Engineering of the start condensation domain with improved N-decanoyl catalytic activity for daptomycin biosynthesis.

Daptomycin, a lipopeptide comprising an N-decanoyl fatty acyl chain and a peptide core, is used clinically as an antimicrobial agent. The start condensation domain (dptC1) is an enzyme that catalyzes the lipoinitiation step of the daptomycin synthesis. In this study, we integrated enzymology, protein engineering, and computer simulation to study the substrate selectivity of the start condensation domain (dptC1) and to screen mutants with improved activity for decanoyl loading. Through molecular docking and computer simulation, the fatty acyl substrate channel and the protein-protein interaction interface of dptC1 are analyzed. Key residues at the protein-protein interface between dptC1 and the acyl carrier were mutated, and a single-point mutant showed more than three-folds improved catalytic efficiency of the target n-decanoyl substrate in comparing with the wild type. Moreover, molecular dynamics simulations suggested that mutants with increased catalytic activity may correlated with a more "open" and contracted substrate binding channel. Our work provides a new perspective for the elucidation of lipopeptide natural products biosynthesis, and also provides new resources to enrich its diversity and optimize the production of important components.

Early Aggregation Mechanism of SOD128-38 Based on Force Field Parameter of 5-Cyano-Tryptophan.

The aggregation of superoxide dismutase 1 (SOD1) results in amyloid deposition and is involved in familial amyotrophic lateral sclerosis, a fatal motor neuron disease. There have been extensive studies of its aggregation mechanism. Noncanonical amino acid 5-cyano-tryptophan (5-CN-Trp), which has been incorporated into the amyloid segments of SOD1 as infrared probes to increase the structural sensitivity of IR spectroscopy, is found to accelerate the overall aggregation rate and potentially modulate the aggregation process. Despite these observations, the underlying mechanism remains elusive. Here, we optimized the force field parameters of 5-CN-Trp and then used molecular dynamics simulation along with the Markov state model on the SOD128-38 dimer to explore the kinetics of key intermediates in the presence and absence of 5-CN-Trp. Our findings indicate a significantly increased probability of protein aggregate formation in 5CN-Trp-modified ensembles compared to wildtype. Dimeric ß-sheets of different natures were observed exclusively in the 5CN-Trp-modified peptides, contrasting with wildtype simulations. Free-energy calculations and detailed analyses of the dimer structure revealed augmented interstrand interactions attributed to 5-CN-Trp, which contributed more to peptide affinity than any other residues. These results explored the key events critical for the early nucleation of amyloid-prone proteins and also shed light on the practice of using noncanonical derivatives to study the aggregation mechanism.

Role of Computer-Aided Drug Design in Drug Development.

The introduction of computational techniques to pharmaceutical chemistry and molecular biology in the 20th century has changed the way people develop drugs [...].

Proteomics-based vaccine targets annotation and design of subunit and mRNA-based vaccines for Monkeypox virus (MPXV) against the recent outbreak.

Monkeypox Virus (MPXV) is a growing public health threat with increasing cases and fatalities globally. To date, no specific vaccine or small molecule therapeutic choices are available for the treatment of MPXV disease. In this work, we employed proteomics and structural vaccinology approaches to design mRNA and multi-epitopes-based vaccines (MVC) against MPXV. We first identified ten proteins from the whole proteome of MPXV as potential vaccine targets. We then employed structural vaccinology approaches to map potential epitopes of these proteins for B cell, cytotoxic T lymphocytes (CTL), and Helper T lymphocytes (HTL). Finally, 9 CTL, 6 B cell, and 5 HTL epitopes were joined together through suitable linkers to construct MVC (multi-epitope vaccine) and mRNA-based vaccines. Molecular docking, binding free energy calculation, and in silico cloning revealed robust interaction of the designed MVC with toll-like receptor 2 (TLR2) and efficient expression in E. Coli K12 strain. The immune simulation results revealed that the antigen titer after the injection reached to the maximum level on the 5th day and an abrupt decline in the antigen titer was observed upon the production of IgM, IgG and IgM + IgG, dendritic cells, IFN-gamma, and IL (interleukins), which suggested the potential of our designed vaccine candidate for inducing an immune response against MPXV.

Central cavity dehydration as a gating mechanism of potassium channels.

The hydrophobic gating model, in which ion permeation is inhibited by the hydrophobicity, rather than a physical occlusion of the nanopore, functions in various ion channels including potassium channels. Available research focused on the energy barriers for ion/water conduction due to the hydrophobicity, whereas how hydrophobic gating affects the function and structure of channels remains unclear. Here, we use potassium channels as examples and conduct molecular dynamics simulations to investigate this problem. Our simulations find channel activities (ion currents) highly correlated with cavity hydration level, implying insufficient hydration as a barrier for ion permeation. Enforced cavity dehydration successfully induces conformational transitions between known channel states, further implying cavity dewetting as a key step in the gating procedure of potassium channels utilizing different activation mechanisms. Our work reveals how the cavity dewetting is coupled to structural changes of potassium channels and how it affects channel activity. The conclusion may also apply to other ion channels.

Visualization of the mechanosensitive ion channel MscS under membrane tension.

Mechanosensitive channels sense mechanical forces in cell membranes and underlie many biological sensing processes1-3. However, how exactly they sense mechanical force remains under investigation4. The bacterial mechanosensitive channel of small conductance, MscS, is one of the most extensively studied mechanosensitive channels4-8, but how it is regulated by membrane tension remains unclear, even though the structures are known for its open and closed states9-11. Here we used cryo-electron microscopy to determine the structure of MscS in different membrane environments, including one that mimics a membrane under tension. We present the structures of MscS in the subconducting and desensitized states, and demonstrate that the conformation of MscS in a lipid bilayer in the open state is dynamic. Several associated lipids have distinct roles in MscS mechanosensation. Pore lipids are necessary to prevent ion conduction in the closed state. Gatekeeper lipids stabilize the closed conformation and dissociate with membrane tension, allowing the channel to open. Pocket lipids in a solvent-exposed pocket between subunits are pulled out under sustained tension, allowing the channel to transition to the subconducting state and then to the desensitized state. Our results provide a mechanistic underpinning and expand on the 'force-from-lipids' model for MscS mechanosensation4,11.

Lipid-protein interactions modulate the conformational equilibrium of a potassium channel.

Cell membranes actively participate in the regulation of protein structure and function. In this work, we conduct molecular dynamics simulations to investigate how different membrane environments affect protein structure and function in the case of MthK, a potassium channel. We observe different ion permeation rates of MthK in membranes with different properties, and ascribe them to a shift of the conformational equilibrium between two states of the channel that differ according to whether a transmembrane helix has a kink. Further investigations indicate that two key residues in the kink region mediate a crosstalk between two gates at the selectivity filter and the central cavity, respectively. Opening of one gate eventually leads to closure of the other. Our simulations provide an atomistic model of how lipid-protein interactions affect the conformational equilibrium of a membrane protein. The gating mechanism revealed for MthK may also apply to other potassium channels.

Phase Separation in Atomistic Simulations of Model Membranes.

Understanding the lateral organization in plasma membranes remains an open problem despite a large body of research. Model membranes with coexisting micrometer-size domains are routinely employed as simplified models of plasma membranes. Many molecular dynamics simulations have investigated phase separation in model membranes at the coarse-grained level, but atomistic simulations remain computationally challenging. We simulate DPPC:DOPC and DPPC:DOPC:cholesterol lipid bilayers to investigate phase transitions at temperatures from 310 to 270 K. In this temperature range, the binary mixture forms a liquid phase (Lα) and a coexistence of Lα and either gel or ripple phases. The ternary mixture forms a liquid disordered (Ld) phase and a coexistence of liquid ordered (Lo) and either Ld or gel phases. We quantify the coexisting phases and discuss their properties against the background of experimental results. We observe partial registration of growing domains in both mixtures. We characterize specific cholesterol-cholesterol and cholesterol-phospholipid interaction geometries underlying its increased partitioning and the smoothed phase transition in the ternary mixture compared to the binary mixture. By comparing coexisting domains with homogeneous bilayers of the same composition, we demonstrate how domain coexistence affects their properties. Our simulations provide important insights into the lipid-lipid interactions in model lipid bilayers and improve our understanding of the lateral organization in plasma membranes with higher compositional complexity.

Cholesterol Flip-Flop in Heterogeneous Membranes.

Cholesterol is the most abundant molecule in the plasma membrane of mammals. Its distribution across the two membrane leaflets is critical for understanding how cells work. Cholesterol trans-bilayer motion (flip-flop) is a key process influencing its distribution in membranes. Despite extensive investigations, the rate of cholesterol flip-flop and its dependence on the lateral heterogeneity of membranes remain uncertain. In this work, we used atomistic molecular dynamics simulations to sample spontaneous cholesterol flip-flop events in a DPPC:DOPC:cholesterol mixture with heterogeneous lateral distribution of lipids. In addition to an overall flip-flop rate at the time scale of sub-milliseconds, we identified a significant impact of local environment on flip-flop rate. We discuss the atomistic details of the flip-flop events observed in our simulations.

Lipid-Protein Interactions Are Unique Fingerprints for Membrane Proteins.

Cell membranes contain hundreds of different proteins and lipids in an asymmetric arrangement. Our current understanding of the detailed organization of cell membranes remains rather elusive, because of the challenge to study fluctuating nanoscale assemblies of lipids and proteins with the required spatiotemporal resolution. Here, we use molecular dynamics simulations to characterize the lipid environment of 10 different membrane proteins. To provide a realistic lipid environment, the proteins are embedded in a model plasma membrane, where more than 60 lipid species are represented, asymmetrically distributed between the leaflets. The simulations detail how each protein modulates its local lipid environment in a unique way, through enrichment or depletion of specific lipid components, resulting in thickness and curvature gradients. Our results provide a molecular glimpse of the complexity of lipid-protein interactions, with potentially far-reaching implications for our understanding of the overall organization of real cell membranes.

Regulation of Shigella Effector Kinase OspG through Modulation of Its Dynamic Properties.

Gram-negative pathogens secrete effector proteins into human cells to modulate normal cellular processes and establish a bacterial replication niche. Shigella and pathogenic Escherichia coli possess homologous effector kinases, OspG and NleH1/2, respectively. Upon translocation, OspG but not NleH binds to ubiquitin and a subset of E2~Ub conjugates, which was shown to activate its kinase activity. Here we show that OspG, having a minimal kinase fold, acquired a novel mechanism of regulation of its activity. Binding of the E2~Ub conjugate to OspG not only stimulates its kinase activity but also increases its optimal temperature for activity to match the human body temperature and stabilizes its labile C-terminal domain. The melting temperature (Tm) of OspG alone is only 31 °C, as compared to 41 °C to NleH1/2 homologs. In the presence of E2~Ub, the Tm of OspG increases to ~42 °C, while Ub by itself increases the Tm to 39 °C. Moreover, OspG alone displays maximal activity at 26 °C, while in the presence of E2~Ub, maximal activity occurs at ~42 °C. Using NMR and molecular dynamics calculations, we have identified the C-terminal lobe and, in particular, the C-terminal helix, as the key elements responsible for lower thermal stability of OspG as compared to homologous effector kinases.

Structure of Transmembrane Helix 8 and Possible Membrane Defects in CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that regulates the flow of anions across epithelia. Mutations in CFTR cause cystic fibrosis. CFTR belongs to the ATP-binding cassette transporter superfamily, and gating is controlled by phosphorylation and ATP binding and hydrolysis. Recently obtained ATP-free and ATP-bound structures of zebrafish CFTR revealed an unwound segment of transmembrane helix (TM) 8, which appears to be a unique feature of CFTR not present in other ATP-binding cassette transporter structures. Here, using µs-long molecular dynamics simulations, we investigate the interactions formed by this TM8 segment with nearby helices in both ATP-free and ATP-bound states. We highlight ATP-dependent interactions as well as the structural role of TM8 in maintaining the functional architecture of the pore via interactions common to both the ATP-bound and ATP-free state. The results of the molecular dynamics simulations are discussed in the context of the gating mechanism of CFTR.

Coarse-grained molecular dynamics simulations reveal lipid access pathways in P-glycoprotein.

P-glycoprotein (P-gp) exports a broad range of dissimilar compounds, including drugs, lipids, and lipid-like molecules. Because of its substrate promiscuity, P-gp is a key player in the development of cancer multidrug resistance. Although P-gp is one of the most studied ABC transporters, the mechanism by which its substrates access the cavity remains unclear. In this study, we perform coarse-grained molecular dynamics simulations to explore possible lipid access pathways in the inward-facing conformation of P-gp embedded in bilayers of different lipid compositions. In the inward-facing orientation, only lipids from the lower leaflet access the cavity of the transporter. We identify positively charged residues at the portals of P-gp that favor lipid entrance to the cavity, as well as lipid-binding sites at the portals and within the cavity, which is in good agreement with previous experimental studies. This work includes several examples of lipid pathways for phosphatidylcholine and phosphatidylethanolamine lipids that help elucidate the molecular mechanism of lipid binding in P-gp.

Inhibition of ß-Amyloid Channels with a Drug Candidate wgx-50 Revealed by Molecular Dynamics Simulations.

Destabilization of cellular ionic homeostasis by toxic ß-amyloid (Aß) channels/barrels, which is a pathogenic mechanism for Alzheimer's disease (AD), is inhibited by a novel anti-AD drug candidate wgx-50 significantly in our previous biological experiments. In this work, molecular dynamics simulations are conducted to investigate wgx-50-Aß channels/barrels interactions, as well as the ion conductance inhibition mechanism. Ion influx from the extracellular side to the central pore, which is found in apo-form simulations, is blocked by wgx-50 ligands that bind to the hydrophobic rings at the entrance of the channels/barrels. The wgx-50 binding results in smaller pore diameter of the channels/barrels; however, the overall morphology of them remains unaffected in accessible simulation time. The wgx-50 binding site in this work is consistent with what we found in our previous simulations of Aß protofibril. Our work not only investigates the ligand-Aß channels/barrels interaction mechanism but also provides insights into the rational drug design of Alzheimer's disease.

The dynamic binding of cholesterol to the multiple sites of C99: as revealed by coarse-grained and all-atom simulations.

It is generally believed that the etiology of Alzheimer's disease (AD) is closely related to the amyloid-ß polypeptides, produced from γ-secretase cleavage of C99. There is preliminary evidence that cholesterol directly activates γ-secretase cleavage of C99 through mechanisms that have not been understood so far. In this article, coarse-grained (CG) and all-atom (AT) simulations were employed to investigate the association between C99 and cholesterol, which is essential for our understanding of the role of cholesterol in the amyloidogenic pathway. Firstly, we find that both the N-terminus and the C-terminus of the C99 transmembrane domain (TMD) show interactions with cholesterol. Secondly, a multi-site dynamic cholesterol binding model was captured from the simulations, where 6 binding sites in the C99 TMD were presented. The analyses of the binding energies show that cholesterol prefers the site no. 1, 2, 4 and 5 over others. The most favorable binding energy of nearly -58.857 kJ mol-1 is from site 1, the repeat GxxxG motif. There are two pathways and two binding states of cholesterol binding to this site. Ser697 and Phe690 contribute most to the stabilization of the tightly binding state and the loosely binding state, respectively. The other binding sites described may also be potential drug targets. Thirdly, the residues GAVILMTKF, especially IVKF play a key role in this association. The C99 model appears to suggest a new mechanism for cholesterol binding. Finally, the multiple-site dynamic cholesterol binding model better explains the hypotheses that cholesterol promotes the amyloidogenic AßPP route. The GxxxA motif in the middle of the C99 transmembrane domain is completely exposed without cholesterol sheltering, which might help γ-secretase identify the cleavage sites and then promote γ-cleavage. Our results provide a detailed picture of dynamic cholesterol binding, which is crucial to our recognition of the potential influence of cholesterol on the C99 process and the etiology of AD.

Conformational flexibility of PL12 family heparinases: structure and substrate specificity of heparinase III from Bacteroides thetaiotaomicron (BT4657).

Glycosaminoglycans (GAGs) are linear polysaccharides comprised of disaccharide repeat units, a hexuronic acid, glucuronic acid or iduronic acid, linked to a hexosamine, N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine. GAGs undergo further modification such as epimerization and sulfation. These polysaccharides are abundant in the extracellular matrix and connective tissues. GAGs function in stabilization of the fibrillar extracellular matrix, control of hydration, regulation of tissue, organism development by controlling cell cycle, cell behavior and differentiation. Niche adapted bacteria express enzymes called polysaccharide lyases (PL), which degrade GAGs for their nutrient content. PL have been classified into 24 sequence-related families. Comparison of 3D structures of the prototypic members of these families allowed identification of distant evolutionary relationships between lyases that were unrecognized at the sequence level, and identified occurrences of convergent evolution. We have characterized structurally and enzymatically heparinase III from Bacteroides thetaiotaomicron (BtHepIII; gene BT4657), which is classified within the PL12 family. BtHepIII is a 72.5 kDa protein. We present the X-ray structures of two crystal forms of BtHepIII at resolution 1.8 and 2.4 Å. BtHepIII contains two domains, the N-terminal α-helical domain forming a toroid and the C-terminal ß-sheet domain. Comparison with recently determined structures of two other heparinases from the same PL12 family allowed us to identify structural flexibility in the arrangement of the domains indicating open-close movement. Based on comparison with other GAG lyases, we identified Tyr301 as the main catalytic residue and confirmed this by site-directed mutagenesis. We have characterized substrate preference of BtHepIII toward sulfate-poor heparan sulfate substrate.

Ganglioside-Lipid and Ganglioside-Protein Interactions Revealed by Coarse-Grained and Atomistic Molecular Dynamics Simulations.

Gangliosides are glycolipids in which an oligosaccharide headgroup containing one or more sialic acids is connected to a ceramide. Gangliosides reside in the outer leaflet of the plasma membrane and play a crucial role in various physiological processes such as cell signal transduction and neuronal differentiation by modulating structures and functions of membrane proteins. Because the detailed behavior of gangliosides and protein-ganglioside interactions are poorly known, we investigated the interactions between the gangliosides GM1 and GM3 and the proteins aquaporin (AQP1) and WALP23 using equilibrium molecular dynamics simulations and potential of mean force calculations at both coarse-grained (CG) and atomistic levels. In atomistic simulations, on the basis of the GROMOS force field, ganglioside aggregation appears to be a result of the balance between hydrogen bond interactions and steric hindrance of the headgroups. GM3 clusters are slightly larger and more ordered than GM1 clusters due to the smaller headgroup of GM3. The different structures of GM1 and GM3 clusters from atomistic simulations are not observed at the CG level based on the Martini model, implying a difference in driving forces for ganglioside interactions in atomistic and CG simulations. For protein-ganglioside interactions, in the atomistic simulations, GM1 lipids bind to specific sites on the AQP1 surface, whereas they are depleted from WALP23. In the CG simulations, the ganglioside binding sites on the AQP1 surface are similar, but ganglioside aggregation and protein-ganglioside interactions are more prevalent than in the atomistic simulations. Using the polarizable Martini water model, results were closer to the atomistic simulations. Although experimental data for validation is lacking, we proposed modified Martini parameters for gangliosides to more closely mimic the sizes and structures of ganglioside clusters observed at the atomistic level.

Conformational Changes of the Antibacterial Peptide ATP Binding Cassette Transporter McjD Revealed by Molecular Dynamics Simulations.

The ATP binding cassette (ABC) transporters form one of the largest protein superfamilies. They use the energy of ATP hydrolysis to transport chemically diverse ligands across membranes. An alternating access mechanism in which the transporter switches between inward- and outward-facing conformations has been proposed to describe the translocation process. One of the main open questions in this process is the degree of opening of the transporter at different stages of the transport cycle, as crystal structures and biochemical data have suggested a wide range of distances between nucleotide binding domains. Recently, the crystal structure of McjD, an antibacterial peptide ABC transporter from Escherichia coli, revealed a new occluded intermediate state of the transport cycle. The transmembrane domain is closed on both sides of the membrane, forming a cavity that can accommodate its ligand, MccJ25, a lasso peptide of 21 amino acids. In this work, we investigate the degree of opening of the transmembrane cavity required for ligand translocation. By means of steered molecular dynamics simulations, the ligand was pulled from the internal cavity to the extracellular side. This resulted in an outward-facing state. Comparison with existing outward-facing crystal structures shows a smaller degree of opening in the simulations, suggesting that the large conformational changes in some crystal structures may not be necessary even for a large substrate like MccJ25.