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A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response. Evidence supporting these various latter hypotheses is indirect or subject to alternative interpretation, and it remains unknown if endosome-localized GPCRs are even present in an active form. Here we describe the application of conformation-specific single-domain antibodies (nanobodies) to directly probe activation of the 2-adrenoceptor, a prototypical GPCR, and its cognate G protein, Gs (ref. 12), in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational change in vivo.
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Copyright: 2020 Liu, Gumbart. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Institutes of Health (R01-GM123169). JL was supported by Office of Undergraduate Education, funded by Tang Aoqing Honors Program in Science. JL is grateful to College of Chemistry of Jilin University for sustaining research training abroad. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
(A) BamA of N. gonorrhoeae in its OM with protein in white. 1 is highlighted in red and 16 is highlighted in purple. (B) Five POTRA domains of BamA of N. gonorrhoeae in tandem with different colors.
With the exact mechanisms of OMP insertion by BamA and TamA unknown, we investigated the structural features of BamAs of different species together with TamA of E. coli using molecular dynamics (MD) simulations with each embedded in its respective native OM. We constructed 12 systems with and without POTRA domains and ran 45.5 s of equilibrium simulations as well as over 14 s of replica-exchange umbrella sampling (REUS) to probe the lateral gating of BamA (see Table 1 for the full list). Our simulations show that the frequency of lateral gating for BamA is species dependent while membrane thinning for BamA is species independent, thus shedding light on potential insertion mechanisms.
An additional system starting from EcBamA was also constructed. In this system, a model of the E. coli peptidoglycan (PG) cell wall [57, 58] was added in the periplasm. The PG was anchored to the OM via five Lpp trimers (PDB 1EQ7 [59]), which are tri-acylated at their N-termini (inserted into the inner leaflet of the OM) and covalently bonded to PG for one out of every three copies at their C-terminal end [60]. The number of Lpp was selected to be commensurate with the roughly 500,000 copies per cell [1].
All systems were equilibrated first in NAMD by releasing system components sequentially (lipid tails for 1 ns, everything except protein for 10 ns, everything except protein backbone for 10 ns, everything for 10 ns). The systems were minimized for 2000 steps before each step. After equilibration, all systems but the PG-containing one were run twice in Amber for 2000 ns each using HMR and a time step of 4 fs [68, 69]. Because systems with bonds crossing periodic boundaries cannot be run in Amber, the PG-containing system was run in NAMD for 1500 ns using HMR.
We used REUS to calculate the potential of mean force (PMF) for lateral gate opening [70]. Targeted Molecular Dynamics (TMD) was used to generate starting states for REUS [71] (see S1 Fig for more details about TMD). The collective variable for BamA of N. gonorrhoeae is defined as the distance between N and O atoms that potentially form hydrogen bonds at the lateral gate projected to the direction that lateral gates open (see S2 Fig). The colvars module of NAMD was used to construct all collective variables [72]. A total of 17-20 unevenly distributed windows were used for REUS, covering a range from 2-12 . Different force constants were used for different windows during REUS: see S2 Table for centers and force constants. The sampling data of REUS was used to calculate the PMF using the weighted histogram analysis method (WHAM) [73]. Each REUS simulation was run until an additional 5 ns changed the PMF endpoint by less than 0.2 kcal/mol at which point it was considered to be converged (see S3 Fig).
(A) Average thickness around the -barrel for the first run of each system (See S6 Fig for all systems). Labeled concentric circles represent the membrane thickness in . (B) Average thickness of the whole membrane (in red) as well as average thickness near 8 and 9 at the back of the -barrel in grey and near the lateral gate in blue.
(A) Snapshots of a closed state of NgBamA (top) and the maximum strand separation in NgBamA (bottom) with 1 in red and 16 in purple. (B) Snapshots of -barrel of NgBamA before (left) and after (right) the sliding. (C) Strand separation (top) and number of hydrogen bonds between backbones of 1 and 16 (bottom) of NgBamA system over time. Strand separation is defined by the average of the distance between O of W432 and N of L788 and the distance between N of W432 and O of L788 in NgBamA. A cutoff distance of 3.5 and an angle of 30 degrees are used to define a hydrogen bond.
One of the most unexpected observations from our equilibrium simulations is sliding of the lateral gate (Fig 3B). In our NgBamA equilibrium simulation, W432 and L788 form hydrogen bonds initially. As the lateral gate opens, the hydrogen bonds break and the distance between W432 and L788 fluctuates. Then the distance sharply increases and stays above 8 while the number of hydrogen bonds rises simultaneously (Fig 3C), which results from F786 sliding down and forming hydrogen bonds with W432. Additionally, we observed sliding of the lateral gate in HdBamAP, which explains the apparent sharp increase in strand separation (S8 Fig). This observed sliding agrees with experimental results in which EcBamA can form disulfide cross-links between lateral gate residues (G433C-T809C and G431C-Q803C) that are 14 apart in the crystal structure [33], demonstrating its flexibility.
To further investigate lateral gate opening, we ran replica-exchange umbrella sampling (REUS) simulations to calculate the potential of mean force (PMF) for opening of all systems with and without POTRA domains. In order to avoid conflating the sliding conformation with the lateral gate opening conformation, we projected the distance of atoms forming hydrogen bonds at the lateral gate on the direction it opens. For example, we projected the vector that the N/O atoms of W432 and O/N atoms of L788 form in NgBamA (and the corresponding residues for other proteins) on their direction at maximum separation, defined as the lateral gate opening coordinate (S2 Fig).
We use FhaC as a control since it is also a member of the Omp85 family but does not assist with OMP insertion [76]. As expected, with or without POTRA domains, the PMFs of BamA and TamA are lower than those of FhaC [34]. Unexpectedly, although all PMFs tend to increase as the strands separate, a well in the PMF appears from 3.5 to 5.5 . This small drop in the PMFs is due to water molecules going between 1 and 16 and forming hydrogen bonds with the backbone, stabilizing the open state slightly (S9 Fig).
(A) PMFs of lateral gate opening for systems with POTRA domains. (B) PMFs of lateral gate opening for barrel-only systems. In both panels, the region over which hydrogen bonds between the N- and C-terminal strands are ruptured is indicated.
Based on our equilibrium simulations, we quantified the interaction propensity of each -barrel residue with lipids, LPS, water, and other protein residues. For each residue, the number of atoms within 4 of its side chain for each type of molecule is accumulated over the two 2-s runs. We plot the percentage for each type in S10 Fig. Unsurprisingly, the 16 -stands primarily interact with lipids while loops and turns interact with water, as previously reported for another OMP, OmpLA [77]. Additionally, it is evident that the -strand residues whose side chains are inside the barrel are interacting more with water, while the -strand residues whose side chains are outside the barrel are interacting with lipid A head group, lipid A tail, PL tail and PL head group periodically.
Briefly, this difference is a Euclidean distance between each set of interaction propensities, averaged over all aligned residues between two proteins. See S11 Fig for further details of the calculation.
To interrogate the hybrid barrel model of OMP insertion and probe the dynamics of the lateral gate in BamA from different species, we ran a series of equilibrium simulations at 310 K with or without POTRA domains to examine the gate separation. In two 2-s simulations for each of the nine BamA/TamA systems, we observed spontaneous lateral gate opening for N. gonorrhoeae BamA and E. coli TamA. No lateral gate opening is observed for E. coli, S. enterica, and H. ducreyi BamA, nor for our control protein, FhaC, on the 2-s time scale. We next calculated the energetic landscape of lateral gate opening, which supports the observations from the equilibrium simulations. NgBamA requires the least energy to open its gate, explaining why it was observed in 2 s. E. coli and S. enterica systems, on the other hand, require the most energy, consistent with no lateral gate opening observed in equilibrium simulations. Also, after 2-4 of separation, all the PMFs of gate opening in BamA and TamA are lower than the respective FhaC systems (with or without POTRA domains; Fig 3). Additional evidence for a functional role of lateral gating in BamA comes from a recent structure in which two BamAs, both with open gates, are in contact [37]. 152ee80cbc
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