Interplay between Mg2+ and Ca2+ at multiple sites of the ryanodine receptor.

RyR1 is an intracellular Ca2+ channel important in excitable cells such as neurons and muscle fibers. Ca2+ activates it at low concentrations and inhibits it at high concentrations. Mg2+ is the main physiological RyR1 inhibitor, an effect that is overridden upon activation. Despite the significance of Mg2+-mediated inhibition, the molecular-level mechanisms remain unclear. In this work we determined two cryo-EM structures of RyR1 with Mg2+ up to 2.8 Å resolution, identifying multiple Mg2+ binding sites. Mg2+ inhibits at the known Ca2+ activating site and we propose that the EF hand domain is an inhibitory divalent cation sensor. Both divalent cations bind to ATP within a crevice, contributing to the precise transmission of allosteric changes within the enormous channel protein. Notably, Mg2+ inhibits RyR1 by interacting with the gating helices as validated by molecular dynamics. This structural insight enhances our understanding of how Mg2+ inhibition is overcome during excitation.

Significance of Zn2+ in RyR1 for Structural Integrity and Ligand Binding: Insight from Molecular Dynamics.

Ryanodine receptor type 1 (RyR1) is a Ca2+-release channel central to skeletal muscle excitation-contraction (EC) coupling. RyR1's cryo-EM structures reveal a zinc-finger motif positioned within the cytoplasmic C-terminal domain (CTD). Yet, owing to limitations in cryo-EM resolution, RyR1 structures lack precision in detailing the metal coordination structure, prompting the need for an accurate model. In this study, we employed molecular dynamics (MD) simulations and the density functional theory (DFT) method to refine the binding characteristics of Zn2+ in the zinc-finger site of the RyR1 channel. Our findings also highlight substantial conformational changes in simulations conducted in the absence of Zn2+. Notably, we observed a loss of contact at the interface between protein domains proximal to the zinc-finger site, indicating a crucial role of Zn2+ in maintaining structural integrity and interdomain interactions within RyR1. Furthermore, this study provides valuable insights into the modulation of ATP, Ca2+, and caffeine binding, shedding light on the intricate relationship between Zn2+ coordination and the dynamic behavior of RyR1. Our integrative approach combining MD simulations and DFT calculations enhances our understanding of the molecular mechanisms governing ligand binding in RyR1.

Deciphering molecular basis of pesticide-induced recurrent pregnancy loss: insights from transcriptomics analysis.

Recent studies have revealed a notable connection between pesticide exposure and Recurrent Pregnancy Loss (RPL), yet the precise molecular underpinning of this toxicity remains elusive. Through the alignment of Differentially Expressed Genes (DEGs) of healthy and RPL patients with the target genes of 9 pesticide components, we identified a set of 12 genes responsible for RPL etiology. Interestingly, biological process showed that besides RPL, those 12 genes also associated with preeclampsia and cardiovascular disease. Enrichment analysis showed the engagement of these genes associated with essential roles in the molecular transport of small molecules, as well as the aldosterone-regulated sodium reabsorption, endocrine and other factor-regulated calcium reabsorption, mineral absorption, ion homeostasis, and ion transport by P-type ATPases. Notably, the crosstalk targets between pesticide components played crucial roles in influencing RPL results, suggesting a role in attenuating pesticide agents that contribute to RPL. It is important to note that non-significant concentration of the pesticide components observed in both control and RPL samples should not prematurely undermine the potential for pesticides to induce RPL in humans. This study emphasizes the complexity of pesticide induced RPL and highlights avenues for further research and precautionary measures.

Exploring polyamine interactions and binding pockets in SARS-CoV-2 ORF3a.

Ongoing global pandemic caused by coronavirus (COVID-19) requires urgent development of vaccines, treatments, and diagnostic tools. Open reading frame 3a (ORF3a) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is considered to be a potential drug target for COVID-19 treatment. ORF3a is an accessory protein that plays a significant role in virus-host interactions and in facilitating host immune responses. Using putrescine, spermidine and spermine, an aliphatic polyamine for the activity suppression of ORF3a appears to be a promising approach in finding new targets for drug design. In this study, we explored the possible binding poses of polyamines to the ORF3a protein using a combination of various computational approaches i.e. pocket prediction, blind and site-specific molecular docking, molecular dynamics and ligand flooding simulations. The results showed that the tip of cytoplasmic domain and the upper tunnel of transmembrane domain of ORF3a provide a suitable binding site specific for the polyamines. MD simulations revealed the stability of spermidine binding in the upper tunnel pocket of ORF3a through salt bridge and hydrogen bond interactions between the amine groups of the ligand and negatively charged residues of ORF3a. These findings can be helpful in designing new therapeutic drugs.

Effect of pH on stability of dimer structure of the main protease of coronavirus-2.

The viral main protease (Mpro) from a novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a key enzyme essential for viral replication and has become an attractive target for antiviral drug development. The Mpro forms a functional dimer and exhibits a pH-dependent enzyme activity and dimerization. Here, we report a molecular dynamics (MD) investigation to gain insights into the structural stability of the enzyme dimer at neutral and acidic pH. Our data shows larger changes in structure of the protein with the acidic pH than that with the neutral pH. Structural analysis of MD trajectories reveals a substantial increase in intersubunit separation, the loss of domain contacts, binding free energy and interaction energy of the dimer which implies the protein instability and tendency of dimer dissociation at acidic pH. The loss in the interaction energy is mainly driven by electrostatic interactions. We have identified the intersubunit hydrogen-bonding residues involved in the decreased dimer stability. These findings may be helpful for rational drug design and target evaluation against COVID-19.

Interaction fingerprint of transmembrane segments in voltage sensor domains.

Voltage sensor domain (VSD) in channel and non-channel membrane proteins shares a common function in the detection of changes in the transmembrane electric potential. The VSD is made of four helical transmembrane segments (S1-S4) that form a structurally conserved scaffold through inter-transmembrane residue-residue interactions. Details about these interactions are yet to be fully understood in the context of the unique structural and physical characteristics of the voltage sensor unit. In this study, molecular dynamics simulations were carried out to investigate transmembrane helix-helix interactions via residue-based nonbonding energies using the activated and resting state conformations of VSD from Hv1, CiVSP, KvAP and NavAb. Inter-transmembrane interaction energies within the VSD were determined. Analysis of electrostatic and van der Waals components revealed the strengths and weaknesses of the interactions between each pair of transmembrane segments. In all cases the S4 helix had the highest electrostatic contribution to favor the key role as the voltage sensitive segment. Electrostatic interactions for the S1-S2 pair as well as the S1-S3 pair were relatively weak. Van der Waal interaction energies between adjacent segments were on average greater than that between diagonally opposite segments. Salt bridge interactions between S4-arginines and the negatively charged residues in other segments appear to contribute more to stabilizing the energy than the van der Waals interactions between nonpolar residues. The overall behavior of residue-residue contacts is similar among the transmembrane domains, reflecting the common inter- transmembrane interaction pattern in the VSD. In addition, analysis of the residue positions suggested that subtle differences in the orientation of the salt-bridges can be attributed to the difference in the inter-transmembrane interaction strengths inside the VSDs.

Self-Organized Morphology and Multiscale Structures of CoVE Proteins.

Self-organizing structures of CoVE proteins have been investigated using a coarse-grained model in Monte Carlo simulations as a function of temperature (T) in a range covering the native (low T) to denatured (high T) phases. The presence of even a few chains accelerates the very slow dynamics of an otherwise free protein chain in the native phase. The radius of gyration depends nonmonotonically on temperature and increases with the protein concentration in both the native and denatured phase. The density of organized morphology over residue-to-sample length scales (λ) is quantified by an effective dimension (D) that varies between ~ 2 at high to ~ 3 at low temperatures at λ ~ R g with an overall lower density (D ~ 2) on larger scales. The magnitude of D depends on temperature, length scale, and concentration of proteins, i.e., D ~ 3.2 at λ ~ Rg, D ~ 2.6 at λ > R g, and D ~ 2.0 at λ ≫ R g, at T = 0.024.

Dynamics and Environmental Characteristics of Spin Labels in a KvAP Voltage Sensor by Molecular Dynamics Simulations.

The nitroxide spin label is the most widely used probe for electron paramagnetic resonance (EPR) spectroscopy studies of the structure and function of biomolecules. However, the role of surrounding environments in determining the dynamics of nitroxide spin labels in biological complex systems remains to be clarified. This study aims to characterize the dynamics and environmental structure of spin labels in the voltage-sensing domain (VSD) of a KvAP potassium channel by means of molecular dynamics (MD) studies. MD simulations for unlabeled and 132 spin-labeled KvAP-VSD models (spin labels introduced at positions 20-151) were carried out in a phospholipid bilayer to evaluate conformational dynamics of nitroxide spin-label side chains in the VSD. Structural flexibility, conformational freedom, and orientation of the spin-label side chains were investigated in relation to their dynamics in different microenvironments. The analysis of MD data showed that the attached spin-label probe did not severely perturb the protein dynamics. The conformational freedoms of the nitroxide side chain vary with the physical structure of the surrounding environments. The two terminal dihedral angles of the nitroxide side chain tend to cluster and adopt several preferred rotameric states. From the nearest-neighbor analysis, the spin label can be exposed to either a homogeneous or heterogeneous environment with various exposure scenarios. The dynamical movement of KvAP-VSD is high at a water-exposed site, moderate in the membrane, and low in the protein core. Understanding the structure and dynamics behaviors of spin labels helps to manage the experimental uncertainty and avoid misleading interpretation in relation to the protein structure.

Thermally induced structural organization of nanodiscs by coarse-grained molecular dynamics simulations.

Membrane scaffold proteins (MSP) nanodiscs have been extensively used in structural study of membrane proteins. In cryo-EM, an incorporation of target proteins into nanodiscs is conducted under a rapid change from cryogenic to ambient temperatures. We present a coarse-grained molecular dynamics (CGMD) study for investigating an effect of temperature on the structural organization of DPPC-nanodisc and POPC-nanodisc. A non-monotonic response of physical quantities (i.e. the lipid order parameter, nanodisc flatness, structural change, solvation property, radius of gyration) with increase in temperature (T = 200-350 K) is found to be associated with the gel-ripple-liquid crystalline phase change within nanodiscs. The reorganization of lipids upon temperature variation induced conformational changes of MSP to minimize hydrophobic exposure of the lipid membrane to an aqueous environment. Structural response to temperature is different to a certain extent between the saturated DPPC and unsaturated POPC.

Conformation, flexibility and hydration of hyaluronic acid by molecular dynamics simulations.

Hyaluronic acid (HA) is a biopolymer of disaccharide with two alternate glycosidic bonds, ß(1,3) and ß(1,4). A molecular dynamics study presented here unveiled conformational variability in association with the flexibility of the glycosidic linkers, which depends on the number of disaccharide units. HA chain maintains a rigid rod-like conformation with short chain lengths. Crossover from a rod-like to a random-coil conformation is observed with increasing the chain length. The conformation with the ß(1,4) linkage is more flexible than that with the ß(1,3) linkage. Variation of the radius of gyration and conformational fluctuation showed that the ß(1,4) linkers along with the HA chain length enhance the overall conformational flexibility and therefore elastic response of the polymer chain. Besides the inter-saccharide hydrogen bonding, Na+ binds preferably at the ß(1,4) site. The hydration number of HA increases as an increase in the chain length. The hydration per disaccharide unit remains constant with the chain length.

Structure and dynamics of aquaporin-1.

Structural response of a AQP1 is examined by a coarse-grained model with a phenomenological interaction potential with a knowledge-based residue-residue interaction (derived from an ensemble of protein structures in PDB). The thermal response of the protein chain exhibits an unexpected characteristics in its native phase where the radius of gyration of the protein decreases on raising the temperature. The radius of gyration of AQP1 increases on increasing the temperature before saturating to a random-coil morphology in denatured phase at high temperatures. Three regions of persistent globularization are identified, toward the end segments 1M-25V and 250V-269K and a narrow region in the middle 155A-163D along the backbone. Varying the temperature leads to a systematic redistribution of self-organizing residues with globular and fibrous morphologies with an effective dimension D~2 (random coil) at high temperature and D~3 (globular conformation) in native phase. A preliminary analysis is also presented on the effect of a crowded membrane environment on the protein structure by incorporating effective solute constituents. Conformation of the protein is found to be pinned by selective binding of solute to specific targets; the matrix directed structure differs considerably from that of a protein in a generic solvent. The structure of AQP1 can be controlled by temperature and constitutive elements of the underlying matrix.

Furofuran lignans as a new series of antidiabetic agents exerting α-glucosidase inhibition and radical scarvenging: Semisynthesis, kinetic study and molecular modeling.

A new series of furofuran lignans containing catechol moiety were prepared from the reactions between lignans and a variety of phenolics. All 22 products obtained were evaluated against three different α-glucosidases (maltase, sucrase and Baker's yeast glucosidase) and DPPH radical. Of furofuran lignans evaluated, ß-14, having two catechol moieties and one acetoxy group, was the most potent inhibitor against Baker's yeast, maltase, and sucrase with IC50 values of 5.3, 25.7, and 12.9 µM, respectively. Of interest, its inhibitory potency toward Baker's yeast was 28 times greater than standard drug, acarbose and its DPPH radical scavenging (SC50 11.2 µM) was 130 times higher than commercial antioxidant BHT. Subsequent investigation on mechanism underlying the inhibitory effect of ß-14 revealed that it blocked Baker's yeast and sucrase functions by mixed-type inhibition while it exerted non-competitive inhibition toward maltase. Molecular dynamics simulation of the most potent furofuran lignans (4, α-8b, α-14, and ß-14) with the homology rat intestinal maltase at the binding site revealed that the hydrogen bond interactions from catechol, acetoxy, and quinone moieties of furofuran lignans were the key interaction to bind tightly to α-glucosidase. The results indicated that ß-14 possessed promising antidiabetic activity through simultaneously inhibiting α-glucosidases and free radicals.

Effect of Ionization State on Voltage-Sensor Structure in Resting State of the Hv1 Channel.

Voltage-gated proton-selective channels (Hv1) mediate proton extrusion during intracellular acidification. Hv1 is gated by the proton electrochemical gradient. Intracellular ionizable residues in Hv1 have been proposed to serve as proton-binding sites for pH-dependent gating, but detailed descriptions remain unclear. Here, molecular dynamics (MD) simulations were performed to investigate the effect of ionization states of charged residues on the X-ray structure of Hv1. Modification of the protonation state of acidic residues affected the resting conformation of Hv1 by disrupting salt bridges between S4 and the other segments. Upon protonation, conformational changes enabled the displacement of the S4 arginines toward the extracellular side and increased the mobility of hydrophobic residues at the gate. The aqueous crevice was considerably wider with increased hydration in the pore. Solvation free energies of the pore residues were low at the extra- and intracellular entrances, whereas the narrowest region exhibited the energy barrier for water translocation. Our MD data showed that water molecules in the upper and lower pore oriented differently. In neutral pH, the pore water oriented its dipole pointing away from the voltage-sensing domain center, whereas the opposite direction of the water dipole was observed in acidic pH.

Quercitol: From a Taxonomic Marker of the Genus Quercus to a Versatile Chiral Building Block of Antidiabetic Agents.

Quercitol is a cyclohexanepentol that has been recognized as a biomarker of plants in genus Quercus, which includes oak. As a result of its glucose-like structure, it has been introduced as an alternative chiral building block in the synthesis of several bioactive compounds. Our continuing investigations on the synthesis of antidiabetic agents from quercitol have demonstrated that this chiral synthon can generate diverse structural features with improved hypoglycemic activity.

Insight into the Role of the Hv1 C-Terminal Domain in Dimer Stabilization.

The voltage-gated proton-selective channel (Hv1) conducts protons in response to changes in membrane potential. The Hv1 protein forms dimers in the membrane. Crystal structures of Hv1 channels have revealed that the primary contacts between the two monomers are in the C-terminal domain (CTD), which forms a coiled-coil structure. The role of Hv1-CTD in channel assembly and activity is not fully understood. Here, molecular dynamics (MD) simulations of full-length and truncated CTD models of human and mouse Hv1 channels reveal a strong contribution of the CTD to the packing of the transmembrane domains. Simulations of the CTD models highlight four fundamental interactions of the key residues contributing to dimer stability. These include salt bridges, hydrophobic interactions, hydrogen bonds, and a disulfide bond across the dimer interface. At neutral pH, salt-bridge interactions increase dimer stability and the dimer becomes less stable at acidic pH. Hydrophobic core packing of the heptad pattern is important for stability, as shown by favorable nonpolar binding free energies rather than by electrostatic components. Moreover, free-energy calculations indicate that a more uniform hydrophobic core in the coiled-coil structure of the Hv1-NIN, a channel carrying the triple mutation M234N-N235I-V236N, leads to an increase in dimer stability with respect to the wild-type. A Cys disulfide bond has a strong impact on dimer stability by holding the dimer together and facilitating the interactions described above. These results are consistent with dissociative temperatures and energy barriers of dimer dissociation obtained from the temperature-accelerated MD.

Asymmetry in structural response of inner and outer transmembrane segments of CorA protein by a coarse-grain model.

Structure of CorA protein and its inner (i.corA) and outer (o.corA) transmembrane (TM) components are investigated as a function of temperature by a coarse-grained Monte Carlo simulation. Thermal response of i.corA is found to differ considerably from that of the outer component, o.corA. Analysis of the radius of gyration reveals that the inner TM component undergoes a continuous transition from a globular conformation to a random coil structure on raising the temperature. In contrast, the outer transmembrane component exhibits an abrupt (nearly discontinuous) thermal response in a narrow range of temperature. Scaling of the structure factor shows a globular structure of i.corA at a low temperature with an effective dimension D ∼ 3 and a random coil at a high temperature with D ∼ 2. The residue distribution in o.corA is slightly sparser than that of i.corA in a narrow thermos-responsive regime. The difference in thermos-response characteristics of these components (i.corA and o.corA) may reflect their unique transmembrane functions.

Voglibose-inspired synthesis of new potent α-glucosidase inhibitors N-1,3-dihydroxypropylaminocyclitols.

Voglibose, an N-1,3-dihydroxypropylaminocyclitol, has widely been used as an effective α-glucosidase inhibitor for diabetes therapy. Several attempts have been made to synthesize closely related analogues through the coupling of various aminocyclitols and propane-1,3-diol; however, most of them showed weaker or no inhibition. In this communication, we synthesized a pair of new N-1,3-dihydroxypropylaminocyclitols (10 and 11) using (+)-proto-quercitol (1) as a cyclitol core structure. The newly synthesized compounds revealed potent rat intestinal α-glucosidases, particularly against maltase, with IC50 values at submicromolar. Subsequent study on mechanisms underlying the inhibition of 11 indicated the competitive manner towards maltase and sucrase. The potent inhibition of these compounds was elaborated by docking study, in which their binding profiles towards key amino acid residues in the active site were similar to that of voglibose. Therefore, introduction of propane-1,3-diol moiety to suitable cyclohexane core structure such as aminoquercitol would be a potential approach to discover a new series of effective α-glucosidase inhibitors.

Inner and Outer Coordination Shells of Mg(2+) in CorA Selectivity Filter from Molecular Dynamics Simulations.

Structural data of CorA Mg(2+) channels show that the five Gly-Met-Asn (GMN) motifs at the periplasmic loop of the pentamer structure form a molecular scaffold serving as a selectivity filter. Unfortunately, knowledge about the cation selectivity of Mg(2+) channels remains limited. Since Mg(2+) in aqueous solution has a strong first hydration shell and apparent second hydration sphere, the coordination structure of Mg(2+) in a CorA selectivity filter is expected to be different from that in bulk water. Hence, this study investigated the hydration structure and ligand coordination of Mg(2+) in a selectivity filter of CorA using molecular dynamics (MD) simulations. The simulations reveal that the inner-shell structure of Mg(2+) in the filter is not significantly different from that in aqueous solution. The major difference is the characteristic structural features of the outer shell. The GMN residues engage indirectly in the interactions with the metal ion as ligands in the second shell of Mg(2+). Loss of hydrogen bonds between inner- and outer-shell waters observed from Mg(2+) in bulk water is mostly compensated by interactions between waters in the first solvation shell and the GMN motif. Some water molecules in the second shell remain in the selectivity filter and become less mobile to support the metal binding. Removal of Mg(2+) from the divalent cation sensor sites of the protein had an impact on the structure and metal binding of the filter. From the results, it can be concluded that the GMN motif enhances the affinity of the metal binding site in the CorA selectivity filter by acting as an outer coordination ligand.

Transmembrane Helix Assembly by Max-Min Ant System Algorithm.

Because of the rapid progress in biochemical and structural studies of membrane proteins, considerable attention has been given on developing efficient computational methods for solving low-to-medium resolution structures using sparse structural data. In this study, we demonstrate a novel algorithm, max-min ant system (MMAS), designed to find an assembly of α-helical transmembrane proteins using a rigid helix arrangement guided by distance constraints. The new algorithm generates a large variety with finite number of orientations of transmembrane helix bundle and finds the solution that is matched with the provided distance constraints based on the behavior of ants to search for the shortest possible path between their nest and the food source. To demonstrate the efficiency of the novel search algorithm, MMAS is applied to determine the transmembrane packing of KcsA and MscL ion channels from a limited distance information extracted from the crystal structures, and the packing of KvAP voltage sensor domain using a set of 10 experimentally determined constraints, and the results are compared with those of two popular used stochastic methods, simulated annealing Monte Carlo method and genetic algorithm.

Shaping the water crevice to accommodate the voltage sensor in a down conformation: a molecular dynamics simulation study.

Voltage sensor domains (VSD) of voltage-dependent ion channels share a basic molecular structure with a voltage-sensing phosphatase and a voltage-gated proton channel. The VSD senses and responds to changes in the membrane potential by undergoing conformational changes associated with the movement of the charged arginines located on the S4 segment. Although several functional and structural studies have provided useful information about the conformational changes in many ion channels, a detailed and unambiguous explanation has not been published. Therefore, understanding the principle of voltage-dependent gating at an atomic level is required. In this study, we took advantage of the available spin labeling electron paramagnetic resonance spectrometry data and computational methods to investigate the structure and dynamic properties of the Up-state (activated) and Down-state (resting) conformations of the VSD by means of all-atom molecular dynamics (MD) simulations. The MD results of the Down conformation determined in bilayers with and without lipid phosphates both revealed a different shape of the aqueous crevice, in which more water molecules surround and fill the intracellular crevice in its Down state than in its Up state. The solvent accessible surface within the crevice has a complementary shape that can account for water-mediated interactions between the voltage sensor and the lipid bilayer. The results support the previously reported experimental data.