The Role of ICL1 and H8 in Class B1 GPCRs; Implications for Receptor Activlation

The first intracellular loop (ICL1) of G protein-coupled receptors (GPCRs) has received little attention, although there is evidence that, with the 8th helix (H8), it is involved in early conformational changes following receptor activation as well as contacting the G protein β subunit. In class B1 GPCRs, the distal part of ICL1 contains a conserved R12.48KLRCxR2.46b motif that extends into the base of the second transmembrane helix; this is weakly conserved as a [R/H]12.48KL[R/H] motif in class A GPCRs. In the current study, the role of ICL1 and H8 in signaling through cAMP, iCa2+ and ERK1/2 has been examined in two class B1 GPCRs, using mutagenesis and molecular dynamics. Mutations throughout ICL1 can either enhance or disrupt cAMP production by CGRP at the CGRP receptor. Alanine mutagenesis identified subtle differences with regard elevation of iCa2+, with the distal end of the loop being particularly sensitive. ERK1/2 activation displayed little sensitivity to ICL1 mutation. A broadly similar pattern was observed with the glucagon receptor, although there were differences in significance of individual residues. Extending the study revealed that at the CRF1 receptor, an insertion in ICL1 switched signaling bias between iCa2+ and cAMP. Molecular dynamics suggested that changes in ICL1 altered the conformation of ICL2 and the H8/TM7 junction (ICL4). For H8, alanine mutagenesis showed the importance of E3908.49b for all three signal transduction pathways, for the CGRP receptor, but mutations of other residues largely just altered ERK1/2 activation. Thus, ICL1 may modulate GPCR bias via interactions with ICL2, ICL4 and the Gβ subunit.

Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates

Enzyme rhodopsins, including cyclase opsins (Cyclops) and rhodopsin phosphodiesterases (RhoPDEs), were recently discovered in fungi, algae and protists. In contrast to the well-developed light-gated guanylyl/adenylyl cyclases as optogenetic tools, ideal light-regulated phosphodiesterases are still in demand. Here, we investigated and engineered the RhoPDEs from Salpingoeca rosetta, Choanoeca flexa and three other protists. All the RhoPDEs (fused with a cytosolic N-terminal YFP tag) can be expressed in Xenopus oocytes, except the AsRhoPDE that lacks the retinal-binding lysine residue in the last (8th) transmembrane helix. An N296K mutation of YFP::AsRhoPDE enabled its expression in oocytes, but this mutant still has no cGMP hydrolysis activity. Among the RhoPDEs tested, SrRhoPDE, CfRhoPDE1, 4 and MrRhoPDE exhibited light-enhanced cGMP hydrolysis activity. Engineering SrRhoPDE, we obtained two single point mutants, L623F and E657Q, in the C-terminal catalytic domain, which showed ~40 times decreased cGMP hydrolysis activity without affecting the light activation ratio. The molecular characterization and modification will aid in developing ideal light-regulated phosphodiesterase tools in the future.

Molecular Mechanism of Hyperactivation Conferred by a Truncated TRPA1 Disease Mutant Suggests New Gating Insights

The wasabi receptor, TRPA1, is a non-selective homotetrameric cation channel expressed in primary sensory neurons of the pain pathway, where it is activated by diverse chemical irritants. A direct role for TRPA1 in human health has been highlighted by the discovery of genetic variants associated with severe pain disorders. One such TRPA1 mutant was identified in a father-son pair with cramp fasciculation syndrome (CFS) and neuronal hyperexcitability-hypersensitivity symptoms that may be caused by aberrant channel activity, though the mechanism of action for this mutant is unknown. Here, we show the CFS-associated R919* TRPA1 mutant is functionally inactive when expressed alone in heterologous cells, which is not surprising since it lacks the 201 C-terminal amino acids that house critical channel gating machinery including the pore-lining transmembrane helix. Interestingly, the R919* mutant confers enhanced agonist sensitivity when co-expressed with wild type (WT) TRPA1. This channel hyperactivation mechanism is conserved in distant TRPA1 species orthologues and can be recapitulated in the capsaicin receptor, TRPV1. Using a combination of ratiometric calcium imaging, immunostaining, surface biotinylation, pulldown assays, fluorescence size exclusion chromatography, and proximity biotinylation assays, we show that the R919* mutant co-assembles with WT subunits into heteromeric channels. Within these heteromers, we postulate that R919* TRPA1 subunits contribute to hyperactivation by lowering energetic barriers to channel activation contributed by the missing regions. Additionally, we show heteromer activation can originate from the R919* TRPA1 subunits, which suggests an unexpected role for the ankyrin repeat and coiled coil domains in concerted channel gating. Our results demonstrate the R919* TRPA1 mutant confers gain-of-function thereby expanding the physiological impact of nonsense mutations, reveals a novel and genetically tractable mechanism for selective channel sensitization that may be broadly applicable to other receptors, and uncovers new gating insights that may explain the molecular mechanism of temperature sensing by some TRPA1 orthologues.

Integrative Study of the Structural and Dynamical Properties of a KirBac3.1 Mutant: Functional Implication of a Highly Conserved Tryptophan in the Transmembrane Domain

ATP-sensitive potassium (K-ATP) channels are ubiquitously expressed on the plasma membrane of cells in several organs, including the heart, pancreas, and brain, and they govern a wide range of physiological processes. In pancreatic β-cells, K-ATP channels composed of Kir6.2 and SUR1 play a key role in coupling blood glucose and insulin secretion. A tryptophan residue located at the cytosolic end of the transmembrane helix is highly conserved in eukaryote and prokaryote Kir channels. Any mutation on this amino acid causes a gain of function and neonatal diabetes mellitus. In this study, we have investigated the effect of mutation on this highly conserved residue on a KirBac channel (prokaryotic homolog of mammalian Kir6.2). We provide the crystal structure of the mutant KirBac3.1 W46R (equivalent to W68R in Kir6.2) and its conformational flexibility properties using HDX-MS. In addition, the detailed dynamical view of the mutant during the gating was investigated using the in silico method. Finally, functional assays have been performed. A comparison of important structural determinants for the gating mechanism between the wild type KirBac and the mutant W46R suggests interesting structural and dynamical clues and a mechanism of action of the mutation that leads to the gain of function.

A comprehensive examination of ACE-2 receptor and prediction of spike glycoprotein and ACE-2 interaction based on in silico analysis of ACE-2 receptor

ACE-2 receptor plays a vital role not only in the SARS-CoV-induced epidemic but also in some diseases. Studies have been carried out on the interactions of ACE-2- SARS-CoV proteins. However, comprehensive research has not been conducted on ACE2 protein by using bioinformatic tools. The present study especially two places, G104 and L108 points, which are effective in protecting the structure of the ACE-2 protein, play a critical role in the biological functioning of this protein, and play an essential role in determining the chemical-physical properties of this protein, and play a crucial role for ACE-2 protein-SARS CoV surface glycoprotein, were determined. It was also found that the G104 and L108 regions were more prone to possible mutations or deletions than the other ACE-2 protein regions. Moreover, it was determined that all possible mutations or deletions in these regions affect the chemical-physical properties, biological functions, and structure of the ACE-2 protein. Having a negative GRAVY value, one transmembrane helix, a significant molecular weight, a long-estimated half-life as well as most having unstable are results of G104 and L108 points mutations or deletions. Finally, it was determined that LQQNGSSVLS, which belong to the ACE-2 protein, may play an active role in binding the spike protein of SARS-CoV. All possible docking score results were estimated. It is thought that this study will bring a different perspective to ACE-2 _SARS-CoV interaction and other diseases in which ACE-2 plays an important role and will also be an essential resource for studies on ACE-2 protein.

The Effect of Ligands and Transducers on the Neurotensin Recep-tor 1 (NTS1) Conformational Ensemble

Using a discrete, intracellular 19F-NMR probe on Neurotensin receptor 1 (NTS1) transmembrane helix (TM) 6, we aim to understand how ligands and transducers modulate the receptors structural ensemble in solution. For apo NTS1, 19F-NMR spectra reveal an ensemble of at least three states (one inactive and two active-like) in equilibrium that exchange on the ms-s timescale. Dynamic NMR experiments reveal that these substates follow a linear three-site exchange process that is both thermodynamically and kinetically remodeled by orthosteric ligands. As previously observed in other GPCRs, the full agonist is insufficient to completely stabilize the active state. Receptor coupling to b-arrestin-1 or the C-terminal helix of Gaq, which comprises >60% of the GPCR/G protein interface surface area, abolishes the inactive substate. But whereas b-arrestin-1 selects for preexisting active-like substates, the Gaq peptide induces two new substates. Both transducer molecules promote substantial line-broadening of active states suggesting contributions from additional us-ms exchange processes. Together, our study suggests i) the NTS1 allosteric activation mechanism is alternatively dominated by induced fit or conformational selection depending on the coupled transducer, and ii) the available static structures do not represent the entire conformational ensemble observed in solution.

Functional analysis of the polar amino acid in TatA transmembrane helix

The twin-arginine translocation (Tat) pathway utilizes the proton-motive force (PMF) to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N-terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, a complete set of 19-amino-acid substitutions was examined. Unexpectedly, although being preferred overall, our experiments suggest that the polar amino acid is not necessary for a functional TatA. Hydrophobicity and helix stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophobicity, which was demonstrated to impact the Tat transport activity. Furthermore, a four-residue motif at the N-terminus of the TatA TMH was identified by sequence alignment. Using a biochemical approach, the N-terminal motif was found to be functionally significant, with evidence indicating a potential role in the preference for utilizing different PMF components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism.

Novel Pyrazolo[3,4-c]pyridine Antagonists with Nanomolar affinity for A1 / A3 Adenosine Receptors: Binding Kinetics and Exploration of their Binding Profile Using Mutagenesis Experiments, MD simulations and TI/MD calculations

Drugs targeting the four adenosine receptor (AR) subtypes can provide “soft" treatment of various significant diseases. Even for the two experimentally resolved AR subtypes the description of the orthosteric binding area and structure-activity relationships of ligands remains a demanding task due to the high similar amino acids sequence but also the broadness and flexibility of the ARs binding area. The identification of new pharmacophoric moieties and nanomolar leads and the exploration of their binding area with mutagenesis and state-of-the-art computational methods useful also for drug design purposes remains a challenging aim for all ARs. Here, we identified several low nanomolar ligands and potent competitive antagonists against A1R / A3R, containing the novel pyrazolo[3,4-c]pyridine pharmacophore for ARs, from a screen of an in-house library of only 52 compounds, originally designed for anti-proliferative activity. We identified L2-L10, A15, A17 with 3-aryl, 7-anilino and a electronegative group at 5-position as low micromolar to low nanomolar A1R / A3R antagonists. A17 has for A1R Kd = 5.62 nM and a residence time (RT) 41.33 min and for A3R Kd = 13.5 nM, RT = 47.23 min. The kinetic data showed that compared to the not potent or mediocre congeners the active compounds have similar association, for example at A1R Kon = 13.97 x106 M-1 (A17) vs Kon = 3.36 x106 M-1 (A26) but much lower dissociation rate Koff = 0.024 min-1 (A17) vs 0.134 min-1 (A26). Using molecular dynamics (MD) simulations and mutagenesis experiments we investigated the binding site of A17 showing that it can interact with an array of residues in transmembrane helix 5 (TM5), TM6, TM7 of A1R or A3R including residues E5.30, E5.28, T7.35 in A1R instead of Q5.28, V5.30 , L7.35 in A3R. A striking observation for drug design purposes is that for L2506.51A the binding affinity of A17 significantly increased at A1R. A17 provides a lead representative of a promising series and by means of the Thermodynamics Integration coupled with MD simulations (TI/MD) method, first applied here on whole GPCR- membrane system and showing a very good agreement between calculated and experimental relative binding free energies for A1R and A3R (spearman rank correlation p = 0.82 and 0.84, respectively), and kinetic experiments can lead to ligands with improved profile against ARs.

Cleavage of PGAM5 by the intramembrane protease PARL is governed by transmembrane helix dynamics and oligomeric state

The intramembrane protease PARL is a crucial mitochondrial safeguard by cleaving the mitophagy regulators PINK1 and PGAM5. PGAM5 substrate determinates have not been rigorously investigated and it is unclear how uncoupling the mitochondrial membrane potential regulates its processing inversely to PINK1. Here we show that in PGAM5 several hydrophilic residues distant from the cleavage site serve as key determinant for PARL-catalyzed cleavage. NMR analysis indicates that a short N-terminal amphipathic helix, followed by a kink and a C-terminal helix harboring the scissile peptide bond, is key for a productive interaction with PARL. In difference to PINK1, PGAM5 is stably inserted into the inner mitochondrial membrane until uncoupling the membrane potential triggers its disassembly into monomers that are vulnerable to PARL-catalyzed processing. We suggest a model in which PGAM5 is a slowly processed substrate with PARL-catalyzed cleavage that is influenced by multiple hierarchical substrate features including a membrane-potential-dependent oligomeric switch.