Competition between inside-out unfolding and pathogenic aggregation in an amyloid-forming ß-propeller.

Studies of folded-to-misfolded transitions using model protein systems reveal a range of unfolding needed for exposure of amyloid-prone regions for subsequent fibrillization. Here, we probe the relationship between unfolding and aggregation for glaucoma-associated myocilin. Mutations within the olfactomedin domain of myocilin (OLF) cause a gain-of-function, namely cytotoxic intracellular aggregation, which hastens disease progression. Aggregation by wild-type OLF (OLFWT) competes with its chemical unfolding, but only below the threshold where OLF loses tertiary structure. Representative moderate (OLFD380A) and severe (OLFI499F) disease variants aggregate differently, with rates comparable to OLFWT in initial stages of unfolding, and variants adopt distinct partially folded structures seen along the OLFWT urea-unfolding pathway. Whether initiated with mutation or chemical perturbation, unfolding propagates outward to the propeller surface. In sum, for this large protein prone to amyloid formation, the requirement for a conformational change to promote amyloid fibrillization leads to direct competition between unfolding and aggregation.

A molecular perspective on identifying TRPV1 thermosensitive regions and disentangling polymodal activation.

TRPV1 is a polymodal receptor ion channel that is best known to function as a molecular thermometer. It is activated in diverse ways, including by heat, protons (low pH), and vanilloid compounds, such as capsaicin. In this review, we summarize molecular studies of TRPV1 thermosensing, focusing on the cross-talk between heat and other activation modes. Additional insights from TRPV1 isoforms and non-rodent/non-human TRPV1 ortholog studies are also discussed in this context. While the molecular mechanism of heat activation is still emerging, it is clear that TRPV1 thermosensing is modulated allosterically, i.e., at a distance, with contributions from many distinct regions of the channel. Similarly, current studies identify cross-talk between heat and other TRPV1 activation modes, such as protons and capsaicin, and that these modes can generally be selectively disentangled. In aggregate, this suggests that future TRPV1 molecular studies should define allosteric pathways and provide mechanistic insight, thereby enabling mode-selective manipulation of the polymodal receptor. These advances are anticipated to have significant implications in both basic and applied biomedical sciences.

Uracil-DNA glycosylase efficiency is modulated by substrate rigidity.

Uracil DNA-glycosylase (UNG) is a DNA repair enzyme that removes the highly mutagenic uracil lesion from DNA using a base flipping mechanism. Although this enzyme has evolved to remove uracil from diverse sequence contexts, UNG excision efficiency depends on DNA sequence. To provide the molecular basis for rationalizing UNG substrate preferences, we used time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations to measure UNG specificity constants (kcat/KM) and DNA flexibilities for DNA substrates containing central AUT, TUA, AUA, and TUT motifs. Our study shows that UNG efficiency is dictated by the intrinsic deformability around the lesion, establishes a direct relationship between substrate flexibility modes and UNG efficiency, and shows that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and UNG activity. The finding that substrate flexibility controls UNG efficiency is likely significant for other repair enzymes and has major implications for the understanding of mutation hotspot genesis, molecular evolution, and base editing.

Modeling long QT syndrome type 2 on-a-chip via in-depth assessment of isogenic gene-edited 3D cardiac tissues.

Long QT syndrome (LQTS) is a cardiovascular disease characterized by QT interval prolongation that can lead to sudden cardiac death. Many mutations with heterogeneous mechanisms have been identified in KCNH2, the gene that encodes for hERG (Kv11.1), which lead to onset of LQTS type 2 (LQTS2). In this work, we developed a LQTS2-diseased tissue-on-a-chip model, using 3D coculture of isogenic stem cell-derived cardiomyocytes (CMs) and cardiac fibroblasts (CFs) within an organotypic microfluidic chip technology. Primarily, we created a hiPSC line with R531W mutation in KCNH2 using CRISPR-Cas9 gene-editing technique and characterized the resultant differentiated CMs and CFs. A deficiency in hERG trafficking was identified in KCNH2-edited hiPSC-CMs, revealing a possible mechanism of R531W mutation in LQTS2 pathophysiology. Following creation of a 3D LQTS2 tissue-on-a-chip, the tissues were extensively characterized, through analysis of calcium handling and response to ß-agonist. Furthermore, attempted phenotypic rescue via pharmacological intervention of LQTS2 on a chip was investigated.

Recombinant SARS-CoV-2 envelope protein traffics to the trans-Golgi network following amphipol-mediated delivery into human cells.

The severe acute respiratory syndrome coronavirus 2 envelope protein (S2-E) is a conserved membrane protein that is important for coronavirus (CoV) assembly and budding. Here, we describe the recombinant expression and purification of S2-E in amphipol-class amphipathic polymer solutions, which solubilize and stabilize membrane proteins, but do not disrupt membranes. We found that amphipol delivery of S2-E to preformed planar bilayers results in spontaneous membrane integration and formation of viroporin cation channels. Amphipol delivery of the S2-E protein to human cells results in plasma membrane integration, followed by retrograde trafficking to the trans-Golgi network and accumulation in swollen perinuclear lysosomal-associated membrane protein 1-positive vesicles, likely lysosomes. CoV envelope proteins have previously been proposed to manipulate the luminal pH of the trans-Golgi network, which serves as an accumulation station for progeny CoV particles prior to cellular egress via lysosomes. Delivery of S2-E to cells will enable chemical biological approaches for future studies of severe acute respiratory syndrome coronavirus 2 pathogenesis and possibly even development of "Trojan horse" antiviral therapies. Finally, this work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.

Structural and in Vitro Functional Characterization of a Menthyl TRPM8 Antagonist Indicates Species-Dependent Regulation.

TRPM8 antagonists derived from its cognate ligand, (-)-menthol, are underrepresented. We determine the absolute stereochemistry of a well-known TRPM8 antagonist, (-)-menthyl 1, using VCD and 2D NMR. We explore 1 for its antagonist effects of the human TRPM8 (hTRPM8) orthologue to uncover species-dependent inhibition versus rat channels. (-)-Menthyl 1 inhibits menthol- and icilin-evoked Ca2+ responses at hTRPM8 with IC50 values of 805 ± 200 nM and 1.8 ± 0.6 µM, respectively, while more potently inhibiting agonist responses at the rat orthologue (rTRPM8 IC50 (menthol) = 117 ± 18 nM, IC50 (icilin) = 521 ± 20 nM). Whole-cell patch-clamp recordings of hTRPM8 confirm the 1 inhibition of menthol-stimulated currents, with an IC50 of 700 ± 200 nM. We demonstrate that 1 possesses ≥400-fold selectivity for hTRPM8 versus hTRPA1/hTRPV1. (-)-menthyl 1 can be used as a novel chemical tool to study hTRPM8 pharmacology and differences in species commonly used in drug discovery.

Hinge-shift mechanism as a protein design principle for the evolution of ß-lactamases from substrate promiscuity to specificity.

TEM-1 ß-lactamase degrades ß-lactam antibiotics with a strong preference for penicillins. Sequence reconstruction studies indicate that it evolved from ancestral enzymes that degraded a variety of ß-lactam antibiotics with moderate efficiency. This generalist to specialist conversion involved more than 100 mutational changes, but conserved fold and catalytic residues, suggesting a role for dynamics in enzyme evolution. Here, we develop a conformational dynamics computational approach to rationally mold a protein flexibility profile on the basis of a hinge-shift mechanism. By deliberately weighting and altering the conformational dynamics of a putative Precambrian ß-lactamase, we engineer enzyme specificity that mimics the modern TEM-1 ß-lactamase with only 21 amino acid replacements. Our conformational dynamics design thus re-enacts the evolutionary process and provides a rational allosteric approach for manipulating function while conserving the enzyme active site.

Delivery of recombinant SARS-CoV-2 envelope protein into human cells.

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.

CryoFold: determining protein structures and data-guided ensembles from cryo-EM density maps.

Cryo-electron microscopy (EM) requires molecular modeling to refine structural details from data. Ensemble models arrive at low free-energy molecular structures, but are computationally expensive and limited to resolving only small proteins that cannot be resolved by cryo-EM. Here, we introduce CryoFold - a pipeline of molecular dynamics simulations that determines ensembles of protein structures directly from sequence by integrating density data of varying sparsity at 3-5 Å resolution with coarse-grained topological knowledge of the protein folds. We present six examples showing its broad applicability for folding proteins between 72 to 2000 residues, including large membrane and multi-domain systems, and results from two EMDB competitions. Driven by data from a single state, CryoFold discovers ensembles of common low-energy models together with rare low-probability structures that capture the equilibrium distribution of proteins constrained by the density maps. Many of these conformations, unseen by traditional methods, are experimentally validated and functionally relevant. We arrive at a set of best practices for data-guided protein folding that are controlled using a Python GUI.

Evidence that the TRPV1 S1-S4 membrane domain contributes to thermosensing.

Sensing and responding to temperature is crucial in biology. The TRPV1 ion channel is a well-studied heat-sensing receptor that is also activated by vanilloid compounds, including capsaicin. Despite significant interest, the molecular underpinnings of thermosensing have remained elusive. The TRPV1 S1-S4 membrane domain couples chemical ligand binding to the pore domain during channel gating. Here we show that the S1-S4 domain also significantly contributes to thermosensing and couples to heat-activated gating. Evaluation of the isolated human TRPV1 S1-S4 domain by solution NMR, far-UV CD, and intrinsic fluorescence shows that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity. Further NMR characterization of the temperature-dependent conformational changes suggests the contribution of the S1-S4 domain to thermosensing shares features with known coupling mechanisms between this domain with ligand and pH activation. Taken together, this study shows that the TRPV1 S1-S4 domain contributes to TRPV1 temperature-dependent activation.

PIRT the TRP Channel Regulating Protein Binds Calmodulin and Cholesterol-Like Ligands.

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.

Hyperthermia induced by transient receptor potential vanilloid-1 (TRPV1) antagonists in human clinical trials: Insights from mathematical modeling and meta-analysis.

Antagonists of the transient receptor potential vanilloid-1 (TRPV1) channel alter body temperature (Tb) in laboratory animals and humans: most cause hyperthermia; some produce hypothermia; and yet others have no effect. TRPV1 can be activated by capsaicin (CAP), protons (low pH), and heat. First-generation (polymodal) TRPV1 antagonists potently block all three TRPV1 activation modes. Second-generation (mode-selective) TRPV1 antagonists potently block channel activation by CAP, but exert different effects (e.g., potentiation, no effect, or low-potency inhibition) in the proton mode, heat mode, or both. Based on our earlier studies in rats, only one mode of TRPV1 activation - by protons - is involved in thermoregulatory responses to TRPV1 antagonists. In rats, compounds that potently block, potentiate, or have no effect on proton activation cause hyperthermia, hypothermia, or no effect on Tb, respectively. A Tb response occurs when a TRPV1 antagonist blocks (in case of hyperthermia) or potentiates (hypothermia) the tonic TRPV1 activation by protons somewhere in the trunk, perhaps in muscles, and - via the acido-antithermogenic and acido-antivasoconstrictor reflexes - modulates thermogenesis and skin vasoconstriction. In this work, we used a mathematical model to analyze Tb data from human clinical trials of TRPV1 antagonists. The analysis suggests that, in humans, the hyperthermic effect depends on the antagonist's potency to block TRPV1 activation not only by protons, but also by heat, while the CAP activation mode is uninvolved. Whereas in rats TRPV1 drives thermoeffectors by mediating pH signals from the trunk, but not Tb signals, our analysis suggests that TRPV1 mediates both pH and thermal signals driving thermoregulation in humans. Hence, in humans (but not in rats), TRPV1 is likely to serve as a thermosensor of the thermoregulation system. We also conducted a meta-analysis of Tb data from human trials and found that polymodal TRPV1 antagonists (ABT-102, AZD1386, and V116517) increase Tb, whereas the mode-selective blocker NEO6860 does not. Several strategies of harnessing the thermoregulatory effects of TRPV1 antagonists in humans are discussed.

Structure-Based Design of Novel Biphenyl Amide Antagonists of Human Transient Receptor Potential Cation Channel Subfamily M Member 8 Channels with Potential Implications in the Treatment of Sensory Neuropathies.

Structure-activity relationship studies of a reported menthol-based transient receptor potential cation channel subfamily M member 8 channel (TRPM8) antagonist, guided by computational simulations and structure-based design, uncovers a novel series of TRPM8 antagonists with >10-fold selectivity versus related TRP subtypes. Spiro[4.5]decan-8-yl analogue 14 inhibits icilin-evoked Ca2+ entry in HEK-293 cells stably expressing human TRPM8 (hTRPM8) with an IC50 of 2.4 ± 1.0 nM, while in whole-cell patch-clamp recordings this analogue inhibits menthol-evoked currents with a hTRPM8 IC50 of 64 ± 2 nM. Molecular dynamics (MD) simulations of compound 14 in our homology model of hTRPM8 suggest that this antagonist forms extensive hydrophobic contacts within the orthosteric site. In the wet dog shakes (WDS) assay, compound 14 dose-dependently blocks icilin-triggered shaking behaviors in mice. Upon local administration, compound 14 dose dependently inhibits cold allodynia evoked by the chemotherapy oxaliplatin in a murine model of peripheral neuropathy at microgram doses. Our findings suggest that 14 and other biphenyl amide analogues within our series can find utility as potent antagonist chemical probes derived from (-)-menthol as well as small molecule therapeutic scaffolds for chemotherapy-induced peripheral neuropathy (CIPN) and other sensory neuropathies.

Competitive Interactions between PIRT, the Cold Sensing Ion Channel TRPM8, and PIP2 Suggest a Mechanism for Regulation.

TRPM8 is a member of the transient receptor potential ion channel family where it functions as a cold and pain sensor in humans and other higher organisms. Previous studies show that TRPM8 requires the signaling phosphoinositide lipid PIP2 to function. TRPM8 function is further regulated by other diverse mechanisms, including the small modulatory membrane protein PIRT (phosphoinositide regulator of TRP). Like TRPM8, PIRT also binds PIP2 and behavioral studies have shown that PIRT is required for normal TRPM8-mediated cold-sensing. To better understand the molecular mechanism of PIRT regulation of TRPM8, solution nuclear magnetic resonance (NMR) spectroscopy was used to assign the backbone resonances of full-length human PIRT and investigate the direct binding of PIRT to PIP2 and the human TRPM8 S1-S4 transmembrane domain. Microscale thermophoresis (MST) binding studies validate the NMR results and identify a competitive PIRT interaction between PIP2 and the TRPM8 S1-S4 domain. Computational PIP2 docking to a human TRPM8 comparative model was performed to help localize where PIRT may bind TRPM8. Taken together, our data suggest a mechanism where TRPM8, PIRT, and PIP2 form a regulatory complex and PIRT modulation of TRPM8 arises, at least in part, by regulating local concentrations of PIP2 accessible to TRPM8.

Structural and Evolutionary Insights Point to Allosteric Regulation of TRP Ion Channels.

The familiar pungent taste of spicy food, the refreshing taste of mint, and many other physiological phenomena are mediated by transient receptor potential (TRP) ion channels. TRP channels are a superfamily of ion channels that are sensitive to diverse chemical and physical stimuli and play diverse roles in biology. In addition to chemical regulation, some family members also sense common physical stimuli, such as temperature or pressure. Since their discovery and cloning in the 1990s and 2000s, understanding the molecular mechanisms governing TRP channel function and polymodal regulation has been a consistent but challenging goal. Until recently, a general lack of high-resolution TRP channel structures had significantly limited a molecular understanding of their function. In the past few years, a flood of TRP channel structures have been released, made possible primarily by advances in cryo-electron microscopy (cryo-EM). The boon of many structures has unleashed unparalleled insight into TRP channel architecture. Substantive comparative studies between TRP structures provide snapshots of distinct states such as ligand-free, stabilized by chemical agonists, or antagonists, partially illuminating how a given channel opens and closes. However, the now ∼75 TRP channel structures have ushered in surprising outcomes, including a lack of an apparent general mechanism underlying channel opening and closing among family members. Similarly, the structures reveal a surprising diversity in which chemical ligands bind TRP channels. Several TRP channels are activated by temperature changes in addition to ligand binding. Unraveling mechanisms of thermosensation has proven an elusive challenge to the field. Although some studies point to thermosensitive domains in the transmembrane region of the channels, results have sometimes been contradictory and difficult to interpret; in some cases, a domain that proves essential for thermal sensitivity in one context can be entirely removed from the channel without affecting thermosensation in another context. These results are not amenable to simple interpretations and point to allosteric networks of regulation within the channel structure. TRP channels have evolved to be fine-tuned for the needs of a species in its environmental niche, a fact that has been both a benefit and burden in unlocking their molecular features. Functional evolutionary divergence has presented challenges for studying TRP channels, as orthologs from different species can give conflicting experimental results. However, this diversity can also be examined comparatively to decipher the basis for functional differences. As with structural biology, untangling the similarities and differences resulting from evolutionary pressure between species has been a rich source of data guiding the field. This Account will contextualize the existing biochemical and functional data with an eye to evolutionary data and couple these insights with emerging structural biology to better understand the molecular mechanisms behind chemical and physical regulation of TRP channels.

Phosphoinositide-interacting regulator of TRP (PIRT) has opposing effects on human and mouse TRPM8 ion channels.

Transient receptor potential melastatin 8 (TRPM8) is a cold-sensitive ion channel with diverse physiological roles. TRPM8 activity is modulated by many mechanisms, including an interaction with the small membrane protein phosphoinositide-interacting regulator of TRP (PIRT). Here, using comparative electrophysiology experiments, we identified species-dependent differences between the human and mouse TRPM8-PIRT complexes. We found that human PIRT attenuated human TPRM8 conductance, unlike mouse PIRT, which enhanced mouse TRPM8 conductance. Quantitative Western blot analysis demonstrates that this effect does not arise from decreased trafficking of TRPM8 to the plasma membrane. Chimeric human/mouse TRPM8 channels were generated to probe the molecular basis of the PIRT modulation, and the effect was recapitulated in a pore domain chimera, demonstrating the importance of this region for PIRT-mediated regulation of TRPM8. Moreover, recombinantly expressed and purified human TRPM8 S1-S4 domain (comprising transmembrane helices S1-S4, also known as the sensing domain, ligand-sensing domain, or voltage sensing-like domain) and full-length human PIRT were used to investigate binding between the proteins. NMR experiments, supported by a pulldown assay, indicated that PIRT binds directly and specifically to the TRPM8 S1-S4 domain. Binding became saturated as the S1-S4:PIRT mole ratio approached 1. Our results have uncovered species-specific TRPM8 modulation by PIRT. They provide evidence for a direct interaction between PIRT and the TRPM8 S1-S4 domain with a 1:1 binding stoichiometry, suggesting that a functional tetrameric TRPM8 channel has four PIRT-binding sites.

A parallel stranded G-quadruplex composed of threose nucleic acid (TNA).

G-rich sequences can adopt four-stranded helical structures, called G-quadruplexes, that self-assemble around monovalent cations like sodium (Na+ ) and potassium (K+ ). Whether similar structures can be formed from xeno-nucleic acid (XNA) polymers with a shorter backbone repeat unit is an unanswered question with significant implications on the fold space of functional XNA polymers. Here, we examine the potential for TNA (α-l-threofuranosyl nucleic acid) to adopt a four-stranded helical structure based on a planar G-quartet motif. Using native polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) and solution-state nuclear magnetic resonance (NMR) spectroscopy, we show that despite a backbone repeat unit that is one atom shorter than the backbone repeat unit found in DNA and RNA, TNA can self-assemble into stable G-quadruplex structures that are similar in thermal stability to equivalent DNA structures. However, unlike DNA, TNA does not appear to discriminate between Na+ and K+ ions, as G-quadruplex structures form equally well in the presence of either ion. Together, these findings demonstrate that despite a shorter backbone repeat unit, TNA is capable of self-assembling into stable G-quadruplex structures.

Structural basis for KCNE3 modulation of potassium recycling in epithelia.

The single-span membrane protein KCNE3 modulates a variety of voltage-gated ion channels in diverse biological contexts. In epithelial cells, KCNE3 regulates the function of the KCNQ1 potassium ion (K(+)) channel to enable K(+) recycling coupled to transepithelial chloride ion (Cl(-)) secretion, a physiologically critical cellular transport process in various organs and whose malfunction causes diseases, such as cystic fibrosis (CF), cholera, and pulmonary edema. Structural, computational, biochemical, and electrophysiological studies lead to an atomically explicit integrative structural model of the KCNE3-KCNQ1 complex that explains how KCNE3 induces the constitutive activation of KCNQ1 channel activity, a crucial component in K(+) recycling. Central to this mechanism are direct interactions of KCNE3 residues at both ends of its transmembrane domain with residues on the intra- and extracellular ends of the KCNQ1 voltage-sensing domain S4 helix. These interactions appear to stabilize the activated "up" state configuration of S4, a prerequisite for full opening of the KCNQ1 channel gate. In addition, the integrative structural model was used to guide electrophysiological studies that illuminate the molecular basis for how estrogen exacerbates CF lung disease in female patients, a phenomenon known as the "CF gender gap."

Structural Insights into Conformation Differences between DNA/TNA and RNA/TNA Chimeric Duplexes.

Threose nucleic acid (TNA) is an artificial genetic polymer capable of heredity and evolution, and is studied in the context of RNA chemical etiology. It has a four-carbon threose backbone in place of the five-carbon ribose of natural nucleic acids, yet forms stable antiparallel complementary Watson-Crick homoduplexes and heteroduplexes with DNA and RNA. TNA base-pairs more favorably with RNA than with DNA but the reason is unknown. Here, we employed NMR, ITC, UV, and CD to probe the structural and dynamic properties of heteroduplexes of RNA/TNA and DNA/TNA. The results indicate that TNA templates the structure of heteroduplexes, thereby forcing an A-like helical geometry. NMR measurement of kinetic and thermodynamic parameters for individual base pair opening events reveal unexpected asymmetric "breathing" fluctuations of the DNA/TNA helix. The results suggest that DNA is unable to fully adapt to the conformational constraints of the rigid TNA backbone and that nucleic acid breathing dynamics are determined from both backbone and base contributions.