Structural basis for cannabinoid-induced potentiation of alpha1-glycine receptors in lipid nanodiscs.

Nociception and motor coordination are critically governed by glycine receptor (GlyR) function at inhibitory synapses. Consequentially, GlyRs are attractive targets in the management of chronic pain and in the treatment of several neurological disorders. High-resolution mechanistic details of GlyR function and its modulation are just emerging. While it has been known that cannabinoids such as Δ9-tetrahydrocannabinol (THC), the principal psychoactive constituent in marijuana, potentiate GlyR in the therapeutically relevant concentration range, the molecular mechanism underlying this effect is still not understood. Here, we present Cryo-EM structures of full-length GlyR reconstituted into lipid nanodisc in complex with THC under varying concentrations of glycine. The GlyR-THC complexes are captured in multiple conformational states that reveal the basis for THC-mediated potentiation, manifested as different extents of opening at the level of the channel pore. Taken together, these structural findings, combined with molecular dynamics simulations and functional analysis, provide insights into the potential THC binding site and the allosteric coupling to the channel pore.

Atomistic mechanisms of human TRPA1 activation by electrophile irritants through molecular dynamics simulation and mutual information analysis.

The ion channel TRPA1 is a promiscuous chemosensor, with reported response to a wide spectrum of noxious electrophilic irritants, as well as cold, heat, and mechanosensation. It is also implicated in the inception of itch and pain and has hence been investigated as a drug target for novel analgesics. The mechanism of electrophilic activation for TRPA1 is therefore of broad interest. TRPA1 structures with the pore in both open and closed states have recently been published as well as covalent binding modes for electrophile agonists. However, the detailed mechanism of coupling between electrophile binding sites and the pore remains speculative. In addition, while two different cysteine residues (C621 and C665) have been identified as critical for electrophile bonding and activation, the bound geometry has only been resolved at C621. Here, we use molecular dynamics simulations of TRPA1 in both pore-open and pore-closed states to explore the allosteric link between the electrophile binding sites and pore stability. Our simulations reveal that an open pore is structurally stable in the presence of open 'pockets' in the C621/C665 region, but rapidly collapses and closes when these pockets are shut. Binding of electrophiles at either C621 or C665 provides stabilisation of the pore-open state, but molecules bound at C665 are shown to be able to rotate in and out of the pocket, allowing for immediate stabilisation of transient open states. Finally, mutual information analysis of trajectories reveals an informational path linking the electrophile binding site pocket to the pore via the voltage-sensing-like domain, giving a detailed insight into the how the pore is stabilized in the open state.

Comment on "On the Functional Annotation of Open-Channel Structures in the Glycine Receptor".

Recently, we reported the simulation of a stable open state of the glycine receptor. Central to the stability of the simulations was the behavior of the highly conserved leucine residues at the 9' gate, which were found to rotate into adjacent pockets, thus providing a structural rationale for decades of biochemical observations. In contrast, a previously reported model from Cerdan et al. (2018) resembled a more collapsed state. However, in support of their model, they draw attention to the agreement between calculated and experimental conductance measurements and argue that our model tends to overestimate ion flow. Here, we argue that there are many pitfalls with this approach and that the apparent agreement most likely reflects a fortuitous cancellation of errors. The computed values are highly sensitive to very small changes in model parameters. It is also likely that polarization effects will be very significant, and these have not yet been considered.

Amphiphilic copolymers change the nature of the ordered-to-disordered phase transition of lipid membranes from discontinuous to continuous.

The phase behaviour as a function of temperature is explored for pure phospholipid (DPPC) and hybrid lipid-polymer (DPPC/Pluronic L64) bilayers with the aid of atomistic MD simulations. The range of the fixed-temperature simulations includes temperatures below and above the known melting temperature (Tm) for DPPC membranes. For the pure lipid bilayer, the main phase transition is discontinuous, as verified by the abrupt changes observed in the membrane structure, elasticity and the lipid diffusivity near the critical temperature Tm, which lies in the region 298.15-303.15 K. A pre-transition step is detected at 298.15 K which has been identified as the ripple phase (Pß'), where ordered and disordered lipids coexist, causing thickness fluctuations. In the ordered gel phase, the positional ordering as assessed by the lipid radial distribution functions is long-range and some degree of hexagonal packing is measured. The hybrid bilayers on the other hand, transform from a more ordered to a disordered phase in a continuous manner, without finite jumps in their properties. No signs of the ripple phase are identified and the ordered phase exhibits very limited hexagonal packing and some positional ordering that decays fast. The effect of the inserted polymers in the two phases is reversed; at low temperatures, they render the membrane thinner, less cohesive and less ordered compared to the pure one, with the lipids assuming faster diffusion rates, whereas at high temperatures, the polymer interaction with the lipids acts reducing their diffusivity, but also increasing the lipid tail ordering and the membrane stiffness. The ability of the amphiphilic L64 copolymers to change the nature of the main phase transition of lipid membranes and their properties both in the ordered and the disordered phase is of vital importance for the prediction of both the efficacy of hybrid lipid/polymer nanoparticles as drug delivery vehicles as well as their potential adverse implications during interactions with healthy cell membranes.

How the Incorporation of Pluronic Block Copolymers Modulates the Response of Lipid Membranes to Mechanical Stress.

We employ atomistic molecular dynamics simulations to investigate the effect that the incorporation of the nonionic amphiphilic copolymer known as Pluronic L64 has on the mechanical stability of a DPPC membrane. The simulations reveal that the incorporation of the polymer chains leads to membranes that can sustain increasing mechanical stresses. Analysis of mechanical, structural, and dynamic properties of the membrane shows that the polymer chains interact strongly with the lipids in the vicinity, restraining their mobility and imparting better mechanical stability to the membrane. The hybrid membranes under tension remain thicker, more ordered, and stiffer in comparison to their lipid analogues. Trans-bilayer lipid movements (flip-flop) are observed and appear to be triggered by the presence of the polymer chains. A careful analysis of the pore formation under high tensions reveals two distinctive mechanisms that depend on the distribution of the hydrophilic polymer blocks in the bilayer. Finally, the rate of growth of the formed membrane defects is slowed down in the presence of polymers. These findings show that Pluronic block copolymers could be exploited for the formation of optimized hybrid nanodevices with controlled elastic and dynamic properties.

Unexpected Like-Charge Self-Assembly of a Biguanide-Based Antimicrobial Polyelectrolyte.

Polyelectrolyte chains dissolved in good solvent are expected to collapse in compact configurations in the presence of multivalent ions. Here, we show that a weakly charged, hydrophilic polyelectrolyte containing biguanide groups self-assembles in water also in the presence of monovalent counterions, even at low salt concentrations. The polymer assembles in a compact, ordered, hairpin-like shape that, with increasing the ionic strength of the solution, can collapse further in three- or five-folded structures. Neither water nor ions mediate the self-assembly which, instead, is driven by the like-charge pairing of the biguanide units. The thermodynamics of the self-assembly show that the self-association is enthalpically driven, is isodesmic (at least at low aggregation number), and is favored by increasing salt concentration. This unique self-assembly behavior may be linked to the well-known polymer's antimicrobial properties and could help in rationalizing its biological activity.