High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875.

Human GPR40 receptor (hGPR40), also known as free fatty-acid receptor 1 (FFAR1), is a G-protein-coupled receptor that binds long-chain free fatty acids to enhance glucose-dependent insulin secretion. Novel treatments for type-2 diabetes mellitus are therefore possible by targeting hGPR40 with partial or full agonists. TAK-875, or fasiglifam, is an orally available, potent and selective partial agonist of hGPR40 receptor, which reached phase III clinical trials for the potential treatment of type-2 diabetes mellitus. Data from clinical studies indicate that TAK-875, which is an ago-allosteric modulator of hGPR40 (ref. 3), demonstrates improved glycaemic control and low hypoglycaemic risk in diabetic patients. Here we report the crystal structure of hGPR40 receptor bound to TAK-875 at 2.3 Å resolution. The co-complex structure reveals a unique binding mode of TAK-875 and suggests that entry to the non-canonical binding pocket most probably occurs via the lipid bilayer. The atomic details of the extensive charge network in the ligand binding pocket reveal additional interactions not identified in previous studies and contribute to a clear understanding of TAK-875 binding to the receptor. The hGPR40-TAK-875 structure also provides insights into the plausible binding of multiple ligands to the receptor, which has been observed in radioligand binding and Ca(2+) influx assay studies. Comparison of the transmembrane helix architecture with other G-protein-coupled receptors suggests that the crystallized TAK-875-bound hGPR40 complex is in an inactive-like state.

Membrane domain structures of three classes of histidine kinase receptors by cell-free expression and rapid NMR analysis.

NMR structural studies of membrane proteins (MP) are hampered by complications in MP expression, technical difficulties associated with the slow process of NMR spectral peak assignment, and limited distance information obtainable for transmembrane (TM) helices. To overcome the inherent challenges in the determination of MP structures, we have developed a rapid and cost-efficient strategy that combines cell-free (CF) protein synthesis, optimized combinatorial dual-isotope labeling for nearly instant resonance assignment, and fast acquisition of long-distance information using paramagnetic probes. Here we report three backbone structures for the TM domains of the three classes of Escherichia coli histidine kinase receptors (HKRs). The ArcB and QseC TM domains are both two-helical motifs, whereas the KdpD TM domain comprises a four-helical bundle with shorter second and third helices. The interhelical distances (up to 12 A) reveal weak interactions within the TM domains of all three receptors. Determined consecutively within 8 months, these structures offer insight into the abundant and underrepresented in the Protein Data Bank class of 2-4 TM crossers and demonstrate the efficiency of our CF combinatorial dual-labeling strategy, which can be applied to solve MP structures in high numbers and at a high speed. Our results greatly expand the current knowledge of HKR structure, opening the doors to studies on their widespread and pharmaceutically important bacterial signaling mechanism.

In Vitro Chondrogenesis Induction by Short Peptides of the Carboxy-Terminal Domain of Transforming Growth Factor ß1.

Τransforming growth factor ß1 (TGF-ß1) comprises a key regulator protein in many cellular processes, including in vivo chondrogenesis. The treatment of human dental pulp stem cells, separately, with Leu83-Ser112 (C-terminal domain of TGF-ß1), as well as two very short peptides, namely, 90-YYVGRKPK-97 (peptide 8) and 91-YVGRKP-96 (peptide 6) remarkably enhanced the chondrogenic differentiation capacity in comparison to their full-length mature TGF-ß1 counterpart either in monolayer cultures or 3D scaffolds. In 3D scaffolds, the reduction of the elastic modulus and viscous modulus verified the production of different amounts and types of ECM components. Molecular dynamics simulations suggested a mode of the peptides' binding to the receptor complex TßRII-ALK5 and provided a possible structural explanation for their role in inducing chondrogenesis, along with endogenous TGF-ß1. Further experiments clearly verified the aforementioned hypothesis, indicating the signal transduction pathway and the involvement of TßRII-ALK5 receptor complex. Real-time PCR experiments and Western blot analysis showed that peptides favor the ERK1/2 and Smad2 pathways, leading to an articular, extracellular matrix formation, while TGF-ß1 also favors the Smad1/5/8 pathway which leads to the expression of the metalloproteinases ADAMTS-5 and MMP13 and, therefore, to a hypertrophic chondrocyte phenotype. Taken together, the two short peptides, and, mainly, peptide 8, could be delivered with a scaffold to induce in vivo chondrogenesis in damaged articular cartilage, constituting, thus, an alternative therapeutic approach for osteoarthritis.

The structure of dihydrodipicolinate reductase (DapB) from Mycobacterium tuberculosis in three crystal forms.

Dihydrodipicolinate reductase (DHDPR, DapB) is an enzyme that belongs to the L-lysine biosynthetic pathway. DHDPR reduces the alpha,beta-unsaturated cyclic imine 2,3-dihydrodipicolinic acid to yield the compound 2,3,4,5-tetrahydrodipicolinic acid in a pyridine nucleotide-dependent reaction. The substrate of this reaction is the unstable product of the preceding enzyme dihydrodipicolinate synthase (DHDPS, DapA). Here, the structure of apo-DHDPR from Mycobacterium tuberculosis is reported in two orthorhombic crystal forms, as well as the structure of DHDPR from M. tuberculosis in complex with NADH in a monoclinic crystal form. A comparison of the results with previously solved structures of this enzyme shows that DHDPR undergoes a major conformational change upon binding of its cofactor. This conformational change can be interpreted as one of the low-frequency normal modes of the structure.

The three-dimensional structure of diaminopimelate decarboxylase from Mycobacterium tuberculosis reveals a tetrameric enzyme organisation.

The three-dimensional structure of the enzyme diaminopimelate decarboxylase from Mycobacterium tuberculosis has been determined in a new crystal form and refined to a resolution of 2.33 A. The monoclinic crystals contain one tetramer exhibiting D(2)-symmetry in the asymmetric unit. The tetramer exhibits a donut-like structure with a hollow interior. All four active sites are accessible only from the interior of the tetrameric assembly. Small-angle X-ray scattering indicates that in solution the predominant oligomeric species of the protein is a dimer, but also that higher oligomers exist at higher protein concentrations. The observed scattering data are best explained by assuming a dimer-tetramer equilibrium with about 7% tetramers present in solution. Consequently, at the elevated protein concentrations in the crowded environment inside the cell the observed tetramer may constitute the biologically relevant functional unit of the enzyme.

Characterization of protein detergent complexes by NMR, light scattering, and analytical ultracentrifugation.

Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent complexes (PDCs) [21], we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably, this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore more effective than centrifugal ultrafiltration devices.

Crystal structure and kinetic study of dihydrodipicolinate synthase from Mycobacterium tuberculosis.

The three-dimensional structure of the enzyme dihydrodipicolinate synthase (KEGG entry Rv2753c, EC 4.2.1.52) from Mycobacterium tuberculosis (Mtb-DHDPS) was determined and refined at 2.28 A (1 A=0.1 nm) resolution. The asymmetric unit of the crystal contains two tetramers, each of which we propose to be the functional enzyme unit. This is supported by analytical ultracentrifugation studies, which show the enzyme to be tetrameric in solution. The structure of each subunit consists of an N-terminal (beta/alpha)(8)-barrel followed by a C-terminal alpha-helical domain. The active site comprises residues from two adjacent subunits, across an interface, and is located at the C-terminal side of the (beta/alpha)(8)-barrel domain. A comparison with the other known DHDPS structures shows that the overall architecture of the active site is largely conserved, albeit the proton relay motif comprising Tyr(143), Thr(54) and Tyr(117) appears to be disrupted. The kinetic parameters of the enzyme are reported: K(M)(ASA)=0.43+/-0.02 mM, K(M)(pyruvate)=0.17+/-0.01 mM and V(max)=4.42+/-0.08 micromol x s(-1) x mg(-1). Interestingly, the V(max) of Mtb-DHDPS is 6-fold higher than the corresponding value for Escherichia coli DHDPS, and the enzyme is insensitive to feedback inhibition by (S)-lysine. This can be explained by the three-dimensional structure, which shows that the (S)-lysine-binding site is not conserved in Mtb-DHDPS, when compared with DHDPS enzymes that are known to be inhibited by (S)-lysine. A selection of metabolites from the aspartate family of amino acids do not inhibit this enzyme. A comprehensive understanding of the structure and function of this important enzyme from the (S)-lysine biosynthesis pathway may provide the key for the design of new antibiotics to combat tuberculosis.

Identification of Novel, Potent, and Orally Available GCN2 Inhibitors with Type I Half Binding Mode.

General control nonderepressible 2 (GCN2) is a master regulator kinase of amino acid homeostasis and important for cancer survival in the tumor microenvironment under amino acid depletion. We initiated studies aiming at the discovery of novel GCN2 inhibitors as first-in-class antitumor agents and conducted modification of the substructure of sulfonamide derivatives with expected type I half binding on GCN2. Our synthetic strategy mainly corresponding to the αC-helix allosteric pocket of GCN2 led to significant enhancement in potency and a good pharmacokinetic profile in mice. In addition, compound 6d, which showed slow dissociation in binding on GCN2, demonstrated antiproliferative activity in combination with the asparagine-depleting agent asparaginase in an acute lymphoblastic leukemia (ALL) cell line, and it also displayed suppression of GCN2 pathway activation with asparaginase treatment in the ALL cell line and mouse xenograft model.

The three-dimensional structure of N-succinyldiaminopimelate aminotransferase from Mycobacterium tuberculosis.

Inhibitors of the enzymes of the lysine biosynthetic pathway are considered promising lead compounds for the design of new antibacterial drugs, because the pathway appears to be indispensable for bacteria and because it is absent in humans. As part of our efforts to structurally characterize all enzymes of this pathway in Mycobacterium tuberculosis (Mtb), we have determined the three-dimensional structure of N-succinyldiaminopimelate aminotransferase (DapC, DAP-AT, Rv0858c) to a resolution of 2.0 A. This structure is the first DAP-AT structure reported to date. The orthorhombic crystals of Mtb-DAP-AT contain one functional dimer exhibiting C(2) symmetry in the asymmetric unit. The homodimer displays the typical S-shape of class I pyridoxal-5'-phosphate (PLP)-binding proteins. The two active sites of the dimer both feature an internal aldimine with the co-factor PLP covalently bound to the Lys232, although neither substrate nor co-factor had been added during protein production, purification and crystallization. Nine water molecules are conserved in the active site and form an intricate hydrogen-bonding network with the co-factor and the surrounding amino acid residues. Together with some residual difference electron density in the active site, this architecture permitted the building of external aldimine models of the enzyme with the substrates glutamate, the amine donor, and N-succinyl-2-amino-6-keto-pimelate, the amine acceptor. Based on these models, the amino acids relevant for substrate binding and specificity can be postulated. Furthermore, in the external aldimine model of N-succinyl-2-amino-6-keto-pimelate, the succinyl group overlaps with a glycerol binding site that has also been identified in both active sites of the Mtb-DAP-AT dimer. A comparison of the structure of Mtb-DAP-AT with other class I PLP-binding proteins, revealed that some inhibitors utilize the same binding site. Thus, the proposed models also provide an explanation for the mode of inhibition of Mtb-DAP-AT and they may be of help in the design of compounds, which are capable of inhibiting the enzyme. Last, but not least, a chloride binding helix exhibiting a peculiar amino acid sequence with a number of exposed hydrophobic side-chains was identified, which may be hypothesized as a putative docking site.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of tetrahydrodipicolinate-N-succinyltransferase (Rv1201c) from Mycobacterium tuberculosis.

Tetrahydrodipicolinate-N-succinyltransferase from Mycobacterium tuberculosis (DapD, Rv1201c) has been cloned, heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized in the cubic space group I23 or I2(1)3. Preliminary diffraction data analysis indicates the presence of five molecules per asymmetric unit. Furthermore, the data exhibit icosahedral point-group symmetry. One possible explanation for this is that the enzyme assembles into a 60-mer exhibiting 235 point-group symmetry and crystallizes as such in space group I23. In this case, the combination of crystallographic and noncrystallographic symmetry elements results in an arrangement of the icosahedrons in the cubic crystal with one pentamer in the asymmetric unit. Another explanation is that the packing of the molecules itself mimics icosahedral symmetry. In this case both space groups I23 and I2(1)3 would be possible.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of isocitrate dehydrogenase 2 (Rv0066c) from Mycobacterium tuberculosis.

Isocitrate dehydrogenase 2 (Icd-2, Rv0066c) from Mycobacterium tuberculosis was cloned and heterologously expressed in Escherichia coli. The protein was purified by affinity and size-exclusion chromatography and crystallized. A complete data set has been collected and reduced to 3.25 A resolution in space group C2. Preliminary diffraction data analysis suggests a complex packing with at least six molecules in the asymmetric unit.

NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes.

BACKGROUND: Structural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs). The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization. RESULTS: Seeking an effective and standardized means applicable to genomic approaches for the characterization of PDCs, we chose 1D-NMR spectroscopic analysis to monitor the detergent content throughout their purification: protein extraction, detergent exchange, and sample concentration. We demonstrate that a single NMR measurement combined with a SDS-PAGE of a detergent extracted sample provides a useful gauge of the detergent's extraction potential for a given protein. Furthermore, careful monitoring of the detergent content during the process of IMP production allows for a high level of reproducibility. We also show that in many cases a simple sedimentation velocity measurement provides sufficient data to estimate both the oligomeric state and the detergent-to-protein ratio in PDCs, as well as to evaluate the homogeneity of the samples prior to crystallization screening. CONCLUSION: The techniques presented here facilitate the screening and selection of the extraction detergent, as well as help to maintain reproducibility in the detergent exchange and PDC concentration procedures. Such reproducibility is particularly important for the optimization of initial crystallization conditions, for which multiple purifications are routinely required.

The high-resolution Structure of LeuB (Rv2995c) from Mycobacterium tuberculosis.

The crystal structure of the enzyme 3-isopropylmalate dehydrogenase (IPMDH) from Mycobacterium tuberculosis (LeuB, Mtb-IPMDH, Rv2995c) without substrate or co-factor was determined at 1.65 A resolution, which is the highest resolution reported for an IPMDH to date. The crystals contain two functional dimers in the asymmetric unit in an arrangement close to a tetramer of D2 symmetry. Despite the absence of a substrate or inhibitor bound to the protein, the structure of the monomer resembles the previously observed closed form of the enzyme more closely than the open form. A comparison with the substrate complex of IPMDH from Thiobacillus ferrooxidans and the co-factor complex of the Thermus thermophilus enzyme revealed a close relationship of the active-site architecture between the various bacterial enzymes. The inhibitor O-isobutenyl oxalylhydroxamate was found to bind to the active site of IPMDH in a mode similar to the substrate isopropylmalate.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DapA (Rv2753c) from Mycobacterium tuberculosis.

Dihydrodipicolinate synthase from Mycobacterium tuberculosis (DHDPS, DapA, Rv2753c) has been cloned, heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized in a monoclinic crystal form. Preliminary diffraction data analysis suggests the presence of two independent tetramers in the asymmetric unit in almost the same relative orientation.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DapC (Rv0858c) from Mycobacterium tuberculosis.

N-Succinyldiaminopimelate aminotransferase from Mycobacterium tuberculosis (DAP-AT; DapC; Rv0858c) has been cloned, heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized in two related crystal forms. Preliminary diffraction data analysis suggests the presence of a monomer in the asymmetric unit of the tetragonal crystal form and a dimer in the asymmetric unit of the orthorhombic crystal form.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of LysA (Rv1293) from Mycobacterium tuberculosis.

Diaminopimelate decarboxylase from Mycobacterium tuberculosis (LysA, DAPDC, Rv1293) has been cloned and heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized. Preliminary diffraction data analysis suggests the presence of a homotetramer in the asymmetric unit.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DapB (Rv2773c) from Mycobacterium tuberculosis.

Dihydrodipicolinate reductase from Mycobacterium tuberculosis (DapB, DHDPR, Rv2773c) has been cloned and heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized in three different crystal forms. Preliminary diffraction data analysis suggests the presence of two tetramers in the asymmetric unit of one crystal form and half a tetramer in the other two crystal forms.

Structures of the OmpF porin crystallized in the presence of foscholine-12.

The endogenous Escherichia coli porin OmpF was crystallized as an accidental by-product of our efforts to express, purify, and crystallize the E. coli integral membrane protein KdpD in the presence of foscholine-12 (FC12). FC12 is widely used in membrane protein studies, but no crystal structure of a protein that was both purified and crystallized with this detergent has been reported in the Protein Data Bank. Crystallization screening for KdpD yielded two different crystals of contaminating protein OmpF. Here, we report two OmpF structures, the first membrane protein crystal structures for which extraction, purification, and crystallization were done exclusively with FC12. The first structure was refined in space group P21 with cell parameters a = 136.7 A, b = 210.5 A, c = 137 A, and beta = 100.5 degrees , and the resolution of 3.8 A. The second structure was solved at the resolution of 4.4 A and was refined in the P321 space group, with unit cell parameters a = 215.5 A, b = 215.5 A, c = 137.5 A, and gamma = 120 degrees . Both crystal forms show novel crystal packing, in which the building block is a tetrahedral arrangement of four trimers. Additionally, we discuss the use of FC12 for membrane protein crystallization and structure determination, as well as the problem of the OmpF contamination for membrane proteins overexpressed in E. coli.

The three-dimensional Structure of a mycobacterial DapD provides insights into DapD diversity and reveals unexpected particulars about the enzymatic mechanism.

The enzyme tetrahydrodipicolinate N-succinyltransferase (DapD) is part of the L-lysine biosynthetic pathway. This pathway is crucial for the survival of the pathogen Mycobacterium tuberculosis (Mtb) and, consequently, the enzymes of the pathway are potential drug targets. We report here the crystal structures of Mtb-DapD and of Mtb-DapD in complex with the co-factor succinyl-CoA (SCoA) at 2.15 A and 1.97 A resolution, respectively. Each subunit of the trimeric enzyme consists of three domains, of which the second, a left-handed, parallel beta-helix (LbetaH domain), is the common structural motif of enzymes belonging to the hexapeptide repeat superfamily. The trimeric quaternary structure is stabilized by Mg(2+) and Na(+) located on the 3-fold axis. The binary complex of Mtb-DapD and SCoA reveals the binding mode(s) of the co-factor and a possible covalent reaction intermediate. The N-terminal domain of Mtb-DapD exhibits a unique architecture, including an interior water-filled channel, which allows access to a magnesium ion located at the 3-fold symmetry axis.