Modeling FRET to investigate the selectivity of lactose permease of Escherichia coli for lipids

Förster resonance energy transfer (FRET) is a photophysical process by which a donor (D) molecule in an electronic excited state transfers its excitation energy to a second species, the acceptor (A). Since FRET efficiency depends on D-A separation, the measurement of donor fluorescence in presence and absence of the acceptor allows determination of this distance, and therefore FRET has been extensively used as a “spectroscopic ruler”. In membranes, interpretation of FRET is more complex, since one D may be surrounded by many A molecules. Such is the case encountered with membrane proteins and lipids in the bilayer. This paper reviews the application of a model built to analyze FRET data between a single tryptophan mutant of the transmembrane protein lactose permease (W151/C154G of LacY), the sugar/H⁺ symporter from Escherichia coli, and different pyrene-labeled phospholipids. Several variables of the system with biological implication have been investigated: The selectivity of LacY for different species of phospholipids, the enhancement of the sensitivity of the FRET modeling, and the mutation of a particular aminoacid (D68C) of the protein. The results obtained support: (i) Preference of LacY for phosphatidylethanolamine (PE) over phosphatidylglycerol (PG); (ii) affinity of LacY for fluid (L_α) phases; and (iii) importance of the aspartic acid in position 68 in the sequence of LacY regarding the interaction with the phospholipid environment. Besides, by exploring the enhancement of the sensitivity by using pure lipid matrices with higher mole fractions of labelled-phospholipid, the dependence on acyl chain composition is unveiled.     

Estimating binding free energy of a putative growth factors EGF–VEGF complex – a computational bioanalytical study

Epidermal growth factor (EGF) and homodimeric vascular endothelial growth factor (VEGF) bind to cell surface receptors. They are responsible for cell growth and angiogenesis, respectively. Docking of the individual proteins as monomeric units using ZDOCK 2.3.2 reveals a partial blocking of the receptor binding site of VEGF by EGF. The receptor binding site of EGF is not affected by VEGF. The calculated binding energy is found to be intermediate between the binding energies calculated for Alzheimer’s Aß42 and the barnase/barstar complex.     

Hierarchical sampling for metastable conformers determines biomolecular recognition: the case of malectin and diglucosylated N-glycan interactions

Structural information over the entire course of binding interactions based on the analyses of energy landscapes is described, which provides a framework to understand the events involved during biomolecular recognition. Conformational dynamics of malectin’s exquisite selectivity for diglucosylated N-glycan (Dig-N-glycan), a highly flexible oligosaccharide comprising of numerous dihedral torsion angles, are described as an example. For this purpose, a novel approach based on hierarchical sampling for acquiring metastable molecular conformations constituting low-energy minima for understanding the structural features involved in a biologic recognition is proposed. For this purpose, four variants of principal component analysis were employed recursively in both Cartesian space and dihedral angles space that are characterized by free energy landscapes to select the most stable conformational substates. Subsequently, k-means clustering algorithm was implemented for geometric separation of the major native state to acquire a final ensemble of metastable conformers. A comparison of malectin complexes was then performed to characterize their conformational properties. Analyses of stereochemical metrics and other concerted binding events revealed surface complementarity, cooperative and bidentate hydrogen bonds, water-mediated hydrogen bonds, carbohydrate–aromatic interactions including CH–π and stacking interactions involved in this recognition. Additionally, a striking structural transition from loop to β-strands in malectin CRD upon specific binding to Dig-N-glycan is observed. The interplay of the above-mentioned binding events in malectin and Dig-N-glycan supports an extended conformational selection model as the underlying binding mechanism.     

Human dihydrofolate reductase and thymidylate synthase form a complex in vitro and co-localize in normal and cancer cells

Enzymes involved in thymidylate biosynthesis, thymidylate synthase (TS), and dihydrofolate reductase (DHFR) are well-known targets in cancer chemotherapy. In this study, we demonstrated for the first time, that human TS and DHFR form a strong complex in vitro and co-localize in human normal and colon cancer cell cytoplasm and nucleus. Treatment of cancer cells with methotrexate or 5-fluorouracil did not affect the distribution of either enzyme within the cells. However, 5-FU, but not MTX, lowered the presence of DHFR-TS complex in the nucleus by 2.5-fold. The results may suggest the sequestering of TS by FdUMP in the cytoplasm and thereby affecting the translocation of DHFR-TS complex to the nucleus. Providing a strong likelihood of DHFR-TS complex formation in vivo, the latter complex is a potential new drug target in cancer therapy. In this paper, known 3D structures of human TS and human DHFR, and some protozoan bifunctional DHFR-TS structures as templates, are used to build an in silico model of human DHFR–TS complex structure, consisting of one TS dimer and two DHFR monomers. This complex structure may serve as an initial 3D drug target model for prospective inhibitors targeting interfaces between the DHFR and TS enzymes.     

Solution structure of the human HSPC280 protein

The human HSPC280 protein belongs to a new family of low molecular weight proteins, which is only present in eukaryotes, and is absent in fungi. The solution structure of HSPC280 was determined using multidimensional NMR spectroscopy. The overall structure consists of three α-helices and four antiparallel β-strands and has a winged helix-like fold. However, HEPC280 is not a typical DNA-binding winged helix protein in that it lacks DNA-binding activity. Unlike most winged-helix proteins, HSPC280 has an unusually long 13-residue (P62-V74) wing 1 loop connecting the β3 and β4 strands of the protein. Molecules of HSPC280 have a positively charged surface on one side and a negatively charged surface on the other side of the protein structure. Comparisons with the C-terminal 80-residue domain of proteins in the Abra family reveal a conserved hydrophobic groove in the HSPC280 family, which may allow HSPC280 to interact with other proteins.     

Self-assembly of mixtures of a dendrimer and lipids: effects of hydrophobicity and electrostatics

Self-assemblies of mixtures of a G7 dendrimer and dimyristoylphosphatidylcholine (DMPC) lipids were simulated using the coarse-grained force fields. A single G7 dendrimer, which consists of either a hydrophilic or a hydrophobic interior, was simulated with the randomly distributed zwitterionic DMPC or anionic lipids. For the dendrimer with hydrophilic interior, its mixture with anionic lipids self-assembles to the dendrimer-encasing liposome, but the one with zwitterionic lipids does not. The liposome diameter agrees with experiment, which is smaller than the typical liposome without dendrimer. This indicates that the strong electrostatic interactions between dendrimer terminals and lipid phosphates induce high curvature of the bilayer, leading to such a small liposome. For the dendrimer with hydrophobic interior, lipids penetrate the dendrimer interior because of the hydrophobic interactions between the dendrimer interior and lipid tails, leading to the dendrimer–lipid micelle with increased dendrimer size. These simulation findings agree qualitatively with the experimentally proposed liposome and micelle models, and indicate that these proposed conformations of the dendrimer–lipid complexes are significantly modulated by dendrimer hydrophobicity and lipid charge.     

Conformational insights on the molecular recognition processes of carbohydrate molecules by proteins and enzymes: A 3D view by using NMR

This review focuses, in a non-exhaustive manner, on the essential structural and conformational features of protein–carbohydrate interactions and on some applications of NMR spectroscopy to deal with this topic from different levels of complexity.     

Mapping the binding sites of challenging drug targets

An increasing number of medically important proteins are challenging drug targets because their binding sites are too shallow or too polar, are cryptic and thus not detectable without a bound ligand or located in a protein–protein interface. While such proteins may not bind druglike small molecules with sufficiently high affinity, they are frequently druggable using novel therapeutic modalities. The need for such modalities can be determined by experimental or computational fragment based methods. Computational mapping by mixed solvent molecular dynamics simulations or the FTMap server can be used to determine binding hot spots. The strength and location of the hot spots provide very useful information for selecting potentially successful approaches to drug discovery.     

3D structure of a Brucella melitensis porin: molecular modelling in lipid membranes

Abstract

Brucella melitensis is a pathogenic bacterium responsible for brucellosis in mammals and humans. Its outer membrane proteins (Omp) control the diffusion of solutes through the membrane, and they consequently have a crucial role in the design of diagnostics and vaccines. Moreover, such proteins have recently revealed their potential for protein-based biomaterials. In the present contribution, the structure of the B. melitensis porin Omp2a is built using the RaptorX threading method. This is a 16-stranded β-barrel with an α-helix on the third loop folding inside the barrel and forming the constriction zone of the channel, a typical feature of general porins such as PhoE and OmpF. The preferential diffusion of cations over anions experimentally observed in anterior studies is evidenced by the presence of distinct clusters of charges in the extracellular loops and in the inner pore. Docking studies support the previously reported hypothesis of Omp2a ability to aid maltotetraose diffusion. The monomer model is then assembled into a homotrimer, stabilized by the L2 loop involved in most of the interface interactions. The stability of the trimer is evaluated in three bilayers: pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and a mixture of 1:1 of POPC/POPE. All-atom molecular dynamics simulations demonstrate the β-barrel-structural stability over time even though a breathing-like motion is observed. Compared to the pure bilayers, the POPC/POPE better preserves the integrity of the protein and its channel. Overall, this work demonstrates the relevancy of the Omp2a model and will help to design new therapeutic agents and bioinspired nanomaterials.     

Lysozyme binding with sulfa group of antibiotics: Comparative binding thermodynamics and computational study

Protein–drug binding study addresses a broad domain of biological problems associating molecular functions to physiological processes composing and modifying safe and coherent drug therapeutics. Comparison of the binding and thermodynamic aspect of sulfa drugs, sulfamethazine (SMZ) and sulfadiazine (SDZ) with the protein, lysozyme (Lyz) was carried out using spectroscopic, molecular docking, and dynamic simulation studies. The fluorescence quenching and apparent binding constant for the binding reaction were calculated to be in the order of 10⁴ M⁻¹, slightly higher for SMZ as compared to that of SDZ and the binding stoichiometry values show 1:1 drug binding with each protein molecule. The binding was an enthalpy-driven spontaneous exothermic reaction favored by a negative enthalpy and a positive entropy contribution for both the complexes. The binding from the fluorescence quenching data suggests a static quenching mechanism dominated by non-polyelectrolytic components. Synchronous fluorescence denoted a conformational change in the tryptophan moiety of the protein. Molecular docking and dynamic simulation study provided a clearer view of the interaction pattern, where the drug resides on the binding pocket of the protein structure. Overall the protein, Lyz binding of SMZ was slightly more favored over SDZ.