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Partial inactivation of the triad core of β2AR in a DOPC membrane. (A) RMSD profile of the conformational changes of the triad core: I 3.40 (red), P 5.50 (green) and F 6.44 (blue) over a 4 μs MD simulation in a DOPC membrane, compared to the active crystal structure (PDB id: 3SN6). (B) Superposition of the triad core observed at 4 μs compared to the active crystal structure triad core (orange, PDB id: 3SN6). Relevant residues are labelled: proline (P), isoleucine (I), phenylalanine (F) on transmembrane (TM) helices 3, 5, 6.
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Lipids are becoming known as essential allosteric modulators of G protein-coupled receptor (GPCRs). However, how they exert their effects on GPCR conformation at the atomic level is still unclear. In light of recent experimental data, we have performed several long-timescale molecular dynamics (MD) simulations, totalling 24 μs, to rigorously map al...
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... DOPC partially inactivates β2-adrenergic receptor. In a 4 μs MD simulation of β2AR in a homogenous membrane of DOPC, ICL3 shows high flexibility and transiently adopts different conformations during the first microsecond (SI Fig. 3). During this time, the loop does not interact with the membrane but gradually moves to an inward position, interacting with ICL2 on the intracellular side of the receptor, as well as intermittently with H8 (SI Fig. 3). From 1 μs onwards, β2AR maintains ICL3 in this inward conformation (SI Fig. 3). The conformational changes of ICL3 ...
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... membrane of DOPC, ICL3 shows high flexibility and transiently adopts different conformations during the first microsecond (SI Fig. 3). During this time, the loop does not interact with the membrane but gradually moves to an inward position, interacting with ICL2 on the intracellular side of the receptor, as well as intermittently with H8 (SI Fig. 3). From 1 μs onwards, β2AR maintains ICL3 in this inward conformation (SI Fig. 3). The conformational changes of ICL3 precede a change in the conformation of TM6, which occurs from 2 μs onwards. In an RMSD profile of TM6 with respect to the inactive-state crystal structure of β2AR (PDB entry: 2RH1), TM6 begins at 7.0 Å but decreases to ...
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... conformations during the first microsecond (SI Fig. 3). During this time, the loop does not interact with the membrane but gradually moves to an inward position, interacting with ICL2 on the intracellular side of the receptor, as well as intermittently with H8 (SI Fig. 3). From 1 μs onwards, β2AR maintains ICL3 in this inward conformation (SI Fig. 3). The conformational changes of ICL3 precede a change in the conformation of TM6, which occurs from 2 μs onwards. In an RMSD profile of TM6 with respect to the inactive-state crystal structure of β2AR (PDB entry: 2RH1), TM6 begins at 7.0 Å but decreases to 3.5-5.5 Å from 2 μs onwards, finishing at 4.7 Å (SI Fig. 4). This suggests that ...
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... in its helical state (Fig. 2). This is despite the receptor containing certain features of the inactive crystal structure such as helical extension at the intracellular end of TM7 (Fig. 2). Likewise, conformational changes are also observed in the triad core, which maintains an active-like conformation of I 3.40 , P 5.50 and F 6.44 until 3.5 μs (Fig. 3), at which point, the RMSD of F 6.44 increases to 4.6 Å, which resembles an inactive-like conformation. Supporting the partial inactivation of β2AR into an intermediate state, the ionic-lock distance gradually decreases to 11.1 Å over 4 μs (SI Fig. 5) and the TM domain reaches an RMSD of 3.4 Å compared to the inactive crystal structure ...
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... DOPE fully deactivates β2-adrenergic receptor. In a 4 μs MD simulation of β2AR in a homog- enous DOPE membrane, like in DOPC, ICL3 shows high flexibility and adopts different conformations during the first 2 μs (SI Fig. 3). During this time, ICL3 does not interact with the membrane but does not reach a stable conformation either. Interestingly, the RMSD profile of TM6 shows faster conformational changes than in DOPC, decreasing from 7.0 Å to 1.9 Å within 1 µs, when comparing with the inactive crystal structure (SI Fig. 4). This suggests an almost ...
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... during this time, the receptor continues to have some active-like features on its intracellular side, such as helical ICL2 and shortened TM7. These continue until 2 μs, at which point they also transition into their inactive con- figurations (Fig. 4, SI Fig. 6). Like in DOPC, ICL3 adopts an inward orientation after 2 μs, interacting with ICL2 ( Fig. 4, SI Fig. 3). However, its exact conformation is a little different as it is even more inward than in DOPC, corresponding with what has been previously described as a "very inactive" conformation 15 and remains mostly stable until the end of the simulation (SI Fig. 3). The receptor finally obtains a full complement of inactive-state features from ...
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... in DOPC, ICL3 adopts an inward orientation after 2 μs, interacting with ICL2 ( Fig. 4, SI Fig. 3). However, its exact conformation is a little different as it is even more inward than in DOPC, corresponding with what has been previously described as a "very inactive" conformation 15 and remains mostly stable until the end of the simulation (SI Fig. 3). The receptor finally obtains a full complement of inactive-state features from 2.5 μs onwards, including a stable triad core (Fig. 5), closed ionic-lock (SI Fig. 5), inward TM6 (SI Fig. 4), disordered ICL2 and extended TM7 (Fig. 4), each of which is in agreement with the inactive-state crystal structure (PDB entry: 2RH1) 13 . In ...
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... At the heart of these differences lie different lipid headgroups. For example, the positively charged headgroup of DOPE lipids create unfavourable interactions with positively charged residues located on TM6, as well as enabling inter-lipid H-bonds between the headgroup of one lipid with the phosphate group of its neighbour (for example, see SI Fig. 13). These charged inter-lipid interactions contribute to the greater density of a DOPE membrane. On the other hand, the more hydrophobic headgroup of DOPC lipids facilitates moderate interaction with TM6, and an absence of inter-lipid H-bonds contributes to lower membrane ...
Citations
... The nature of surrounding lipids may also affect GPCR functions and oligomerization, either directly by specific interactions, or indirectly by modification of the environment fluidity [12,13]. For example, studies based on experimental approaches [14,15] or molecular dynamics (MD) simulations [16,17] report that anionic lipids maintain or favor the active state of different receptors. This may affect GPCR responses in various cardiovascular cell types such as cardiomyocytes, endothelium, and vascular smooth muscle cells, which contain anionic lipids [18][19][20][21]. ...
Environmental factors, including mechanical stress and surrounding lipids, can influence the response of GPCRs, such as the mechanosensitive angiotensin II type 1 receptor (AT1). To investigate the impact of these factors on AT1 activation, we developed a steered molecular dynamics simulations protocol based on quaternion formalism. In this protocol, a pulling force was applied to the N-terminus of transmembrane helix 6 (TM6) to induce the TM6 opening characteristic of activation. Subsequently, the simulations were continued without constraints to allow the receptor to relax around the novel TM6 conformation under different conditions. We analyzed the responses of AT1 to membrane stretching, modeled by applying surface tension, in different bilayers. In phosphocholine bilayers without surface tension, we could observe a transient atypical structure of AT1, with an outward TM7 conformation, at the beginning of the activation process. This atypical structure then evolved toward a pre-active structure with outward TM6 and inward TM7. Strikingly, the presence of anionic phosphoglycerol lipids and application of surface tension synergistically favored the atypical structure, which led to an increase in the cross-section area of the receptor intracellular domain. Lipid internalization and H-bonds between lipid heads and the receptor C-terminus increased in phosphoglycerol vs phosphocholine bilayers, but did not depend on surface tension. The difference in the cross-section area of the atypical and pre-active conformations makes the conformational transition sensitive to lateral pressure, and favors the atypical conformation upon surface tension. Anionic lipids act as allosteric modulators of the conformational transition, by stabilizing the atypical conformation. These findings contribute to decipher the mechanisms underlying AT1 activation, highlighting the influence of environmental factors on GPCR responses. Moreover, our results reveal the existence of intermediary conformations that depend on receptor environment and could be targeted in drug design efforts.
... GPCRs display a unique lipid-protein interaction profile influenced not only by the GPCR itself, but also by the specific conformation of the receptor. These bound lipids not only modify the receptor's surface, but can also act as allosteric modulators of receptor con-formation [68]. The molecular signatures of receptor-lipid interactions and their functions for the majority of GPCRs, including dopamine receptors, are not yet fully understood. ...
Numerous studies highlight the therapeutic potential of G protein-coupled receptor (GPCR) heterodimers, emphasizing their significance in various pathological contexts. Despite extensive basic research and promising outcomes in animal models, the translation of GPCR heterodimer-targeting drugs into clinical use remains limited. The complexities of in vivo conditions, particularly within thecomplex central nervous system, pose challenges in fully replicating physiological environments, hindering clinical success. This review discusses examples of the most studied heterodimers, their involvement in nervous system pathology, and the available data on their potential ligands. In addition, this review highlights the intricate interplay between lipids and GPCRs as a potential key factor in understanding the complexity of cell signaling. The multifaceted role of lipids in modulating the dynamics of GPCR dimerization is explored, shedding light on the elaborate molecular mechanisms governing these interactions.
... Detergents provide a simple and universal way to solubilize MPs, but they also strip away certain lipids that are crucial for fundamental aspects of protein biogenesis and function. [10][11][12][13][14] Several studies show that these membrane lipids, termed endogenous or sometimes annular, associate specifically with MPs to exert effects on their structure, stability or activity. [15][16][17] However, the exact role of these allosteric modulators remains difficult to assess because their associations with MPs are highly dynamic, exchange rapidly and are not always resolved by current structural biology techniques. ...
Compared to protein–protein and protein–nucleic acid interactions, our knowledge of protein-lipid interactions remains limited. This is primarily due to the inherent insolubility of membrane proteins (MPs) in aqueous solution. The traditional use of detergents to overcome the solubility barrier destabilizes MPs and strips away certain lipids that are increasingly recognized as crucial for protein function. Recently, membrane mimetics have been developed to circumvent the limitations. In this study, using the peptidisc, we find that MPs in different lipid states can be isolated based on protein purification and reconstitution methods, leading to observable effects on MP activity and stability. Peptidisc also enables re-incorporating specific lipids to fine-tune the protein microenvironment and assess the impact on downstream protein associations. This study offers a first look at the illusive protein-lipid interaction specificity, laying the path for a systematic evaluation of lipid identity and contributions to membrane protein function.
... The oriented structure was then embedded into a lipid bilayer consisting of 256 POPC molecules with 128 lipids in the upper leaflet and 128 lipids in the lower leaflet. The system was solvated with TIP3P water and neutralised with 0.15 M of potassium chloride ions (Bruzzese et al., 2018;Im & Roux, 2002). The potassium ions, chloride ions and water molecules were added accordingly to the system. ...
The G-protein-coupled receptors are a part of the largest and most physiologically relevant family of membrane proteins. One third of the medications, now on the market, target the GPCR receptor family, which is one of the most important therapeutic targets for many disorders. In the reported work, we have focused on orphan GPR88 receptor which is a part of the GPCR protein family and a potential target for central nervous system disorders. GPR88 is known to show the highest expression in the striatum, which is a key region in motor control and cognitive functions. Recent studies have reported that GPR88 is activated by two agonists, 2-PCCA and RTI-13951-33. In this study, we have predicted the three-dimensional protein structure for the orphan GPR88 using the homology modeling approach. We then used shape-based screening techniques based on known agonists and structure-based virtual screening methods employing docking to uncover novel GPR88 ligands. The screened GPR88-ligand complexes were further subjected to molecular dynamics simulation studies. The selected ligands could fasten the development of novel treatments for the vast list of movement disorders.
... Our data confirm the trend reported previously for MexB and MexY and show a more marked difference in the number of MFSs at DP T , whose average values also increased in both proteins with respect to ref. (Ramaswamy et al., 2018). These differences could be ascribed to the different MD simulations protocol employed (new water model and protein force field, see Materials and Methods) and/or the more complex lipid bilayer environment (i.e., the protein is inserted into a POPE:POPG (4:3) lipid membrane, in which neutral and, especially, negatively charged lipids can interact with the protein and affect its conformation in a way different from in a pure POPE lipid bilayer (Bruzzese et al., 2018;Liko et al., 2018). Moreover, our observation of MFSs in the transmembrane domain of each transporter (Supplementary Figure S8 L and T protomers of MexB, MexF, and MexY best representative structures highlighting MFSs obtained with the FTMap server fragment-based mapping. ...
The secondary transporters of the resistance-nodulation-cell division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria like Pseudomonas aeruginosa. Among these RND transporters, MexB, MexF, and MexY, with partly overlapping specificities, have been implicated in pathogenicity. Only the structure of the former has been resolved experimentally, which together with the lack of data about the functional dynamics of the full set of transporters, limited a systematic investigation of the molecular determinants defining their peculiar and shared features. In a previous work (Ramaswamy et al., Front. Microbiol., 2018, 9, 1144), we compared at an atomistic level the two main putative recognition sites (named access and deep binding pockets) of MexB and MexY. In this work, we expand the comparison by performing extended molecular dynamics (MD) simulations of these transporters and the pathologically relevant transporter MexF. We employed a more realistic model of the inner phospholipid membrane of P. aeruginosa and more accurate force-fields. To elucidate structure/dynamics-activity relationships we performed physico-chemical analyses and mapped the binding propensities of several organic probes on all transporters. Our data revealed the presence, also in MexF, of a few multifunctional sites at locations equivalent to the access and deep binding pockets detected in MexB. Furthermore, we report for the first time about the multidrug binding abilities of two out of five gates of the channels deputed to peripheral (early) recognition of substrates. Overall, our findings help to define a common “recognition topology” characterizing Mex transporters, which can be exploited to optimize transport and inhibition propensities of antimicrobial compounds.
... Few years later, the Giraldo team has investigated by long-timescale (total of 24 μs) molecular dynamics simulations (MDS), the allosteric modulation and conformational changes of the β2AR that occur as a result of interactions with anionic (DOPG) and zwitterionic phospholipids dioleoyl phosphatidylcoline (DOPC) and DOPE. Their studies show that net negatively charged lipids stabilize an active-like state of the receptor that is able to bind to G proteins, while net-neutral zwitterionic lipids inactivate the receptor, generating a fully inactive or intermediate states with kinetics depending on the lipid headgroup charge distribution [8]. Their studies revealed that such lipid modulation of the receptor activity is governed by the chemistry of protein-interacting lipid headgroups, as modeling studies are performed in absence of ligand and G proteins. ...
G protein coupled receptors (GPCRs) are a class of membrane proteins that sense extracellular signals ranging from light to odorants and small molecules and activate intracellular signaling pathways that control important physiological responses. Being composed of 7 transmembrane helices linked by extracellular and intracellular loops, the great majority of the sequence of these receptors is embedded in the lipid membrane. Therefore, it is expected GPCR structure and function to be impacted by the surrounding lipid environment and the lipid membrane physico-chemical and mechanical properties. A large number of examples from the literature is provided to highlight the role of the lipid nature (lipid headgroup, membrane polyunsaturation and cholesterol) and membrane physical and mechanical properties (curvature elastic stress, membrane thickness and hydrophobic mismatch, fluidity) in the activity of different GPCRs. In addition, lipids are important co-factors being identified in very specific locations in several GPCR structures. GPCRs and G proteins can also be lipid post-translationally modified and such events can significantly impact membrane binding, trafficking and signaling. These aspects are all treated in this review. Understanding how the lipid can modulate GPCR activity is important not only from a fundamental point of view but also due to the fact that certain pathologies, where GPCRs are central targets, have been associated with important lipid imbalance. Establishing a link between the lipid pathological imbalance and the receptor functioning in such environment is thus essential as it can open avenues to potentially innovative therapeutic strategies.
... Our previous results report a key role of the G protein subtype in the activation of the dopamine D2 receptor [33]. Some studies indicate that the neutral lipid DOPC can induce partial deactivation of the β2-adrenergic receptor [34,35]. The behaviour of this lipid in our simulations may be related to our previous research which showed the dopamine D2LONG receptor in a complex with the Gi1 protein is partially deactivated. ...
The dopamine D2 receptor, belonging to the class A G protein-coupled receptors (GPCRs), is an important drug target for several diseases, including schizophrenia and Parkinson's disease. The D2 receptor can be activated by the natural neurotransmitter dopamine or by synthetic ligands, which in both cases leads to the receptor coupling with a G protein. In addition to receptor modulation by orthosteric or allosteric ligands, it has been shown that lipids may affect the behaviour of membrane proteins. We constructed a model of a D2 receptor with a long intracellular loop (ICL3) coupled with Giα1 or Giα2 proteins, embedded in a complex asymmetric membrane, and simulated it in complex with positive, negative or neutral allosteric ligands. In this study, we focused on the influence of ligand binding and G protein coupling on the membrane-receptor interactions. We show that there is a noticeable interplay between the cell membrane, G proteins, D2 receptor and its modulators.
... In fact, rhodopsin is up to date the only GPCR with a completely resolved structure (Okada et al., 2004). However, improvements in computational efficiency over the last decades have made molecular dynamics (MD) simulations capable of capturing the unstructured loop regions (Srivastava et al., 2020) and helped unravelling the importance of ICL3 for the active state of the β 2 -adrenergic receptor (β 2 AR), a prominent GPCR responsive to adrenaline (Ozcan et al., 2013;Bruzzese et al., 2018). Such structural plasticity and adaptability of the ICL3 and C-terminus could explain the promiscuous binding of 20 different α subunits of G proteins, 7 GRKs and only two arrestins to the cytosolic binding pocket of more than 800 different GPCRs. ...
... Firstly, negatively charged lipids can intercalate between transmembrane helices (TM) 6 and 7 and block the ionic lock interaction that stabilizes the inactive state of the receptor (Neale et al., 2015;Bruzzese et al., 2020). Alternatively, attachment of ICL3 to a negatively charged membrane surface was suggested to contribute to the stabilization of the outward tilt of TM6 in the active state of the receptor (Bruzzese et al., 2018;Díaz et al., 2019). Moreover, acidic lipids were proven to be important for the binding of arrestin to rhodopsin (Sommer et al., 2006). ...
... The comparison of membrane binding probabilities obtained in free and REST MD simulations is shown in Supplementary Figure S1. Figure 1A, top shows that the nonphosphorylated ICL3 of the β 2 AR in complex with the agonist adrenaline (β 2 AR*) binds to the negatively charged membrane composed of negatively charged 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol (36:54:10 molar ratio). This finding agrees with previous MD simulations of the β 2 AR, adenosine A1 receptor, and cannabinoid receptor type 1 in single-component lipid membranes (Bruzzese et al., 2018;Díaz et al., 2019;Bruzzese et al., 2020). Interestingly, only the TM6-proximal half of ICL3 is membrane attached. ...
G protein-coupled receptors (GPCRs) are the largest class of human membrane proteins that bind extracellular ligands at their orthosteric binding pocket to transmit signals to the cell interior. Ligand binding evokes conformational changes in GPCRs that trigger the binding of intracellular interaction partners (G proteins, G protein kinases, and arrestins), which initiate diverse cellular responses. It has become increasingly evident that the preference of a GPCR for a certain intracellular interaction partner is modulated by a diverse range of factors, e.g., ligands or lipids embedding the transmembrane receptor. Here, by means of molecular dynamics simulations of the β 2 -adrenergic receptor and β-arrestin2, we study how membrane lipids and receptor phosphorylation regulate GPCR-arrestin complex conformation and dynamics. We find that phosphorylation drives the receptor’s intracellular loop 3 (ICL3) away from a native negatively charged membrane surface to interact with arrestin. If the receptor is embedded in a neutral membrane, the phosphorylated ICL3 attaches to the membrane surface, which widely opens the receptor core. This opening, which is similar to the opening in the G protein-bound state, weakens the binding of arrestin. The loss of binding specificity is manifested by shallower arrestin insertion into the receptor core and higher dynamics of the receptor-arrestin complex. Our results show that receptor phosphorylation and the local membrane composition cooperatively fine-tune GPCR-mediated signal transduction. Moreover, the results suggest that deeper understanding of complex GPCR regulation mechanisms is necessary to discover novel pathways of pharmacological intervention.
... It is also important to highlight the crucial role of lipid composition and/or the structure of cell membranes in the conformational state of GPCRs: any alteration in receptor conformation may modify the receptor binding "pocket" altering the way GPCRs are expected to function. In accordance, it has been shown that negatively charged lipids can stabilize the active state of receptors such as β 2 -adrenoceptors, enabling the docking of G αs protein while neutral zwitterionic lipids seem to inactivate the receptor [59]. Therefore, the GPCRs localized in the nuclear membrane may also be influenced by these phenomena and be involved in the pathophysiology and development of noncommunicable diseases [60,61]. ...
G-protein-coupled receptors (GPCRs) comprise a large protein superfamily divided into six classes, rhodopsin-like (A), secretin receptor family (B), metabotropic glutamate (C), fungal mating pheromone receptors (D), cyclic AMP receptors (E) and frizzled (F). Until recently, GPCRs signaling was thought to emanate exclusively from the plasma membrane as a response to extracellular stimuli but several studies have challenged this view demonstrating that GPCRs can be present in intracellular localizations, including in the nuclei. A renewed interest in GPCR receptors’ superfamily emerged and intensive research occurred over recent decades, particularly regarding class A GPCRs, but some class B and C have also been explored. Nuclear GPCRs proved to be functional and capable of triggering identical and/or distinct signaling pathways associated with their counterparts on the cell surface bringing new insights into the relevance of nuclear GPCRs and highlighting the nucleus as an autonomous signaling organelle (triggered by GPCRs). Nuclear GPCRs are involved in physiological (namely cell proliferation, transcription, angiogenesis and survival) and disease processes (cancer, cardiovascular diseases, etc.). In this review we summarize emerging evidence on nuclear GPCRs expression/function (with some nuclear GPCRs evidencing atypical/disruptive signaling pathways) in non-communicable disease, thus, bringing nuclear GPCRs as targets to the forefront of debate.
... Previous studies have provided insights into the interactions of the receptor and the G-protein with anionic lipids present in the intracellular leaflet. Anionic lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-Lserine (POPS), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myoinositol-4',5'-bisphosphate) (PIP2) and 1,2-dioleoyl-snglycero-3-phosphoglycerol (POPG) in the intracellular leaflet are electrostatically coupled to helix 8 of the receptor and mediate interactions with the G-protein [13][14][15]. Presence of intracellular anionic lipids into the crevices between the transmembrane helices of the receptor have been shown to stabilize the activated state of the receptor [15][16][17]. Free energy simulations show strengthening of G-protein-receptor binding by anionic lipids in the intracellular leaflet [13]. ...
... Anionic lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-Lserine (POPS), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myoinositol-4',5'-bisphosphate) (PIP2) and 1,2-dioleoyl-snglycero-3-phosphoglycerol (POPG) in the intracellular leaflet are electrostatically coupled to helix 8 of the receptor and mediate interactions with the G-protein [13][14][15]. Presence of intracellular anionic lipids into the crevices between the transmembrane helices of the receptor have been shown to stabilize the activated state of the receptor [15][16][17]. Free energy simulations show strengthening of G-protein-receptor binding by anionic lipids in the intracellular leaflet [13]. In vitro experiments show that anionic lipids stabilize the folding of neuropeptides PACAP and VIP [18,19]. ...
Vasoactive intestinal polypeptide receptor (VIP1R) is a class B G-protein coupled receptor (GPCR) that is widely distributed throughout the central nervous system, T-lymphocytes, and peripheral tissues of organs like lungs and liver. Critical functions of these receptors render them potential pharmacological targets for the treatment of a broad spectrum of inflammatory and neurodegenerative diseases. Here we use atomistic studies to show that phospholipids can act as potent regulators of peptide binding on to the receptor. We simulated the binding of neuropeptide pituitary adenylate cyclase-activating peptide (PACAP27) into the transmembrane bundle of the receptor. The simulations reveal two lipid binding sites on the peptidic ligand for the negatively charged phosphodiester of phospholipids in the extracellular leaflet which lower the peptide-receptor binding free energy by ∼ 8 kB T . We further simulated the effect of anionic lipids phosphatidylinositol-4,5-bisphosphate (PIP2). These lipids show much stronger interaction, lowering the peptide-receptor binding energy by an additional ∼ 7kBT compared to POPC lipids. These findings suggest that lipids can play an active role in catalyzing peptide-receptor binding and activating vasoactive intestinal polypeptide receptors.
