A Possible Binding Path of Ergosterol Within Elicitins Revealed by Molecular Dynamics

Abstract

Elicitins, produced by most of the phytopathogenic fungi of the genus Phytophthora, provoke in the tobacco plant both remote leaf necrosis and the induction of a resistance against subsequent attack by various micro-organisms. The crystal structure of b-cryptogein (CRY), secreted by Phytophthora cryptogea, was previously reported as well as the first structure of a SCP/sterol complex, the ergosterol-complexed, mutated CRY (K13H). In K13H, the ergosterol molecule is encapsulated in a large internal hydrophobic cavity which is not present in CRY. This binding induces a minor conformational change in the protein structure. Molecular dynamics studies were undertaken to precise the structural behaviour of CRY and K13H with respect to the complexation of the ergosterol.

Although it is not possible to simulate the entrance of the ergosterol in the protein, we assume that capture and release of the ligand possibly both occur following the same path. Our results show that, in the complex K13H, the ergosterol molecule is pushed towards the residue 13 which play a key role in the necrotic activity of the protein. It is likely that the polarity of residue 13, favouring the binding of the hydroxy l of the ligand, would be involved in the recognition of the sterol and in an optimisation of its orientation. Thus, in a first step, the molecule of ergosterol would be rotated around itself to a position which makes possible, in a second step, its translation to the internal cavity, as a key in a keyhole.     

Binding mechanism of nonspecific lipid transfer proteins and their role in plant defense

Plant nonspecific lipid transfer proteins (nsLTPs) are small basic proteins that transport phospholipids between membranes. On the basis of molecular mass, nsLTPs are subdivided into nsLTP1 and nsLTP2. NsLTPs are all helical proteins stabilized by four conserved disulfide bonds. The existence of an internal hydrophobic cavity, running through the molecule, is a typical characteristic of nsLTPs that serves as the binding site for lipid-like substrates. NsLTPs are known to participate in plant defense, but the exact mechanism of their antimicrobial action against fungi or bacteria is still unclear. To trigger plant defense responses, a receptor at the plant surface needs to recognize the complex of a fungal protein (elicitin) and ergosterol. NsLTPs share high structural similarities with elicitin and need to be associated with a hydrophobic ligand to stimulate a defense response. In this study, binding of sterol molecules with rice nsLTPs is analyzed using various biophysical methods. NsLTP2 can accommodate a planar sterol molecule, but nsLTP1 binds only linear lipid molecules. Although the hydrophobic cavity of rice nsLTP2 is smaller than that of rice nsLTP1, it is flexible enough to accommodate the voluminous sterol molecule. The dissociation constant for the nsLTP2/cholesterol complex is approximately 71.21 microM as measured by H/D exchange and mass spectroscopic detection. Schematic models of the nsLTP complex structure give interesting clues about the reason for differential binding modes. Comparisons of NMR spectra of the sterol/rice nsLTP2 complex and free nsLTP2 revealed the residues involved in binding.     

Mutagenesis study of rice nonspecific lipid transfer protein 2 reveals residues that contribute to structure and ligand binding

Plant nonspecific lipid transfer protein 2 (nsLTP2) is a small (7 kDa) protein that binds lipid-like ligands. An inner hydrophobic cavity surrounded by alpha-helices is the defining structural feature of nsLTP2. Although nsLTP2 structures have been reported earlier, the detailed mechanisms of ligand binding and lipid transfer remain unclear. In this study, we used site-directed mutagenesis to determine the role of various hydrophobic residues (L8, I15, F36, F39, Y45, Y48, and V49) in the structure, stability, ligand binding, and lipid transfer activity of rice nsLTP2. Three single mutations (L8A, F36A, and V49A) drastically alter the native tertiary structure and perturb ligand binding and lipid transfer activity. Therefore, these three residues are structurally important. The Y45A mutant, however, retains a native-like structure but has decreased lipid binding affinity and lipid transfer activity, implying that this aromatic residue is critical for these biological functions. The mutants, I15A and Y48A, exhibit quite different ligand binding affinities. Y48 is involved in planar sterol binding but not linear lysophospholipid association. As for I15A, it had the highest dehydroergosterol binding affinity in spite of the lower lipid binding and transfer abilities. Our results suggest that the long alkyl side chain of I15 would restrict the flexibility of loop I (G13-A19) for sterol entry. Finally, F39A can markedly increase the exposed hydrophobic surface to maintain its transfer efficiency despite reduced ligand binding affinity. These findings suggest that the residues forming the hydrophobic cavity play various important roles in the structure and function of rice nsLTP2.     

Solution structure of ileal lipid binding protein in complex with glycocholate.

Ileal lipid binding protein (ILBP) is a cytosolic lipid-binding protein that binds both bile acids and fatty acids. We have determined the solution structure of porcine ILBP in complex with glycocholate by homonuclear and heteronuclear two-dimensional NMR spectroscopy. The conformation of the protein-ligand complex was determined by restrained energy minimization and simulated annealing calculations after docking the glycocholate ligand into the protein structure. The overall tertiary structure of ILBP is highly analogous to the three-dimensional structures of several other intracellular lipid binding proteins (LBPs). Like the apo-structure, the bile-acid complex of ILBP is composed of 10 anti-parallel beta-strands that form a water-filled clam-shell structure, and two short alpha-helices. Chemical shift data indicated that the bile acid ligand is bound inside the protein cavity. Furthermore, 13C-edited heteronuclear single-quantum correlation-NOESY experiments showed NOE contacts between several aromatic residues located in the proposed bile acid portal region and the 13C-labeled ligand. A single bile acid molecule is bound inside the protein, with the steroid moiety penetrating deep into the water-accessible internal cavity, such that ring A is located right above the plane of the Trp49 indole ring. The carboxylate tail of the ligand is protruding from the proposed bile acid portal into the surrounding aqueous solution. The body of the steroid moiety is oriented with the nonpolar face in contact with the mostly hydrophobic residues of beta-strands C, D and E, while the polar face shows contacts with the side-chains of Tyr97, His99, Glu110 and Arg121 in beta-strands H, I and J. Thus, the conformational arrangement of the ligand complex suggests that the binding affinity of ILBP for bile acid molecules is based mainly on strong hydrophobic interactions inside the protein cavity. Furthermore, this binding mode explains how ILBP can transport unconjugated and conjugated bile acids.     

Y132H substitution in Candida albicans sterol 14alpha-demethylase confers fluconazole resistance by preventing binding to haem

Fungal cytochrome P450 sterol 14alpha-demethylase (CYP51) is required for ergosterol biosynthesis and is the target for azole antifungal compounds. The amino acid substitution Y132H in CYP51 from clinical isolates of Candida albicans can cause fluconazole resistance by a novel change in the protein. Fluconazole binding to the mutant protein did not involve normal interaction with haem as shown by inducing a Type I spectral change. This contrasted to the wild-type protein where fluconazole inhibition was reflected in coordination to haem as a sixth ligand and where the typical Type II spectrum was obtained. The Y132H substitution occurred without drastic perturbation of the haem environment or activity allowing resistant mutants to produce ergosterol and retain fitness, an efficient strategy for resistance in nature.     

Sequence and structural analysis of fibronectin‐binding protein reveals importance of multiple intrinsic disordered tandem repeats

The location of certain amino acid sequences like repeats along the polypeptide chain is very important in the context of forming the overall shape of the protein molecule which in fact determines its function. In gram‐positive bacteria, fibronectin‐binding protein (FnBP) is one such repeat containing protein, and it is a cell wall‐attached protein responsible for various acute infections in human. Several studies on sequence, structure, and function of fibronectin‐binding regions of FnBPs were reported; however, no detailed study was carried out on the full‐length protein sequence. In the present study, we have made a thorough sequence and structure analysis on FnBP_A of Staphylococcus aureus and explored the presence of dual ligand‐binding ability of fibrinogen (fg)‐binding region and its molecular recognition processes. Multiple sequence alignment and protein‐protein docking analysis reveal the regions which are likely involved in dual ligand binding. Further analysis of docking of FnBP_A fg‐binding region and fn N‐terminal modules suggests that if the latter binds to the fg‐binding region of FnBP_A, it would inhibit the subsequent binding of fg because of steric hindrance. The sequence analysis further suggests that the abundance of disorder promoting residue glutamic acid and dual personality (both order/disorder promoting) residue threonine in tandem repeats of FnBP_A and B proteins possibly would help the molecule to undergo a conformational change while binding with fn by β‐zipper mechanism. The segment‐based power spectral analysis was carried out which helps to understand the distribution of hydrophobic residues along the sequence particularly in intrinsic disordered tandem repeats. The results presented here will help to understand the role of internal repeats and intrinsic disorder in the molecular recognition process of a pathogenic cell surface protein.     

Investigations of Takeout proteins’ ligand binding and release mechanism using molecular dynamics simulation

Takeout (To) proteins exist in a diverse range of insect species. They are involved in many important processes of insect physiology and behaviors. As the ligand carriers, To proteins can transport the small molecule to the target tissues. However, ligand release mechanism of To proteins is unclear so far. In this contribution, the process and pathway of the ligand binding and release are revealed by conventional molecular dynamics simulation, steered molecular dynamics simulation and umbrella sampling methods. Our results show that the α4-side of the protein is the unique gate for the ligand binding and release. The structural analysis confirms that the internal cavity of the protein has high rigidity, which is in accordance with the recent experimental results. By using the potential of mean force calculations in combination with residue cross correlation calculation, we concluded that the binding between the ligand and To proteins is a process of conformational selection. Furthermore, the conformational changes of To proteins and the hydrophobic interactions both are the key factors for ligand binding and release.     

Testing of the portal hypothesis: analysis of a V32G, F57G, K58G mutant of the fatty acid binding protein of the murine adipocyte

The portal region of fatty acid binding proteins is hypothesized to function as a dynamic aperture, controlling accessibility of external ligands to the internal fatty acid binding cavity. To test this hypothesis, a triple mutant of the murine FABP4 has been developed (V32G, F57G, K58G, referred to as the portal mutant) that is predicted to constitutively enlarge the opening due to a reduction in the molecular dimensions of the side chains of key portal amino acids. The portal mutant was purified from expressing Escherichia coli, its stability was evaluated, and the thermodynamics and kinetics of ligand binding were compared to that of wild-type protein. Introduction of the three amino acid substitutions caused no significant change in the stability of the protein with a free energy of unfolding of 13.7 kJ/mol as compared to 14.0 kJ/mol for the wild-type protein. The portal mutant exhibited a modest decrease (4-fold) in ligand binding affinity using the fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) as a surrogate ligand. 1,8-ANS displacement assays revealed that the binding affinity for oleate increased from a K0.5 of 196 +/- 15 nM for the wild-type protein to 165 +/- 8 for the portal mutant, while that for arachidonate decreased from the wild type of 186 +/- 11 nM to 418 +/- 26 nM for the portal mutant. To evaluate cavity accessibility, rate of 1,8-ANS binding was assessed between the portal and wild-type protein. Using equimolar amounts of ligand and protein at 4 degrees, 1,8-ANS bound within the cavity to 95% saturation (t0.95) in 750 ms, while the mutant protein was fully modified in less than 1.4 ms. To independently evaluate cavity accessibility, modification of the sole protein cysteine residue, C117 residing within the cavity near C2-C4 of the bound ligand, was monitored using 5,5'-dithio-bis(2-nitrobenzoic acid) modification. The half time for modification (t0.5) for the wild-type protein was approximately 20 s, while that for V32G F57G K58G occurred in less than a second. As such, enlargement of the portal region of FABP4 markedly increased the accessibility of ligands to the cavity while having only modest effects on ligand affinity. Taken together, these data provide support for the portal region hypothesis and suggest dynamic fluctuations in this region regulate cavity access, but not ligand affinity or selectivity.     

The effect of replacing the axial methionine ligand with a lysine residue in cytochrome c-550 from Paracoccus versutus assessed by X-ray crystallography and unfolding

The structure of cytochrome c-550 from the nonphotosynthetic bacteria Paraccocus versutus has been solved by X-ray crystallography to 1.90 A resolution, and reveals a high structural homology to other bacterial cytochromes c(2). The effect of replacing the axial heme-iron methionine ligand with a lysine residue on protein structure and unfolding has been assessed using the M100K variant. From X-ray structures at 1.95 and 1.55 A resolution it became clear that the amino group of the lysine side chain coordinates to the heme-iron. Structural differences compared to the wild-type protein are confined to the lysine ligand loop connecting helices four and five. In the heme cavity an additional water molecule is found which participates in an H-bonding interaction with the lysine ligand. Under cryo-conditions extra electron density in the lysine ligand loop is revealed, leading to residues K97 to T101 being modeled with a double main-chain conformation. Upon unfolding, dissociation of the lysine ligand from the heme-iron is shown to be pH dependent, with NMR data consistent with the occurrence of a ligand exchange mechanism similar to that seen for the wild-type protein.     

Ergosterol elicits oxidative burst in tobacco cells via phospholipase A2 and protein kinase C signal pathway.

Ergosterol, a typical fungal sterol, induced in tobacco (Nicotiana tabacum L. cv. Xanthi) suspension cells the synthesis of reactive oxygen species and alkalization of the external medium that are dependent on the mobilization of calcium from internal stores. We used specific inhibitors to elucidate the signal pathway triggered by ergosterol compared with cryptogein, a proteinaceous elicitor of Phytophthora cryptogea. Herbimycin A and genistein, inhibitors of tyrosine protein kinases, had no effect on the oxidative burst and pH changes induced by both elicitors. Similarly, H-89, an inhibitor of protein kinase A, had no effect on the induction of these defense reactions. However, the response to both elicitors was completely blocked by NPC-15437, a specific inhibitor of animal protein kinase C (PKC). The responses induced by cryptogein but not those induced by ergosterol were inhibited by U73122 and neomycin, inhibitors of phospholipase C (PLC). On the other hand, the activity of phospholipase A2 (PLA2) measured using a fluorogenic substrate was stimulated by ergosterol and not by cholesterol and cryptogein. A specific inhibitor of PLA2, arachidonic acid trifluoromethyl ketone (AACOCF3), inhibited the pathway stimulated by ergosterol but not that induced by cryptogein. These results suggest that the cryptogein-induced signal pathway leading to the oxidative burst and DeltapH changes includes PLC and PKC, whereas this response induced by ergosterol includes PLA2 and PKC.     

Variation of protein binding cavity volume and ligand volume in protein–ligand complexes

We have systematically analyzed the variation of protein binding cavity volume of 200 protein–ligand complexes belonging to eight protein families. Wide variation in protein binding cavity volume for the same protein is observed on binding different ligands. Analysis of individual protein families shows high correlation between atom–atom interactions in binding site and ligand volume. This study implies the significance of protein flexibility in docking small molecule inhibitors on the basis of protein binding cavity volume with respect to ligand volume.     

Computational studies of the interaction of myoglobin and xenon

Computational studies are used to investigate the energies of xenon binding to myoglobin and to describe pathways through the protein interior for a metmyoglobin-xenon complex. Empirical energy calculations indicate a favorable enthalpic contribution of 0.6 to 4.2 kcal/mol to xenon binding for four experimentally determined xenon sites. These calculated enthalpies help to explain the different xenon occupancies observed experimentally. A fifth site, modeled in place of the iron co-ordinated water molecule in the distal cavity, is also predicted to bind xenon. The largest contribution to the binding energy is from van der Waals' interactions with smaller contributions from polarization and protein strain terms. Ligand trajectory calculations as well as a new geometric algorithm define a connecting network of channel-like pathways through the static protein structure. One or two pathways appear to lead most easily from each major internal cavity to the protein surface. The importance of these channels in protein dynamics and their implications as routes for ligand motion are discussed.     

Three-dimensional solution structure of beta cryptogein, a beta elicitin secreted by a phytopathogenic fungus Phytophthora cryptogea.

Cryptogein belongs to a new family of 10-kDa proteins called elicitins. Elicitins are necrotic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive-like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The solution structure of beta cryptogein from Phytophthora cryptogea fungus was determined by using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. A set of 18 structures was calculated using 1360 NOE-derived distance restraints and 40 dihedral angle restraints obtained from 3JHNH alpha couplings. The RMS deviation from the mean structure is 0.87 +/- 0.14 A for backbone atoms and 1.34 +/- 0.14 A for all the non-hydrogen atoms of residues 2 to 98. The structure of beta cryptogein reveals a novel protein fold, with five helices and a double-stranded beta-sheet facing an omega-loop. One edge of the beta-sheet and the adjacent face of the omega-loop form a hydrophobic cavity. This cavity made of highly conserved residues represents a plausible binding site. Residue 13, which has been identified from directed mutagenesis and natural sequence comparison studies as a key amino acid involved in the differential control of necrosis, is surface exposed and could contribute to the binding to a ligand or a receptor. The solution structure is close to the X-ray structure, with slight differences lightly due to the crystal packing.     

The major birch allergen, Bet v 1, shows affinity for a broad spectrum of physiological ligands

Bet v 1 is a 17-kDa protein abundantly present in the pollen of the White birch tree and is the primary cause of birch pollen allergy in humans. Its three-dimensional structure is remarkable in that a solvent-accessible cavity traverses the core of the molecule. The biological function of Bet v 1 is unknown, although it is homologous to a family of pathogenesis-related proteins in plants. In this study we first show that Bet v 1 in the native state is able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS). ANS binds to Bet v 1 with 1:1 stoichiometry, and NMR data indicate that binding takes place in the cavity. Using an ANS displacement assay, we then identify a range of physiologically relevant ligands, including fatty acids, flavonoids, and cytokinins, which generally bind with low micromolar affinity. The ability of these ligands to displace ANS suggests that they also bind in the cavity, although the exact binding sites seem to vary among different ligands. The cytokinins, for example, seem to bind at a separate site close to ANS, because they increase the fluorescence of the ANS.Bet v 1 complex. Also, the fluorescent sterol dehydroergosterol binds to Bet v 1 as demonstrated by direct titrations. This study provides the first qualitative and quantitative data on the ligand binding properties of this important pollen allergen. Our findings indicate that ligand binding is important for the biological function of Bet v 1.     

A computational study of ion binding and protonation states in the KcsA potassium channel

We report results from microscopic molecular dynamics and free energy perturbation simulations of the KcsA potassium channel based on its experimental atomic structure. Conformational properties of selected amino acid residues as well as equilibrium positions of K(+) ions inside the selectivity filter and the internal water cavity are examined. Positions three and four (counting from the extracellular site) in the experimental structure correspond to distinctly separate binding sites for K(+) ions inside the selectivity filter. The protonation states of Glu71 and Asp80, which are close to each other and to the selectivity filter, as well as K(+) binding energies are determined using free energy perturbation calculations. The Glu71 residue which is buried inside a protein cavity is found to be most stable in the neutral form while the solvent exposed Asp80 is ionized. The channel altogether exothermically binds up to three ions, where two of them are located inside the selectivity filter and one in the internal water cavity. Ion permeation mechanisms are discussed in relation to these results.     

Crystal structure of axolotl (Ambystoma mexicanum) liver bile acid-binding protein bound to cholic and oleic acid

The family of the liver bile acid-binding proteins (L-BABPs), formerly called liver basic fatty acid-binding proteins (Lb-FABPs) shares fold and sequence similarity with the paralogous liver fatty acid-binding proteins (L-FABPs) but has a different stoichiometry and specificity of ligand binding. This article describes the first X-ray structure of a member of the L-BABP family, axolotl (Ambystoma mexicanum) L-BABP, bound to two different ligands: cholic and oleic acid. The protein binds one molecule of oleic acid in a position that is significantly different from that of either of the two molecules that bind to rat liver FABP. The stoichiometry of binding of cholate is of two ligands per protein molecule, as observed in chicken L-BABP. The cholate molecule that binds buried most deeply into the internal cavity overlaps well with the analogous bound to chicken L-BABP, whereas the second molecule, which interacts with the first only through hydrophobic contacts, is more external and exposed to the solvent.     

Symmetry-based ligand design and evaluation of small molecule inhibitors of programmed cell death-1/programmed death-ligand 1 interaction

The development of small molecule inhibitors of PD-1/PD-L1 is eagerly anticipated for treatment of cancer. We focused on the symmetry of the ternary complex structure of reported small molecule ligands and hPD-L1 homodimers, and designed partially- or fully-symmetric compounds for more potent inhibitors. The design of the new compounds was guided by our hypothesis that the designed symmetric compound would induce a flip of sidechain of _ATyr56 protein residue to form a new cavity. The designed compound 4 exhibited substantially increased binding affinity to hPD-L1, as well as PD-1/PD-L1 inhibitory activity in physiological conditions. Compound 4 also showed a dose-dependent increase in IFN-γ secretion levels in a mixed lymphocyte reaction assay. These results not only indicate the feasibility of targeting the PD-1/PD-L1 pathway with small molecules, but illustrate the applicability of the symmetry-based ligand design as an attractive methodology for targeting protein-protein interaction stabilizers.     

A selective cholesterol-dependent induction of H+/OH- currents in phospholipid vesicles by amphotericin B

The effect of amphotericin B on the proton/hydroxide permeability of small unilamellar vesicles has been investigated by using potential-dependent paramagnetic probes. Amphotericin B at 1-10 molecules/vesicle causes a modest 4-8-fold increase in the background H+/OH- permeability of egg phosphatidylcholine (egg PC) vesicles. However, in the presence of cholesterol, amphotericin B promotes a dramatic increase in the H+/OH- permeability of more than 2 orders of magnitude. Surprisingly, this is not observed in vesicle membranes containing ergosterol. In membranes composed of 5-15 mol% ergosterol, amphotericin B is even less effective at promoting H+/OH- currents than in pure egg PC vesicles. The K+ current promoted by amphotericin B in vesicles formed from egg PC and from egg PC plus cholesterol or ergosterol was measured. No significant sterol dependence was found for the K+ current. These results strongly suggest that different mechanisms, or amphotericin B/sterol complexes, are responsible for the induction of H+/OH- and K+ currents. These results have important implications for understanding the therapeutic and toxic effects of amphotericin B.     

Characterization of crystal water molecules in a high-affinity inhibitor and hematopoietic prostaglandin D synthase complex by interaction energy studies

Hematopoietic prostaglandin D synthase (H-PGDS) is one of the two enzymes that catalyze prostaglandin D₂ synthesis and a potential therapeutic target of allergic and inflammatory responses. To reveal key molecular interactions between a high-affinity ligand and H-PGDS, we designed and synthesized a potent new inhibitor (K_D: 0.14?nM), determined the crystal structure in complex with human H-PGDS, and quantitatively analyzed the ligand–protein interactions by the fragment molecular orbital calculation method. In the cavity, 10 water molecules were identified, and the interaction energy calculation indicated their stable binding to the surface amino acids in the cavity. Among them, 6 water molecules locating from the deep inner cavity to the peripheral part of the cavity contributed directly to the ligand binding by forming hydrogen bonding interactions. Arg12, Gly13, Gln36, Asp96, Trp104, Lys112 and an essential co-factor glutathione also had strong interactions with the ligand. A strong repulsive interaction between Leu199 and the ligand was canceled out by forming a hydrogen bonding network with the adjacent conserved water molecule. Our quantitative studies including crystal water molecules explained that compounds with an elongated backbone structure to fit from the deep inner cavity to the peripheral part of the cavity would have strong affinity to human H-PGDS.