, of the WT AT1 receptor and mutants at each signaling pathway99,100.

The equation for the “Operational Model” was defined by the following parameters:

Equation Type - Explicit Equation: Y = a function of X and parameters

Definition: A = 10^X

operate1 = ((1 + A)/((10^LogR)*A))^n

operate2 = ((1 + A/(10^LogKA))/((10^LogR)*A))^n

Y1 = basal + (Emax − basal)/(1 + operate1)

Y2 = basal + (Emax − basal)/(1 + operate2)

<A:O>Y = Y1

<~A:O>Y = Y2

The relative activities [Δlog(τ/KA)] of AT1 receptor mutants in each signaling pathway was determined by the Eq. (6):

$$\Delta {{\log }}\left(\frac{\tau }{{K}_{A}}\right)={{{\log }}\left(\frac{\tau }{{K}_{A}}\right)}_{{{{{{\rm{mutant}}}}}}}-{{{\log }}\left(\frac{\tau }{{K}_{A}}\right)}_{{{{{{\rm{WT}}}}}}}$$

(6)

The SEM of the Δlog(τ/KA) was calculated according to Eq. (7):

$${{{{{{\rm{SEM}}}}}}}_{\Delta {{\log }}(\frac{\tau }{{K}_{A}})}=\sqrt{{({{{{{{\rm{SEM}}}}}}}_{{{{{{\rm{mutant}}}}}}})}^{2}+{({{{{{{\rm{SEM}}}}}}}_{{{{{{\rm{WT}}}}}}})}^{2}}$$

(7)

Intramolecular FlAsH-BRET assay

The AT1R conformation sensor was generated by fusing Rluc to the C-terminus of the AT1 receptor and inserting a TC-tag (CCPGCC) between K224 and A225 at ICL3. HEK293 cells were transfected with the AT1R conformation sensor without or with different mutations. After 48 h, the cells were labeled with a 2.5-μM FlAsH-ETD2 from a TC-FlAsH II In-Cell Tetracysteine Tag Detection August 1 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The cells were then stimulated with varying concentrations of AngII and BRET signal was measured after addition of 5 μM RLuc substrate coelenterazine-h using a Mithras LB940 microplate reader. The BRET signal was determined as the ratio of luminescence at 530/485 nm.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.