Promiscuous binding of ligands by beta-lactoglobulin involves hydrophobic interactions and plasticity

Bovine beta-lactoglobulin (betaLG) binds a variety of hydrophobic ligands, though precisely how is not clear. To understand the structural basis of this promiscuous binding, we studied the interaction of betaLG with palmitic acid (PA) using heteronuclear NMR spectroscopy. The titration was monitored using tryptophan fluorescence and a HSQC spectrum confirmed a 1:1 stoichiometry for the PA-betaLG complex. Upon the binding of PA, signal disappearances and large changes in chemical shifts were observed for the residues located at the entrance and bottom of the cavity, respectively. This observation indicates that the lower region makes a rigid connection with PA whereas the entrance is more flexible. The result is in contrast to the binding of PA to intestinal fatty acid-binding protein, another member of the calycin superfamily, in which structural consolidation occurs upon ligand binding. On the other hand, the ability of betaLG to accommodate various hydrophobic ligands resembles that of GroEL, in which a large hydrophobic cavity and flexible binding site confer the ability to bind various hydrophobic substrates. Considering these observations, it is suggested that, in addition to the presence of the hydrophobic cavity, the plasticity of the entrance region makes possible the binding of hydrophobic ligands of various shapes. Thus, in contrast to the specific binding seen for many enzymes, betaLG provides an example of binding with low specificity but high affinity, which may play an important role in protein-ligand and protein-protein networks.     

Synthesis and membrane behavior of a new class of unnatural phospholipid analogs useful as phospholipase A2 degradable liposomal drug carriers

A new and unnatural type of lipid analogs with the phosphocholine and phosphoglycerol head groups linked to the C-2 position of the glycerol moiety have been synthesized and the thermodynamic lipid membrane behavior has been investigated using differential scanning calorimetry. From the heat capacity measurements, it was observed that the pre-transition was abolished most likely due to the central position of the head groups providing better packing properties in the low temperature ordered gel phase. Activity measurements of secretory phospholipase A2 (PLA2) on unilamellar liposomal membranes revealed that the unnatural phospholipids are excellent substrates for PLA2 catalyzed hydrolysis. This was manifested as a minimum in the PLA2 lag time in the main phase transition temperature regime and a high degree of lipid hydrolysis over a broad temperature range. The obtained results provide new information about the interplay between the molecular structure of phospholipids and the lipid membrane packing constrains that govern the pre-transition. In addition, the PLA2 activity measurements are useful for obtaining deeper insight into the molecular details of the catalytic site of PLA2. The combined results also suggest new approaches to rationally design liposomal drug carries that can undergo a triggered activation in diseased tissue by overexpressed PLA2.     

Vanadyl as a Probe of the Function of the F1-ATPase-Mg2+ Cofactor

The Mg²⁺ dependent asymmetry of the F₁-ATPase catalytic sites leads to the differences in affinity for nucleotides and is an essential component of the binding-change mechanism. Changes in metal ligands during the catalytic cycle responsible for this asymmetry were characterized by vanadyl (V ^IV + O)²⁺, a functional surrogate for Mg²⁺. The ⁵¹V-hyperfine parameters derived from EPR spectra of VO²⁺ bound to specific sites on F₁ provide a direct probe of the metal ligands. Site-directed mutations of metal ligand residues cause measurable changes in the ⁵¹V-hyperfine parameters of the bound VO²⁺, thereby providing a means to identification. Initial binding of the metal–nucleotide to the low-affinity catalytic site conformation results in metal coordination by hydroxyl groups from the P-loop threonine and catch-loop threonine. Upon conversion to the high-affinity conformation, carboxyl groups from the Walker homology B aspartate and MF₁E197 become ligands in lieu of the hydroxyl groups.     

Solution structure and backbone dynamics of human Raf-1 kinase inhibitor protein

Human Raf-1 kinase inhibitor protein (hRKIP) is a small multi-functional protein of 187 residues. It contains a conserved pocket, which binds a wide range of ligands from various small molecules to distinct proteins. To provide a structural basis for the ligand diversity of RKIP, we herein determined the solution structure of hRKIP, and analyzed its structural dynamics. In solution, hRKIP mainly comprises two antiparallel β sheets, two α helices and two 3₁₀ helices. NMR dynamic analysis reveals that the overall structure of hRKIP is rigid, but its C-terminal helix which is close to the ligand-binding site is mobile. In addition, residues around the ligand-binding pocket exhibit significant conformational exchange on the μs–ms timescale. Conformational flexibility may allow the ligand-binding pocket and the C-terminal helix to adopt various conformations to interact with different substrates. This work may shed light on the underlying molecular mechanisms of how hRKIP recognizes and binds diverse substrate ligands.     

Secondary and tertiary structures of the transmembrane domains of the translocator protein TSPO determined by NMR. Stabilization of the TSPO tertiary fold upon ligand binding

Numerous biological functions are attributed to the peripheral-type benzodiazepine receptor (PBR) recently renamed translocator protein (TSPO). The best characterized function is the translocation of cholesterol from the outer to inner mitochondrial membrane, which is a rate-determining step in steroid biosynthesis. TSPO drug ligands have been shown to stimulate pregnenolone formation by inducing TSPO-mediated translocation of cholesterol. Until recently, no direct structural data on this membrane protein was available. In a previous paper, we showed that a part of the mouse TSPO (mTSPO) C-terminal region adopts a helical conformation, the side-chain distribution of which provides a groove able to fit a cholesterol molecule. We report here on the overall structural properties of mTSPO. This study was first undertaken by dissecting the protein sequence and studying the conformation of five peptides encompassing the five putative transmembrane domains from ¹H-NMR data. The secondary structure of the recombinant protein in micelles was then studied using CD spectroscopy. In parallel, the stability of its tertiary fold was probed using ¹H-¹⁵N NMR. This study provides the first experimental evidence for a five-helix fold of mTSPO and shows that the helical conformation of each transmembrane domain is mainly formed through local short-range interactions. Our data show that, in micelles, mTSPO exhibits helix content close to what is expected but an unstable tertiary fold. They reveal that the binding of a drug ligand that stimulates cholesterol translocation is able to stabilize the mTSPO tertiary structure.     

Probing the mechanism of GSH activation in Schistosoma haematobium glutathione-S-transferase by site-directed mutagenesis and X-ray crystallography

During turnover, the catalytic tyrosine residue (Tyr10) of the sigma class Schistosoma haematobium wild-type glutathione-S-transferase is expected to switch alternately in and out of the reduced glutathione-binding site (G-site). The Tyrout10 conformer forms a pi-cation interaction with the guanidinium group of Arg21. As in other similar glutathione-S-transferases, the catalytic Tyr has a low pKa of 7.2. In order to investigate the catalytic role of Tyr10, and the structural and functional roles of Arg21, we carried out structural studies on two Arg21 mutants (R21L and R21Q) and a Tyr10 mutant, Y10F. Our crystallographic data for the two Arg21 mutants indicate that only the Tyrout10 conformation is populated, thereby excluding a role of Arg21 in the stabilisation of the out conformation. However, Arg21 was confirmed to be catalytically important and essential for the low pKa of Tyr10. Upon comparison with structural data generated for reduced glutathione-bound and inhibitor-bound wild-type enzymes, it was observed that the orientations of Tyr10 and Arg35 are concerted and that, upon ligand binding, minor rearrangements occur within conserved residues in the active site loop. These rearrangements are coupled to quaternary rigid-body movements at the dimer interface and alterations in the localisation and structural order of the C-terminal domain.     

Crystallographic and in silico analysis of the sialoside-binding characteristics of the Siglec sialoadhesin

The Siglec family of receptors mediates cell-surface interactions through recognition of sialylated glycoconjugates. Previously reported structures of the N-terminal domain of the Siglec sialoadhesin (SnD1) in complex with various sialic acid analogs revealed the structural template for sialic acid binding. To characterize further the carbohydrate-binding properties, we have determined the crystal structures of SnD1 in the absence of ligand, and in complex with 2-benzyl-Neu5NPro and 2-benzyl-Neu5NAc. These structures reveal that SnD1 undergoes very few structural changes on ligand binding and detail how two novel classes of sialic acid analogs bind, one of which unexpectedly can induce Siglec dimerization. In conjunction with in silico analysis, this set of structures informs us about the design of putative ligands with enhanced binding affinities and specificities to different Siglecs, and provides data with which to test the effectiveness of different computational drug design protocols.     

Kinetics and thermodynamics of ligand binding to a molten globular enzyme and its native counterpart

An engineered monomeric chorismate mutase (mMjCM) has been found to combine high catalytic activity with the characteristics of a molten globule. To gain insight into the dramatic structural changes that accompany binding of a transition-state analog, we examined mMjCM by isothermal calorimetry and compared it with its dimeric parent protein, MjCM (CM from Methanococcus jannaschii), a thermostable and conventionally folded enzyme. As expected for a ligand-induced ordering process, there is a large entropic penalty for binding to the monomer relative to the dimer (− TΔΔS = 5.1 ± 0.5 kcal/mol, at 20 °C). However, this unfavorable entropy term is largely offset by enthalpic gains (ΔΔH = − 3.5 ± 0.4 kcal/mol), presumably arising from tightening of non-covalent interactions throughout the monomeric complex. Stopped-flow kinetic measurements further reveal that the catalytic molten globule binds and releases ligands significantly faster than its natural counterpart, demonstrating that partial structural disorder can speed up molecular recognition. These results illustrate how structural plasticity may strongly perturb the thermodynamics and kinetics of transition-state recognition while negligibly affecting catalytic efficiency.     

Regulation on the expression and N-glycosylation of integrins by N-acetylglucosaminyltransferase V

The expressions of integrin alpha5, beta1, and alpha6 were studied in H7721 cells by means of flow cytometric and RT-PCR method after transfected with sense and antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V). The transfected cells were characterized by Northern blot. It was found that the order of expression from high to low was beta1>alpha5>alpha6. Transfection of sense GnT-V up-regulated alpha5 and alpha6, but not beta1 subunit, while antisense GnT-V down-regulated alpha5 and beta1, but not alpha6. The alterations of surface integrin subunits were quite compatible with the changes of their mRNAs. Using enzyme-labeled lectin analysis, it was shown that alpha5 subunit contained only C(2)C(2) biantennary N-glycan, which was not regulated by sense and antisense GnT-V. In contrast, beta1 subunit contained both biantennary and tri-/tetra-antennary N-glycans with GlcNAcbeta1,6Manalpha1,6-branch, and the latter was up- and down-regulated by the sense and antisense GnT-V, respectively. Therefore, the amount of biantennary N-glycans on beta1 subunit, but not the integrin protein, was correlated to the cell adhesion to fibronectin and laminin, which was reduced and elevated in the sense and antisense GnT-V-transfected cells, respectively, as we previously reported.     

Thermostabilisation of an agonist-bound conformation of the human adenosine A(2A) receptor

The adenosine A_2A receptor (A_2AR) is a G-protein-coupled receptor that plays a key role in transmembrane signalling mediated by the agonist adenosine. The structure of A_2AR was determined recently in an antagonist-bound conformation, which was facilitated by the T4 lysozyme fusion in cytoplasmic loop 3 and the considerable stabilisation conferred on the receptor by the bound inverse agonist ZM241385. Unfortunately, the natural agonist adenosine does not sufficiently stabilise the receptor for the formation of diffraction-quality crystals. As a first step towards determining the structure of A_2AR bound to an agonist, the receptor was thermostabilised by systematic mutagenesis in the presence of the bound agonist [³H]5'-N-ethylcarboxamidoadenosine (NECA). Four thermostabilising mutations were identified that when combined to give mutant A_2AR-GL26, conferred a greater than 200-fold decrease in its rate of unfolding compared to the wild-type receptor. Pharmacological analysis suggested that A_2AR-GL26 is stabilised in an agonist-bound conformation because antagonists bind with up to 320-fold decreased affinity. None of the thermostabilising mutations are in the ZM241385 binding pocket, suggesting that the mutations affect ligand binding by altering the conformation of the receptor rather than through direct interactions with ligands. A_2AR-GL26 shows considerable stability in short-chain detergents, which has allowed its purification and crystallisation.     

Structure–function analysis of cytochromes P450 2B

In the last 4 years, breakthroughs were made in the field of P450 2B (CYP2B) structure–function through determination of one ligand-free and two inhibitor-bound X-ray crystal structures of CYP2B4, which revealed many of the structural features required for binding ligands of different size and shape. Large conformational changes of several plastic regions of CYP2B4 can dramatically reshape the active site of the enzyme to fit the size and shape of the bound ligand without perturbing the overall P450 fold. Solution biophysical studies using isothermal titration calorimetry (ITC) have revealed the large difference in the thermodynamic parameters of CYP2B4 in binding inhibitors of different ring chemistry and side chains. Other studies have revealed that the effects of site-specific mutations on steady-state kinetic parameters and mechanism-based inactivation are often substrate dependent. These findings agree with the structural data that the enzymes adopt different conformations to bind various ligands. Thus, the substrate specificity of an individual enzyme is determined not only by active site residues but also non-active site residues that modulate conformational changes that are important for substrate access and rearrangement of the active site to accommodate the bound substrate.     

Exploring the Linkage Dependence of Polyubiquitin Conformations Using Molecular Modeling

Posttranslational modification of proteins by covalent attachment of a small protein ubiquitin (Ub) or a polymeric chain of Ub molecules (called polyubiquitin) is involved in controlling a vast variety of processes in eukaryotic cells. The question of how different polyubiquitin signals are recognized is central to understanding the specificity of various types of polyubiquitination. In polyubiquitin, monomers are linked to each other via an isopeptide bond between the C-terminal glycine of one Ub and a lysine of the other. The functional outcome of polyubiquitination depends on the particular lysine involved in chain formation and appears to rely on linkage-dependent conformation of polyubiquitin. Thus, K48-linked chains, a universal signal for proteasomal degradation, under physiological conditions adopt a closed conformation where functionally important residues L8, I44, and V70 are sequestered at the interface between two adjacent Ub monomers. By contrast, K63-linked chains, which act as a nonproteolytic regulatory signal, adopt an extended conformation that lacks hydrophobic interubiquitin contact. Little is known about the functional roles of the so-called “noncanonical” chains (linked via K6, K11, K27, K29, or K33, or linked head-to-tail), and no structural information on these chains is available, except for information on the crystal structure of the head-to-tail-linked diubiquitin (Ub₂). In this study, we use molecular modeling to examine whether any of the noncanonical chains can adopt a closed conformation similar to that in K48-linked polyubiquitin. Our results show that the eight possible Ub₂ chains can be divided into two groups: chains linked via K6, K11, K27, or K48 are predicted to form a closed conformation, whereas chains linked via K29, K33, or K63, or linked head-to-tail are unable to form such a contact due to steric occlusion. These predictions are validated by the known structures of K48-, K63-, and head-to-tail-linked chains. Our study also predicts structural models for Ub₂ chains linked via K6, K11, or K27. The implications of these findings for linkage-selective recognition of noncanonical polyubiquitin signals by various receptors are discussed.     

Structural Evidence that Peroxiredoxin Catalytic Power Is Based on Transition-State Stabilization

Peroxiredoxins (Prxs) are important peroxidases associated with both antioxidant protection and redox signaling. They use a conserved Cys residue to reduce peroxide substrates. The Prxs have a remarkably high catalytic efficiency that makes them a dominant player in cell-wide peroxide reduction, but the origins of their high activity have been mysterious. We present here a novel structure of human PrxV at 1.45 Å resolution that has a dithiothreitol bound in the active site with its diol moiety mimicking the two oxygens of a peroxide substrate. This suggests diols and similar di-oxygen compounds as a novel class of competitive inhibitors for the Prxs. Common features of this and other structures containing peroxide, peroxide-mimicking ligands, or peroxide-mimicking water molecules reveal hydrogen bonding and steric factors that promote its high reactivity by creating an oxygen track along which the peroxide oxygens move as the reaction proceeds. Key insights include how the active-site microenvironment activates both the peroxidatic cysteine side chain and the peroxide substrate and how it is exquisitely well suited to stabilize the transition state of the in-line S_N2 substitution reaction that is peroxidation.     

Crystal structure of the catalytic domain of human MAP kinase phosphatase 5: structural insight into constitutively active phosphatase

MAP kinase phosphatase 5 (MKP5) is a member of the mitogen-activated protein kinase phosphatase (MKP) family and selectively dephosphorylates JNK and p38. We have determined the crystal structure of the catalytic domain of human MKP5 (MKP5-C) to 1.6 A. In previously reported MKP-C structures, the residues that constitute the active site are seriously deviated from the active conformation of protein tyrosine phosphatases (PTPs), which are accompanied by low catalytic activity. High activities of MKPs are achieved by binding their cognate substrates, representing substrate-induced activation. However, the MKP5-C structure adopts an active conformation of PTP even in the absence of its substrate binding, which is consistent with the previous results that MKP5 solely possesses the intrinsic activity. Further, we identify a sequence motif common to the members of MKPs having low catalytic activity by comparing structures and sequences of other MKPs. Our structural information provides an explanation of constitutive activity of MKP5 as well as the structural insight into substrate-induced activation occurred in other MKPs.     

Enzymatic activity assay of D-hydantoinase by isothermal titration calorimetry. Determination of the thermodynamic activation parameters for the hydrolysis of several substrates

Isothermal titration calorimetry (ITC) has been applied to the determination of the activity of D-hydantoinase (EC 3.5.2.2) with several substrates by monitoring the heat released during the reaction. The method is based on the proportionality between the reaction rate and the thermal power (heat/time) generated. Microcalorimetric assays carried out at different temperatures provided the dependence of the catalytic rate constant on temperature. We show that ITC assay is a nondestructive method that allows the determination of the catalytic rate constant (kcat), Michaelis constant (KM), activation energy and activation Gibbs energy, enthalpy and entropy of this reaction.     

Elevated expression of N-acetylglucosaminyltransferase V in first trimester human placenta

In early pregnancy, placental trophoblast cells rapidly grow and invade into maternal uterine tissue. N-Acetylglucosaminyltransferase V (GnT-V) and its product, beta1-6-GlcNAc branching glycan, are known to correlate with tumor invasion and metastasis. Since the placentation process resembles invasion of cancer cells, we examined the expression of beta1-6-GlcNAc branching glycan and GnT-V in human placenta. Placentas derived from the first trimester contained a larger amount of beta1-6-GlcNAc branching glycan, detected by leukoagglutinating phytohemagglutinin lectin blotting, than those at term. Immunohistochemical study revealed that beta1-6-GlcNAc branching glycans and GnT-V protein were localized in the trophoblast layer. Both protein expression and the enzyme activity of GnT-V in first trimester placentas were higher than those at term. These results suggest that GnT-V would contribute to placentation in the early phase of pregnancy, possibly regulating the process of invasion of trophoblast cells.     

Adaptability of the Vitamin D nuclear receptor to the synthetic ligand Gemini: remodelling the LBP with one side chain rotation

The crystal structure of the ligand binding domain (LBD) of the wild-type Vitamin D receptor (VDR) of zebrafish bound to Gemini, a synthetic agonist ligand with two identical side chains branching at carbon 20 reveals a ligand-dependent structural rearrangement of the ligand binding pocket (LBP). The rotation of a Leu side chain opens the access to a channel that can accommodate the second side chain of the ligand. The 25% increase of the LBP's volume does not alter the essential agonist features of VDR. The possibility to adapt the LBP to novel ligands with different chemistry and/or structure opens new perspectives in the design of more specifically targeted ligands.