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Transient receptor potential (TRP) ion channels are polymodal receptors that have been implicated in a variety of pathophysiologies, including pain, obesity, and cancer. The capsaicin and heat sensor TRPV1, and the menthol and cold sensor TRPM8, have been shown to be modulated by the membrane protein PIRT (Phosphoinositide-interacting regulator of TRP). The emerging mechanism of PIRT-dependent TRPM8 regulation involves a competitive interaction between PIRT and TRPM8 for the activating phosphatidylinositol 4,5-bisphosphate (PIP2) lipid. As many PIP2 modulated ion channels also interact with calmodulin, we investigated the possible interaction between PIRT and calmodulin. Using microscale thermophoresis (MST), we show that calmodulin binds to the PIRT C-terminal α-helix, which we corroborate with a pull-down experiment, nuclear magnetic resonance-detected binding study, and Rosetta-based computational studies. Furthermore, we identify a cholesterol-recognition amino acid consensus (CRAC) domain in the outer leaflet of the first transmembrane helix of PIRT, and with MST, show that PIRT specifically binds to a number of cholesterol-derivatives. Additional studies identified that PIRT binds to cholecalciferol and oxytocin, which has mechanistic implications for the role of PIRT regulation of additional ion channels. This is the first study to show that PIRT specifically binds to a variety of ligands beyond TRP channels and PIP2.

Photosynthetic reaction centers (RCs) evolved > 3 billion years ago and have diverged into Type II RCs reducing quinones and Type I RCs reducing soluble acceptors via iron–sulfur clusters. Photosystem I (PSI), the exemplar Type I RC, uses modified menaquinones as intermediate electron transfer cofactors, but it has been controversial if the Type I RC of heliobacteria (HbRC) uses its two bound menaquinones in the same way. The sequence of the quinone-binding site in PSI is not conserved in the HbRC, and the recently solved crystal structure of the HbRC does not reveal a quinone in the analogous site. We found that illumination of heliobacterial membranes resulted in reduction of menaquinone to menaquinol, suggesting that the HbRC can perform a function thought restricted to Type II RCs. Experiments on membranes and live cells are consistent with the hypothesis that the HbRC preferentially reduces soluble electron acceptors (e.g., ferredoxins) in low light, but switches to reducing lipophilic quinones in high light, when the soluble acceptor pool becomes full. Thus, the HbRC may represent a functional evolutionary intermediate between PSI and the Type II RCs.

Ordered membrane nanodomains colloquially known as \"lipid rafts\" have many proposed cellular functions. However, pharmacological tools to modulate protein affinity for rafts and to manipulate raft formation are currently lacking. We screened 24,000 small molecules for compounds that impact the raft affinity of a known raft-preferring model protein, peripheral myelin protein 22 (PMP22), in giant plasma membrane vesicles (GPMVs). Hits were tested against another model raft protein, MAL, and also tested for their impact on raft stability. We identified three chemically distinct tools for manipulating lipid rafts. Two compounds were found to destabilize ordered domains (VU0607402 and VU0519975) while a third (primaquine diphosphate) increased PMP22 partitioning and stabilized ordered domains. While discovered in a PMP22-focused screen, all three were seen to modulate raft formation in a protein-independent manner by altering lipid-lipid interactions and membrane fluidity. Acute treatment of live cells with the raft destabilizing compound, VU0607402 was seen to modulate TRPM8 channel function, highlighting the utility of this compound in live-cell experiments for dissecting the role that membrane order and fluidity play in cell signaling. These compounds provide pharmacological tools for probing lipid raft properties and function in biophysical experiments and in living cells.

Ordered membrane nanodomains colloquially known as \"lipid rafts\" have many proposed cellular functions. However, pharmacological tools to modulate protein affinity for rafts and to manipulate raft formation are currently lacking. We screened 24,000 small molecules for compounds that impact the raft affinity of a known raft-preferring protein, peripheral myelin protein 22 (PMP22), in giant plasma membrane vesicles (GPMVs). Hits were counter-screened against another raft protein, MAL, and also tested for their impact on raft stability. We identified three chemically distinct tools for manipulating lipid rafts. Two compounds were seen to both decrease PMP22 raft partitioning and to destabilize ordered domains (VU0607402 and VU0519975) while a third (primaquine diphosphate) increased PMP22 partitioning and stabilized ordered domains. While discovered in a PMP22-focused screen, all three were seen to modulate raft formation in a protein-independent manner by altering lipid-lipid interactions and membrane fluidity. Acute treatment of live cells with the raft destabilizing compound, VU0607402 was seen to modulate TRPM8 channel function, highlighting the utility of this compound in live-cell experiments for dissecting the role that membrane order and fluidity play in cell signaling. These compounds provide novel pharmacological tools for probing lipid raft properties and function in biophysical experiments and in living cells.

Protein dynamics have emerged as a key feature associated with function in various systems. Here, NMR-based studies coupled with computational cheminformatics and cellular function are leveraged to identify a relationship between the human cold and menthol receptor TRPM8 dynamics, chemical structure, and cellular potency. TRPM8 is a validated target for a variety of pain indications but generally has been clinically limited by on-target side effects impacting thermosensing and thermoregulation. This study shows that cheminformatic analysis of a TRPM8 regulating small molecule ligand library correlates with cellular function. Electrophysiology studies further validate the relationship and show a correlation between chemical structure and functional features such as compound potency. Solution NMR studies of the TRPM8 voltage sensing-like domain, which houses the canonical menthol ligand binding site, show that ligand binding conformationally selects NMR-detected TRPM8 dynamics in a manner that quantitatively correlates with chemical structure. The relationship between chemical structure and protein dynamics can be used predictively, where a chemical structure is predictive of dynamics in a latent reduced dimensionality space. Moreover, the robustness of the conformational selection of the dynamic ensemble is evaluated by varying related and divergent chemotypes, signal-to-noise sensitivity, and sample bias. Taken together, this study identifies that protein dynamics can serve as a quantifiable bridge between chemical structure and cellular function, which has implications for drug discovery in difficult systems.

Development of an understanding of membrane nanodomains colloquially known as \"lipid rafts\" has been hindered by a lack of pharmacological tools to manipulate rafts and protein affinity for rafts. We screened 24,000 small molecules for modulators of the affinity of peripheral myelin protein 22 (PMP22) for rafts in giant plasma membrane vesicles (GPMVs). Hits were counter-screened against another raft protein, MAL, and tested for impact on raft , leading to two classes of compounds. Class I molecules altered the raft affinity of PMP22 and MAL and also reduced raft formation in a protein-dependent manner. Class II molecules modulated raft formation in a protein-independent manner. This suggests independent forces work collectively to stabilize lipid rafts. Both classes of compounds altered membrane fluidity in cells and modulated TRPM8 channel function. These compounds provide new tools for probing lipid raft function in cells and for furthering our understanding of raft biophysics.

Transient receptor potential (TRP) ion channels are polymodal receptors that have been implicated in a variety of pathophysiologies, including pain, obesity, and cancer. The capsaicin and heat sensor TRPV1, and the menthol and cold sensor TRPM8, have been shown to be modulated by the membrane protein PIRT (Phosphoinositide-interacting regulator of TRP). The emerging mechanism of PIRT-dependent TRPM8 regulation involves a competitive interaction between PIRT and TRPM8 for the activating phosphatidylinositol 4,5-bisphosphate (PI[P.sub.2]) lipid. As many PI[P.sub.2] modulated ion channels also interact with calmodulin, we investigated the possible interaction between PIRT and calmodulin. Using microscale thermophoresis (MST), we show that calmodulin binds to the PIRT C-terminal [alpha]-helix, which we corroborate with a pull-down experiment, nuclear magnetic resonance-detected binding study, and Rosetta-based computational studies. Furthermore, we identify a cholesterol-recognition amino acid consensus (CRAC) domain in the outer leaflet of the first transmembrane helix of PIRT, and with MST, show that PIRT specifically binds to a number of cholesterol-derivatives. Additional studies identified that PIRT binds to cholecalciferol and oxytocin, which has mechanistic implications for the role of PIRT regulation of additional ion channels. This is the first study to show that PIRT specifically binds to a variety of ligands beyond TRP channels and [PIP.sub.2].

Abstract SARS-CoV-2 envelope protein (S2-E) is a conserved membrane protein that is essential to coronavirus assembly and budding. Here, we describe the recombinant expression and purification of S2-E into amphipol-class amphipathic polymer solutions. The physical properties of amphipols underpin their ability to solubilize and stabilize membrane proteins without disrupting membranes. Amphipol delivery of S2-E to pre-formed planar bilayers results in spontaneous membrane integration and formation of viroporin ion channels. Amphipol delivery of the S2-E protein to human cells results in membrane integration followed by retrograde trafficking to a location adjacent to the endoplasmic reticulum-to-Golgi intermediate compartment (ERGIC) and the Golgi, which are the sites of coronavirus replication. Delivery of S2-E to cells enables both chemical biological approaches for future studies of SARS-CoV-2 pathogenesis and development of “Trojan Horse” anti-viral therapies. This work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells. Competing Interest Statement The authors have declared no competing interest. * Abbreviations (SARS-CoV-2) Severe acute respiratory syndrome 2 virus (S2-E) SARS-CoV-2 envelope protein (ERGIC) endoplasmic reticulum-to-Golgi intermediate compartment (CoV) coronavirus (VLP) virus-like particle (ARDS) acute respiratory distress syndrome (NBD) nitrobenzoxadiazole (UPR) unfolded protein response