Protein-lipid acyl chain interactions: Depth-dependent changes of segmental mobility of phospholipid in contact with bacteriorhodopsin.

Boundary lipids surrounding membrane proteins play an essential role in protein function and structure. These protein-lipid interactions are mainly divided into electrostatic interactions between the polar amino acids of proteins and polar heads of phospholipids, and hydrophobic interactions between protein transmembrane sites and phospholipid acyl chains. Our previous report (Kawatake et al., Biochim. Biophys. Acta 1858 [2016] 2106-2115) covered a method for selectively analyzing boundary lipid interactions and showed differences in membrane protein-peripheral lipid interactions due to differences in their head group. Interactions in the hydrophobic acyl chains of phospholipids are relatively consistent among proteins, but the details of these interactions have not been elucidated. In this study, we reconstituted bacteriorhodopsin as a model protein into phospholipid membranes labeled with 2H and 13C for solid-state NMR measurement to investigate the depth-dependent effect of the head group structure on the lipid bilayer. The results showed that the position of the phospholipid near the carbonyl carbon was affected by the head group in terms of selectivity for protein surfaces, whereas in the deep interior of the bilayer near the leaflet interface, there was little difference between the head groups, indicating that the dependence of their interactions on the head group was much reduced.

Discovery of New Quinolone-Based Diarylamides as Potent B-RAFV600E/C-RAF Kinase Inhibitors Endowed with Promising In Vitro Anticancer Activity.

The emergence of cancer resistance to targeted therapy represents a significant challenge in cancer treatment. Therefore, identifying new anticancer candidates, particularly those addressing oncogenic mutants, is an urgent medical demand. A campaign of structural modifications has been conducted to further optimize our previously reported 2-anilinoquinoline-diarylamides conjugate VII as a B-RAFV600E/C-RAF inhibitor. Considering the incorporation of a methylene bridge between the terminal phenyl and cyclic diamine, focused quinoline-based arylamides have been tailored, synthesized, and biologically evaluated. Among them, the 5/6-hydroxyquinolines 17b and 18a stood out as the most potent members, with IC50 values of 0.128 µM, 0.114 µM against B-RAFV600E, and 0.0653 µM, 0.0676 µM against C-RAF. Most importantly, 17b elicited remarkable inhibitory potency against the clinically resistant B-RAFV600K mutant with an IC50 value of 0.0616 µM. The putative binding mode of 17b and 18a were studied by molecular docking and molecular dynamics (MD). Moreover, the antiproliferative activity of all target compounds has been examined over a panel of NCI-60 human cancer cell lines. In agreement with cell-free assays, the designed compounds exerted superior anticancer impact over the lead quinoline VII against all cell lines at a 10 µM dose. Notably, both 17b and 18b showed highly potent antiproliferative activity against melanoma cell lines with growth percent under -90% (SK-MEL-29, SK-MEL-5, and UACC-62) at a single dose, while 17b maintained potency with GI50 values of 1.60-1.89 µM against melanoma cell lines. Taken together, 17b, a promising B-RAFV600E/V600K and C-RAF kinase inhibitor, may serve as a valuable candidate in the arsenal of anticancer chemotherapeutics.

Binding of human ACE2 and RBD of Omicron enhanced by unique interaction patterns among SARS-CoV-2 variants of concern.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19, has continued to mutate and spread worldwide despite global vaccination efforts. In particular, the Omicron variant, first identified in South Africa in late November 2021, has become the dominant strain worldwide. Compared to the original strain identified in Wuhan, Omicron features 50 genetic mutations, with 15 mutations in the receptor-binding domain (RBD) of the spike protein, which binds to the human angiotensin-converting enzyme 2 (ACE2) receptor for viral entry. However, it is not completely understood how these mutations alter the interaction and binding strength between the Omicron RBD and ACE2. In this study, we used a combined steered molecular dynamics (SMD) simulation and experimental microscale thermophoresis (MST) approach to quantify the interaction between Omicron RBD and ACE2. We report that the Omicron brings an enhanced RBD-ACE2 interface through N501Y, Q498R, and T478K mutations; the changes further lead to unique interaction patterns, reminiscing the features of previously dominated variants, Alpha (N501Y) and Delta (L452R and T478K). Among the Q493K and Q493R, we report that Q493R shows stronger binding to ACE2 than Q493K due to increased interactions. Our MST data confirmed that the Omicron mutations in RBD are associated with a five-fold higher binding affinity to ACE2 compared to the RBD of the original strain. In conclusion, our results could help explain the Omicron variant's prevalence in human populations, as higher interaction forces or affinity for ACE2 likely promote greater viral binding and internalization, leading to increased infectivity.

Analysis of the docking property of host variants of hACE2 for SARS-CoV-2 in a large cohort.

The recent novel coronavirus disease (COVID-19) outbreak, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is threatening global health. However, an understanding of the interaction of SARS-CoV-2 with human cells, including the physical docking property influenced by the host's genetic diversity, is still lacking. Here, based on germline variants in the UK Biobank covering 502,543 individuals, we revealed the molecular interactions between human angiotensin-converting enzyme 2 (hACE2), which is the representative receptor for SARS-CoV-2 entry, and COVID-19 infection. We identified six nonsense and missense variants of hACE2 from 2585 subjects in the UK Biobank covering 500000 individuals. Using our molecular dynamics simulations, three hACE2 variants from 2585 individuals we selected showed higher levels of binding free energy for docking in the range of 1.44-3.69 kcal/mol. Although there are diverse contributors to SARS-CoV-2 infections, including the mobility of individuals, we analyzed the diagnosis records of individuals with these three variants of hACE2. Our molecular dynamics simulations combined with population-based genomic data provided an atomistic understanding of the interaction between hACE2 and the spike protein of SARS-CoV-2.

Amphotericin B assembles into seven-molecule ion channels: An NMR and molecular dynamics study.

Amphotericin B, an antifungal drug with a long history of use, forms fungicidal ion-permeable channels across cell membranes. Using solid-state nuclear magnetic resonance spectroscopy and molecular dynamics simulations, we experimentally elucidated the three-dimensional structure of the molecular assemblies formed by this drug in membranes in the presence of the fungal sterol ergosterol. A stable assembly consisting of seven drug molecules was observed to form an ion conductive channel. The structure is somewhat similar to the upper half of the barrel-stave model proposed in the 1970s but substantially different in the number of molecules and in their arrangement. The present structure explains many previous findings, including structure-activity relationships of the drug, which will be useful for improving drug efficacy and reducing adverse effects.

SPICA Force Field for Proteins and Peptides.

A coarse-grained (CG) model for peptides and proteins was developed as an extension of the Surface Property fItting Coarse grAined (SPICA) force field (FF). The model was designed to examine membrane proteins that are fully compatible with the lipid membranes of the SPICA FF. A preliminary version of this protein model was created using thermodynamic properties, including the surface tension and density in the SPICA (formerly called SDK) FF. In this study, we improved the CG protein model to facilitate molecular dynamics (MD) simulations with a reproduction of multiple properties from both experiments and all-atom (AA) simulations. An elastic network model was adopted to maintain the secondary structure within a single chain. The side-chain analogues reproduced the transfer free energy profiles across the lipid membrane and demonstrated reasonable association free energy (potential of mean force) in water compared to those from AA MD. A series of peptides/proteins adsorbed onto or penetrated into the membrane simulated by the CG MD correctly predicted the penetration depths and tilt angles of peripheral and transmembrane peptides/proteins as comparable to those in the orientations of proteins in membranes (OPM) database. In addition, the dimerization free energies of several transmembrane helices within a lipid bilayer were comparable to those from experimental estimation. Application studies on a series of membrane protein assemblies, scramblases, and poliovirus capsids demonstrated the good performance of the SPICA FF.

Effects of aquaporin-lipid molar ratio on the permeability of an aquaporin Z-phospholipid membrane system.

Aquaporins are water-permeable membrane-channel proteins found in biological cell membranes that selectively exclude ions and large molecules and have high water permeability, which makes them promising candidates for water desalination systems. To effectively apply the properties of aquaporins in the desalination process, many studies have been conducted on aquaporin-lipid membrane systems using phospholipids, which are the main component of cell membranes. Many parametric studies have evaluated the permeability of such systems with various aquaporin types and lipid compositions. In this study, we performed molecular dynamics simulations for four cases with different protein-lipid molar ratios (1:50, 1:75, 1:100, and 1:150) between aquaporin Z and the phospholipids, and we propose a possibility of the existence of optimal protein-lipid molar ratio to maximize water permeability. Elucidating these simulation results from a structural viewpoint suggests that there is a relationship between the permeability and changes in the hydrophobic thickness of the lipid membrane adjacent to the aquaporin as a structural parameter. The results of this study can help optimize the design of an aquaporin-lipid membrane by considering its molar ratio at an early stage of development.

Pivotal Role of Interdigitation in Interleaflet Interactions: Implications from Molecular Dynamics Simulations.

The asymmetric lipid composition in plasma membranes within the inner leaflet is not typically suitable for domain formation. Thus elucidation of the likelihood of the formation or stability of a raft-like domain in the inner leaflet is necessary. Herein we investigated the phase behavior of asymmetric membranes using coarse-grained molecular dynamics simulations. The lipid leaflet comprising dioleoylphosphatidylcholine (DOPC) and cholesterol (Chol) does not typically show well-developed domains in symmetric bilayer membranes; however, it does separate into liquid ordered (Lo) and liquid disordered (Ld) phases when the opposing leaflet containing sphingomyelin (SM), DOPC, and Chol demonstrates domain formation. We determine that interdigitated acyl chains modulated the partitioning of Chol in the opposing leaflet, resulting in phase separation. Similarly, the acyl chain length of SM within the opposing leaflet affected the phase behavior of the leaflet. Our results reveal the crucial role of interdigitation in determining the phase status in asymmetric membranes.

The Amphotericin B-Ergosterol Complex Spans a Lipid Bilayer as a Single-Length Assembly.

Amphotericin B (AmB) is a polyene macrolide antibiotic clinically used as an antifungal drug. Its preferential complexation with ergosterol (Erg), the major sterol of fungal membranes, leads to the formation of a barrel-stave-like ion channel across a lipid bilayer. To gain a better understanding of the mechanism of action, the mode of lipid bilayer spanning provides essential information. However, because of the lack of methodologies to observe it directly, it has not been revealed for the Erg-containing channel assembly for many years. In this study, we disclosed that the AmB-Erg complex spans a lipid bilayer with a single-molecule length, using solid-state nuclear magnetic resonance (NMR) experiments. Paramagnetic relaxation enhancement by Mn2+ residing near the surface of lipid bilayers induced the depth-dependent decay of 13C NMR signals for individual carbon atoms of AmB. We found that both terminal segments, the 41-COOH group and C38-C40 methyl groups, come close to the lipid bilayer surfaces, suggesting that the AmB-Erg complex spans a palmitoyloleoylphosphatidylcholine (POPC) bilayer with a single-molecule length. Molecular dynamics simulation experiments further confirmed the stabilization of the AmB-Erg complex as a single-length spanning complex. These results provide experimental evidence of the single-length complex incorporated in the membrane by making thinner a POPC-Erg bilayer that mimics fungal membranes.

Average Conformation of Branched Chain Lipid PGP-Me That Accounts for the Thermal Stability and High-Salinity Resistance of Archaeal Membranes.

The average conformation of the methyl-branched chains of archaeal lipid phosphatidyl glycerophosphate methyl ester (PGP-Me) was examined in a hydrated bilayer membrane based on the 2H nuclear magnetic resonance (NMR) of enantioselectively 2H-labeled compounds that were totally synthesized for the first time in this study. The NMR results in combination with molecular dynamics simulations revealed that the PGP-Me chain appeared to exhibit behavior different from that of typical membrane lipids such as dimyristoylphosphatidylcholine (DMPC). The C-C bonds of the PGP-Me chain adopt alternative parallel and tilted orientations to the membrane normal as opposed to a DMPC chain where all of the C-C bonds tilt in the same way on average. This characteristic orientation causes the intertwining of PGP-Me chains, which plays an important role in the excellent thermal and high-salinity stabilities of archaeal lipid bilayers and membrane proteins.

Cholesterol-Induced Conformational Change in the Sphingomyelin Headgroup.

Sphingomyelin (SM) and cholesterol (Cho) are the important lipids for the formation of biologically functional membrane domains, lipid rafts. However, the interaction between Cho and the headgroup of SM remains unclear. In this study, we performed solid-state NMR experiments to reveal the Cho effects on the headgroup conformation using 2H-labeled stearoyl-SM (SSM). Deuterated SSMs at the Cα, Cß, and Cγ positions of a choline moiety were separately prepared and subjected to NMR measurements to determine the quadrupolar splitting of 2H signals in hydrated SSM unitary and SSM/Cho (1:1) bilayers. Using 2H NMR and 13C-31P REDOR data, the conformation and orientation of the choline moiety were deduced and compared with those derived from molecular dynamics simulations. In SSM unitary bilayers, three torsional angles in the phosphocholine moiety, P-O-Cα-Cß, were found to be consecutive +gauche(g)/+g/+g or -g/-g/-g. The orientation and conformation of the SSM headgroup were consistent with the results of our molecular dynamics simulations and the previous results on phosphatidylcholines. The quadrupolar coupling at the α methylene group slightly increased in the presence of Cho, and those at the Cß and Cγ decreased more significantly, thus suggesting that Cho reduced the gauche conformation at the Cα-Cß torsion. The conformational ensemble in the presence of Cho may enhance the so-called umbrella effect of the SSM headgroup, resulting in the stabilization of Cho near the SM molecules by concealing the hydrophobic Cho core from interfacial water. We also examined the effect of the chiral centers at the sphingosine chain to the headgroup conformation by determining the enantiomeric excess between the diastereomeric +g/+g/+g and -g/-g/-g conformers using (S)-Cα-deuterated and (R)-Cα-deuterated SSMs. Their 2H NMR measurements showed that the chiral centers induced the slight diastereomeric excess in the SM headgroup conformation.

SPICA Force Field for Lipid Membranes: Domain Formation Induced by Cholesterol.

Heterogeneity is essential for multicomponent lipid membranes. Especially, sterol-induced domain formation in membranes has recently attracted attention because of its biological importance. To investigate such membrane domains at the molecular level, coarse-grained molecular dynamics (CG-MD) simulations are a promising approach since they allow one to consider the temporal and spatial scales involved in domain formation. In this work, we present a new CG force field, named SPICA, which can accurately predict domain formation within various lipids in membranes. The SPICA force field was developed as an extension of a previous CG model, known as SDK (Shinoda-DeVane-Klein), in which membrane properties such as tension, elasticity, and structure are well reproduced. By examining domain formation in a series of ternary lipid bilayers, we observed a separation into liquid-ordered and liquid-disordered phases fully consistent with experimental observations. Importantly, it is shown that the SPICA force field can detect the different phase behavior that results from subtle differences in the lipid composition of the bilayer.

Normal mode-guided transition pathway generation in proteins.

The biological function of proteins is closely related to its structural motion. For instance, structurally misfolded proteins do not function properly. Although we are able to experimentally obtain structural information on proteins, it is still challenging to capture their dynamics, such as transition processes. Therefore, we need a simulation method to predict the transition pathways of a protein in order to understand and study large functional deformations. Here, we present a new simulation method called normal mode-guided elastic network interpolation (NGENI) that performs normal modes analysis iteratively to predict transition pathways of proteins. To be more specific, NGENI obtains displacement vectors that determine intermediate structures by interpolating the distance between two end-point conformations, similar to a morphing method called elastic network interpolation. However, the displacement vector is regarded as a linear combination of the normal mode vectors of each intermediate structure, in order to enhance the physical sense of the proposed pathways. As a result, we can generate more reasonable transition pathways geometrically and thermodynamically. By using not only all normal modes, but also in part using only the lowest normal modes, NGENI can still generate reasonable pathways for large deformations in proteins. This study shows that global protein transitions are dominated by collective motion, which means that a few lowest normal modes play an important role in this process. NGENI has considerable merit in terms of computational cost because it is possible to generate transition pathways by partial degrees of freedom, while conventional methods are not capable of this.

Opening and closing of a toroidal group II chaperonin revealed by a symmetry constrained elastic network model.

Recently, the atomic structures of both the closed and open forms of Group 2 chaperonin protein Mm-cpn were revealed through crystallography and cryo-electron microscopy. This toroidal-like chaperonin is composed of two eightfold rings that face back-to-back. To gain a computational advantage, we used a symmetry constrained elastic network model (SCENM), which requires only a repeated subunit structure and its symmetric connectivity to neighboring subunits to simulate the entire system. In the case of chaperonin, only six subunits (i.e., three from each ring) were used out of the eight subunits comprising each ring. A smooth and symmetric pathway between the open and closed conformations was generated by elastic network interpolation (ENI). To support this result, we also performed a symmetry-constrained normal mode analysis (NMA), which revealed the intrinsic vibration features of the given structures. The NMA and ENI results for the representative single subunit were duplicated according to the symmetry pattern to reconstruct the entire assembly. To test the feasibility of the symmetry model, its results were also compared with those obtained from the full model. This study allowed the folding mechanism of chaperonin Mm-cpn to be elucidated by SCENM in a timely manner.

Efficient prediction of protein conformational pathways based on the hybrid elastic network model.

Various computational models have gained immense attention by analyzing the dynamic characteristics of proteins. Several models have achieved recognition by fulfilling either theoretical or experimental predictions. Nonetheless, each method possesses limitations, mostly in computational outlay and physical reality. These limitations remind us that a new model or paradigm should advance theoretical principles to elucidate more precisely the biological functions of a protein and should increase computational efficiency. With these critical caveats, we have developed a new computational tool that satisfies both physical reality and computational efficiency. In the proposed hybrid elastic network model (HENM), a protein structure is represented as a mixture of rigid clusters and point masses that are connected with linear springs. Harmonic analyses based on the HENM have been performed to generate normal modes and conformational pathways. The results of the hybrid normal mode analyses give new physical insight to the 70S ribosome. The feasibility of the conformational pathways of hybrid elastic network interpolation (HENI) was quantitatively evaluated by comparing three different overlap values proposed in this paper. A remarkable observation is that the obtained mode shapes and conformational pathways are consistent with each other. Our timing results show that HENM has some advantage in computational efficiency over a coarse-grained model, especially for large proteins, even though it takes longer to construct the HENM. Consequently, the proposed HENM will be one of the best alternatives to the conventional coarse-grained ENMs and all-atom based methods (such as molecular dynamics) without loss of physical reality.

A mass weighted chemical elastic network model elucidates closed form domain motions in proteins.

An elastic network model (ENM), usually Cα coarse-grained one, has been widely used to study protein dynamics as an alternative to classical molecular dynamics simulation. This simple approach dramatically saves the computational cost, but sometimes fails to describe a feasible conformational change due to unrealistically excessive spring connections. To overcome this limitation, we propose a mass-weighted chemical elastic network model (MWCENM) in which the total mass of each residue is assumed to be concentrated on the representative alpha carbon atom and various stiffness values are precisely assigned according to the types of chemical interactions. We test MWCENM on several well-known proteins of which both closed and open conformations are available as well as three α-helix rich proteins. Their normal mode analysis reveals that MWCENM not only generates more plausible conformational changes, especially for closed forms of proteins, but also preserves protein secondary structures thus distinguishing MWCENM from traditional ENMs. In addition, MWCENM also reduces computational burden by using a more sparse stiffness matrix.

KOSMOS: a universal morph server for nucleic acids, proteins and their complexes.

KOSMOS is the first online morph server to be able to address the structural dynamics of DNA/RNA, proteins and even their complexes, such as ribosomes. The key functions of KOSMOS are the harmonic and anharmonic analyses of macromolecules. In the harmonic analysis, normal mode analysis (NMA) based on an elastic network model (ENM) is performed, yielding vibrational modes and B-factor calculations, which provide insight into the potential biological functions of macromolecules based on their structural features. Anharmonic analysis involving elastic network interpolation (ENI) is used to generate plausible transition pathways between two given conformations by optimizing a topology-oriented cost function that guarantees a smooth transition without steric clashes. The quality of the computed pathways is evaluated based on their various facets, including topology, energy cost and compatibility with the NMA results. There are also two unique features of KOSMOS that distinguish it from other morph servers: (i) the versatility in the coarse-graining methods and (ii) the various connection rules in the ENM. The models enable us to analyze macromolecular dynamics with the maximum degrees of freedom by combining a variety of ENMs from full-atom to coarse-grained, backbone and hybrid models with one connection rule, such as distance-cutoff, number-cutoff or chemical-cutoff. KOSMOS is available at http://bioengineering.skku.ac.kr/kosmos.

DNA nanotube formation based on normal mode analysis.

Ever since its inception, a popular DNA motif called the cross tile has been recognized to self-assemble into addressable 2D templates consisting of periodic square cavities. Although this may be conceptually correct, in reality certain types of cross tiles can only form planar lattices if adjacent tiles are designed to bind in a corrugated manner, in the absence of which they roll up to form 3D nanotube structures. Here we present a theoretical study on why uncorrugated cross tiles self-assemble into counterintuitive 3D nanotube structures and not planar 2D lattices. Coarse-grained normal mode analysis of single and multiple cross tiles within the elastic network model was carried out to expound the vibration modes of the systems. While both single and multiple cross tile simulations produce results conducive to tube formations, the dominant modes of a unit of four cross tiles (one square cavity), termed a quadruplet, fully reflect the symmetries of the actual nanotubes found in experiments and firmly endorse circularization of an array of cross tiles.