Crystal structure of the multiple antibiotic resistance regulator MarR from Clostridium difficile.

Regulators of multiple antibiotic resistance (MarRs) are key players against toxins in prokaryotes. MarR homologues have been identified in many bacterial and archaeal species which pose daunting antibiotic resistance issues that threaten public health. The continuous prevalence of Clostridium difficile infection (CDI) throughout the world is associated with the abuse of antibiotics, and antibiotic treatments of CDI have limited effect. In the genome of C. difficile strain 630, the marR gene (ID 4913953) encodes a MarR protein. Here, MarR from C. difficile (MarRC.difficile) was subcloned and crystallized for the first time. MarRC.difficile was successfully expressed in Escherichia coli in a soluble form and was purified to near-homogeneity (>95%) by a two-step purification protocol. The structure of MarRC.difficile has been solved at 2.3 Šresolution. The crystal belonged to the monoclinic space group P43212, with unit-cell parameters a = b = 66.569, c = 83.654 Å. The structure reported reveals MarRC.difficile to be a dimer, with each subunit consisting of six α-helices and three antiparallel ß-hairpins. MarRC.difficile shows high structural similarity to the MarR proteins from E. coli and Staphylococcus aureus, indicating that MarRC.difficile might be a DNA-binding protein.

Single-agent capecitabine as maintenance therapy after induction of XELOX (or FOLFOX) in first-line treatment of metastatic colorectal cancer: randomized clinical trial of efficacy and safety.

BACKGROUND: The optimal strategy of maintenance therapy for patients with mCRC is controversial. This study was to evaluate the efficacy and safety of maintenance therapy with capecitabine versus observation following inductive chemotherapy in patients with metastatic colorectal cancer. PATIENTS AND METHODS: In this randomized, open-label, multicenter, phase III trial, patients who received 18-24 weeks of induction chemotherapy with XELOX or FOLFOX and achieved disease control were randomly assigned centrally (1:1) to receive maintenance therapy of capecitabine or only observation until disease progression. The primary end point was progression-free survival (PFS) from randomization; the secondary end points included overall survival (OS), PFS from induction treatment (PFS2) and safety. Analyses were done by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT02027363. RESULTS: Between 30 July 2010 and 15 September 2013, 274 patients were enrolled in the study from 11 sites in China and randomly assigned to maintenance group (n = 136) or observation group (n = 138). Clinicopathological characteristics were balanced in two groups. The median follow-up time from randomization was 29.0 months [interquartile range (IQR) 21-36 months]. The primary end point of PFS was statistically significantly longer in capecitabine maintenance group than in observation group {6.43 [95% confidence interval (CI) 5.26-7.71] versus 3.43 (2.83-4.16) months, HR 0.54 (0.42-0.70), P < 0.001}. The median OS of capecitabine maintenance group was longer than that of observation group, but not statistically significant [25.63 (22.46-27.80) versus 23.30 (19.68-26.92) months; HR 0.85 (0.64-1.11), P = 0.2247]. Similar safety profiles were observed in both arms. The most common grade 3 or 4 toxicities in capecitabine maintenance group versus observation group were neutropenia, hand-foot syndrome, and mucositis. CONCLUSIONS: Maintenance therapy with a single agent of capecitabine can be considered an appropriate option following the induction of XELOX or FOLFOX in mCRC patients with acceptable toxicities. CLINICAL TRIALS NUMBER: NCT02027363.

Exposing the Moving Parts of Proteins with NMR Spectroscopy.

Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful tool for investigating the dynamics of biomolecules since it provides a description of motion that is comprehensive, site-specific, and relatively non-invasive. In particular, the study of protein dynamics has benefited from sustained methodological advances in NMR that have expanded the scope and time scales of accessible motion. Yet, many of these advances may not be well known to the more general physical chemistry community. Accordingly, this Perspective provides a glimpse of some of the more powerful methods in liquid state NMR that are helping reshape our understanding of functional motions of proteins.

Silica Induces Plasminogen Activator Inhibitor-1 Expression through a MAPKs/AP-1-Dependent Mechanism in Human Lung Epithelial Cells.

ABSTRACT Plasminogen activator inhibitor-1 (PAI-1) plays an important role in the silica-induced pulmonary fibrosis. The effect of silica on the expression of PAI-1 was investigated in human lung epithelial cells (A549). Silica induced PAI-1 expression in a concentration-(50-200 mug/mL) and time-(4-24 h) dependent manner in A549 cells. Furthermore, the roles of mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) signaling pathways in silica-induced PAI-1 expression were examined. We found that silica (200 mug/mL) treatment for 4 to 24 h resulted in AP-1 activation in A549 cells. Cells were pretreated with the AP-1 inhibitor curcumin (10, 25, 50 muM), and silica-induced PAI-1 expression was reduced by 20%, 63%, and 65%, respectively. In addition, dominant-negative mutant c-Jun (TAM67) down-regulated silica-induced PAI-1 expression by 59%. P38 kinase inhibitor SB203580 (20 muM) and Erk inhibitor PD98059 (50 muM) suppressed silica-induced PAI-1 expression by 35% and 51%, respectively. Additionally, PD98059 but not SB203580 inhibited the AP-1 DNA binding activity induced by silica. The results suggest that the PAI-1 expression induced by silica may be involved in the activation of MAPKs/AP-1 signaling pathways in human lung epithelial cells.

Cross-correlated (19)F relaxation measurements for the study of fluorinated ligand-receptor interactions.

Fluorine is often used in drug-design efforts to enhance the pharmacokinetic properties of biologically active compounds. Additionally fluorine nuclei ((19)F) have properties that are well suited to current pharmaceutical NMR screening programs. Together, these considerations have motivated our interest in the utility of fluorine relaxation parameters to study ligand-receptor interactions. Here, we investigate the potential for cross-correlated relaxation effects between the (19)F anisotropic chemical-shift and (19)F-(1)H dipole-dipole relaxation mechanisms to help pinpoint and quantify exchange processes. Methods are proposed and demonstrated in which the magnitude ratio of the transverse cross-correlation rate constant eta(xy) and the fluorine transverse relaxation rate constant, R(2), help estimate the exchange rate constant for ligand-binding equilibria. These exchange rate constants provide estimates of the ligand dissociation rate constants k(off) and can thus provide a means for rank-ordering the binding affinities of ligands identified in pharmaceutical screens.

Simulation study of the structure and dynamics of the Alzheimer's amyloid peptide congener in solution.

The amyloid Abeta(10-35)-NH2 peptide is simulated in an aqueous environment on the nanosecond time scale. One focus of the study is on the validation of the computational model through a direct comparison of simulated statistical averages with experimental observations of the peptide's structure and dynamics. These measures include (1) nuclear magnetic resonance spectroscopy-derived amide bond order parameters and temperature-dependent H(alpha) proton chemical shifts, (2) the peptide's radius of gyration and end-to-end distance, (3) the rates of peptide self-diffusion in water, and (4) the peptide's hydrodynamic radius as measured by quasielastic light scattering experiments. A second focus of the study is the identification of key intrapeptide interactions that stabilize the central structural motif of the peptide. Particular attention is paid to the structure and fluctuation of the central LVFFA hydrophobic cluster (17-21) region and the VGSN turn (24-27) region. There is a strong correlation between preservation of the structure of these elements and interactions between the cluster and turn regions in imposing structure on the peptide monomer. The specific role of these interactions in relation to proposed mechanisms of amyloidosis is discussed.

The Alzheimer's peptide a beta adopts a collapsed coil structure in water.

The self-assembly of the soluble peptide Abeta into Alzheimer's disease amyloid is believed to involve a conformational change. Hence the solution conformation of Abeta is of significant interest. In contrast to studies in other solvents, in water Abeta is collapsed into a compact series of loops, strands, and turns and has no alpha-helical or beta-sheet structure. Conformational stabilization is primarily attributed to van der Waals and electrostatic forces. A large conspicuous uninterrupted hydrophobic patch covers approximately 25% of the surface. The compact coil structure appears meta-stable, and because fibrillization leads to formation of intermolecular beta-sheet secondary structure, a global conformational rearrangement is highly likely. A molecular hypothesis for amyloidosis includes at least two primary driving forces, changes in solvation thermodynamics during formation of amyloid deposits and relief of internal conformational stress within the soluble precursor during formation of lower-energy amyloid fibrils.

Restoration of cytotoxic T lymphocyte function in malignant pleural effusion: interleukin-15 vs. interleukin-2.

The present study attempts to define the role of interleukin-15 (IL-15), as compared with IL-2, in generating cytotoxic T lymphocytes (CTL) from the malignant effusions of cancer patients. Effusion-associated lymphocytes (EAL) from malignant effusion were incubated with IL-15 or IL-2 with or without alphaCD3. Proliferation and cytotoxicity assays were performed. IL-15 was found to have at least an equivalent, if not higher, activity to IL-2 in terms of lymphocyte proliferation and generation of CTL from EAL. The proliferative response of EAL, cocultured with IL-15, with or without alphaCD3, was partly inhibited by pretreatment with an anti-IL2 receptor beta chain monoclonal antibody (mAb). The proliferative response of EAL, cocultured with alphaCD3, IL-2, or both, was partly inhibited by pretreatment with an anti-IL-2 receptor alpha chain mAb. Overnight [5lCr] release assays against K562, Daudi, and the patients' autologous tumor cells were done to evaluate EAL's cytolytic activity. MHC class I Ab blocked the stimulated cytolytic activity of EAL against autologous tumors. An mAb depletion assay showed that the phenotype of the restored EAL was CD16-CD4-CD8+; thus, the restored activity of EAL was CTL activity. The results suggest that both IL-15 and IL-2 can restore CTL activity from EAL in the presence of T cell receptor (TCR)-CD3 engagement, but the effect of IL-15 was superior.

The SHAPES strategy: an NMR-based approach for lead generation in drug discovery.

BACKGROUND: Recently, it has been shown that nuclear magnetic resonance (NMR) may be used to identify ligands that bind to low molecular weight protein drug targets. Recognizing the utility of NMR as a very sensitive method for detecting binding, we have focused on developing alternative approaches that are applicable to larger molecular weight drug targets and do not require isotopic labeling. RESULTS: A new method for lead generation (SHAPES) is described that uses NMR to detect binding of a limited but diverse library of small molecules to a potential drug target. The compound scaffolds are derived from shapes most commonly found in known therapeutic agents. NMR detection of low (microM-mM) affinity binding is achieved using either differential line broadening or transferred NOE (nuclear Overhauser effect) NMR techniques. CONCLUSIONS: The SHAPES method for lead generation by NMR is useful for identifying potential lead classes of drugs early in a drug design program, and is easily integrated with other discovery tools such as virtual screening, high-throughput screening and combinatorial chemistry.

Solution structure of core binding factor beta and map of the CBF alpha binding site.

The core binding factor beta subunit (CBF beta) is the non-DNA binding subunit of the core-binding factors, transcription factors essential for multiple developmental processes including hematopoiesis and bone development. Chromosomal translocations involving the human CBFB gene are associated with a large percentage of human leukemias. The N-terminal 141 amino acids of CBF beta contains the heterodimerization domain for the DNA-binding CBF alpha subunits, and is sufficient for CBF beta function in vivo. Here we present the high-resolution solution structure of the CBF beta heterodimerization domain. It is a novel alpha/beta structure consisting of two three-stranded beta-sheets packed on one another in a sandwich arrangement, with four peripheral alpha-helices. The CBF alpha binding site on CBF beta has been mapped by chemical shift perturbation analysis.

Dynamic NMR studies of ligand-receptor interactions: design and analysis of a rapidly exchanging complex of FKBP-12/FK506 with a 24 kDa calcineurin fragment.

Dynamic NMR methods, such as differential line broadening and transferred NOE spectroscopy, are normally reserved for the study of small molecule ligand interactions with large protein receptors. Using a combination of isotope labeling and isotope edited NMR, we have extended these techniques to characterize interactions of a much larger protein/drug complex, FKBP-12/ FK506 with its receptor protein, calcineurin. In order to examine this multicomponent system by dynamic NMR methods, the 93 kDa, tightly bound FKBP-12/FK506/Cn complex was replaced with a lower affinity, rapidly exchanging system consisting of FKBP-12/FK506 (13 kDa), recombinant calcineurin subunit B (CnB) (20 kDa), and a synthetic peptide (4 kDa) corresponding to the B binding domain (BBD) of calcineurin catalytic subunit A (CnA). Analysis of 1H-13C HSQC data acquired for the FKBP-12/ 13C-FK506 and FKBP-12/13C-FK506/CnB/BBD complexes indicates that FKBP-12/FK506 and CnB/BBD are in fast exchange in the quaternary complex. Comparison of proton line widths shows significant broadening of resonances along the macrocycle backbone at 13-CH, 13-OMe, 15-OMe, 18-CH2, 20-CH, 21-CH, and 25-Me, as well as moderate broadening on the macrocycle backbone at 17-Me, 24-CH, and the pyranose 12-CH2 protons. The tri-substituted olefin and cyclohexyl groups also show moderate broadening at the 27-Me, 28-CH, and 30-CH2 positions, respectively. Unexpectedly, little line broadening was observed for the allyl resonances of FK506 in the quaternary complex, although 13C longitudinal relaxation measurements suggest this group also makes contacts with calcineurin. In addition, intermolecular transfer NOE peaks were observed for the allyl 37-CH2, 21-CH, 30-CH2, 13-OMe, 15-OMe, 17-Me, 25-Me, and 27-Me groups, indicating that these are potential sites on the FK506 molecule that interact with calcineurin.

Internal mobility in the partially folded DNA binding and dimerization domains of GAL4: NMR analysis of the N-H spectral density functions.

The DNA binding domain (residues 1--65) of the yeast transcriptional activator GAL4 is only partially folded. While residues 10-41, the DNA recognition domain, form a well-defined structure in the free protein, the whole polypeptide folds up and dimerizes upon binding DNA. In order to describe the mobility of the protein, we have characterized the frequency spectrum of the motions of N-H bond vectors of GAL4(1-65) using a reduced spectral density mapping approach (an approximation of the full spectral density mapping technique) [Peng, J. W., & Wagner, G. (1992a) J. Magn. Reson. 98, 308-332; Peng. J. W., & Wagner, G. (1992b) Biochemistry 31, 8571-8586]. 15N spin-lattice relaxation [Rn(Nz)], spin-spin relaxation [Rn(Nx,y)], cross-relaxation [RN(Hz-->Nz)], two-spin order [RNH(2HzNz)], and antiphase [RNH(2HzNx,y)] rates were determined for 52 of the 65 backbone amide groups at 10 degrees C and ph 6.5 at 11.74 T. Calculations of the spectral density functions using a reduced set of RN(Nz),RN(Nx,y),RN(Hz-->Nz), and RNH(2HzNz) gave excellent agreement with those calculated using all six sets. The reduced method has the added advantage that the errant behavior seen at high field values is circumvented. A linear correlation was found between J(omega N) and J(0) with a limited and clearly defined range of J(0) values which defines the range of rates for internal motions in GAL4(1-65). It appears that all residues experience a combination of two movements: one of the overall tumbling (correlation time, 8.65 ns) and the other of fast internal fluctuations of the structure. The respective weights of these contributions vary with the primary sequence and faithfully mirror the secondary and tertiary elements of the protein. The position on the correlation line of J(omega N) versus J(0) indicates the amount of angular averaging relative to the overall motion of the protein. A spectral density function for internal motions can be described.

Investigations of peptide hydration using NMR and molecular dynamics simulations: A study of effects of water on the conformation and dynamics of antamanide.

The influence of water binding on the conformational dynamics of the cyclic decapeptide antamanide dissolved in the model lipophilic environment chloroform is investigated by NMR relaxation measurements. The water-peptide complex has a lifetime of 35 µs at 250 K, which is longer than typical lifetimes of water-peptide complexes reported in aqueous solution. In addition, there is a rapid intracomplex mobility that probably involves librational motions of the bound water or water molecules hopping between different binding sites. Water binding restricts the flexibility of antamanide. The experimental findings are compared with GROMOS molecular dynamics simulations of antamanide with up to eight bound water molecules. Within the simulation time of 600 ps, no water molecule leaves the complex. Additionally, the simulations show a reduced flexibility for the complex in comparison with uncomplexed antamanide. Thus, there is a qualitative agreement between the experimental NMR results and the computer simulations.

Frequency spectrum of NH bonds in eglin c from spectral density mapping at multiple fields.

The internal mobility of the protein eglin c is characterized with spectral density functions of the NH vectors obtained from heteronuclear NMR relaxation at multiple field strengths (7.04, 11.74, and 14.1 T). The spectral density functions, J(omega), describe the frequency spectrum of the rotational fluctuations of the XH bond vectors (15N-1H and 13C-1H). The spectral density-mapping approach [Peng, J. W., & Wagner, G. (1992a) J. Magn. Reson. 98, 308-332; Peng, J. W., & Wagner, G (1992b) Biochemistry 31, 8571-8586] permits the direct evaluation of J(omega) at the five frequencies 0, omega N, magnitude of omega H - magnitude of omega X, omega H, and magnitude of omega H + magnitude of omega X. The 15N-1H relaxation measurements from three field strengths on 15N-enriched eglin c resulted in 18 relaxation rate constants per NH bond and 13 unique evaluations of each NH spectral density function. Dynamic heterogeneity along the protein backbone is manifested most clearly in spectral density values at lower frequencies (< 100 MHz). The effective value of J(0), J(eff)(0), is the most sensitive probe of dynamics as it is affected by both rapid internal motions and slow chemical exchange processes. Low J(eff)(0) and J(omega N) values are correlated with fast amide proton-deuteron exchange rates; the converse, however, is not observed. Anomalies in J(omega H) and J(magnitude of omega H +/- magnitude of omega N) observed in the first applications of the spectral-mapping approach are now attributable to the high sensitivity of these values to small errors in the rate constants. These anomalies can be reduced by the use of a reduced spectral-mapping procedure. The use of multiple field strengths allows the identification of slow exchange processes manifested as an increase of J(eff)(0) with spectrometer field strength.

Mapping of the spectral densities of N-H bond motions in eglin c using heteronuclear relaxation experiments.

A new strategy is used for studying the internal motions of proteins based on measurements of NMR relaxation parameters. The strategy yields values of the so-called spectral density functions J(omega) for N-H bond vectors. The spectral density functions are related to the distribution of frequencies contained in the rotational (overall and internal) motions of these NH bond vectors. No a priori model assumptions about the dynamics are required in this approach. The method involves measurements of six relaxation parameters consisting of 15N longitudinal relaxation rates, transverse relaxation rates of in-phase and antiphase coherence, the relaxation rates of heteronuclear 1H-15N two-spin order, the heteronuclear 1H-15N nuclear Overhauser effects, and longitudinal relaxation rates of the amide protons. The values of the spectral density functions at the five frequencies 0, omega N, omega H + omega N, omega H, and omega H - omega N are determined from the relaxation parameters using analytical relations derived previously [Peng & Wagner (1992) J. Magn. Reson. 98, 308-332]. Here, the method is applied to characterize the backbone dynamics of the 15N-enriched proteinase inhibitor eglin c, a protein of 70 residues. The values for J(0) and J(omega N = 50 MHz) vary significantly with the amino acid sequence, whereas the spectral densities at higher frequencies, J(450 MHz), J(500 MHz), and J(550 MHz), are typically much smaller and show no significant variation with the sequence. The collective behavior of the J(omega) values indicate greater internal motion for the proteinase binding loop residues and the first eight N-terminal residues. The additional internal motion in these regions is in the rate range below 450 MHz. The values of J(omega) are also compared with root mean square deviations (rmsds) of backbone atoms as obtained in NMR structure determinations. Low values of J(0) and J(omega N) are correlated with high rmsds. Spectral densities at higher frequencies, J(450 MHz), J(500 MHz), and J(550 MHz), are small and show no correlation with rmsds. A comparison with the spectral density functions obtained by fitting the experimental data to the functional dependence of the Lipari and Szabo formalism [Lipari & Szabo (1982a) J. Am. Chem. Soc. 104, 4546-4559] is made.

Changing the inhibitory specificity and function of the proteinase inhibitor eglin c by site-directed mutagenesis: functional and structural investigation.

Amino acids in the serine proteinase inhibitor eglin c important for its inhibitory specificity and activity have been investigated by site-directed mutagenesis. The specificity of eglin c could be changed from elastase to trypsin inhibition by the point mutation Leu45----Arg (L45R) in position P1 [nomenclature according to Schechter and Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162]. Model building studies based on the crystal structure of mutant L45R [Heinz et al. (1991) J. Mol. Biol. 217, 353-371] were used to rationalize this specificity change. Surprisingly, the double mutant L45R/D46S was found to be a substrate of trypsin and various other serine proteinases. Multidimensional NMR studies show that wild-type eglin c and the double mutant have virtually identical conformations. In the double mutant L45R/D46S, however, the N-H bond vector of the scissile peptide bond shows a much higher mobility, indicating that the internal rigidity of the binding loop is significantly weakened due to the loss or destabilization of the internal hydrogen bond of the P1' residue. Mutant T44P was constructed to examine the role of a proline in position P2, which is frequently found in serine proteinase inhibitors [Laskowski and Kato (1980) Annu. Rev. Biochem. 49, 593-626]. The mutant remains a potent elastase inhibitor but no longer inhibits subtilisin, which could be explained by model building. Both Arg51 and Arg53, located in the core of the molecule and participating in the hydrogen bonding network with residues in the binding loop to maintain rigidity around the scissile bond, were individually replaced with the shorter but equally charged amino acid lysine. Both mutants showed a decrease in their inhibitory potential. The crystal structure of mutant R53K revealed the loss of two hydrogen bonds between the core and the binding loop of the inhibitor, which are partially restored by a solvent molecule, leading to a decrease in inhibition of elastase by 2 orders of magnitude.

NMR studies of structure and dynamics of isotope enriched proteins.

Structural studies of globular proteins by nmr can be enhanced by the use of isotope enrichment. We have been working with proteins enriched with 15N, and with both 15N and 13C. Due to the isotope enrichment we could assign several large proteins with up to 186 residues and could address structural questions. Furthermore, we can accurately measure heteronuclear and homonuclear vicinal coupling constants. This involves in part multidimensional multiple resonance experiments. This is important for characterization of minor conformational changes caused by mutations. We have also made use of isotope enrichment to study the internal mobility of proteins. We also have developed novel methods for measuring accurately 15N relaxation parameters, in particular transverse relaxation rates. This has led us toward a method for directly mapping spectral density functions of the rotational motions of N-H bond vectors in proteins. The protein systems that are discussed include the unlabeled proteins kistrin and cytochrome c551, and the labeled proteins eglin c, a flavodoxin, and human dihydrofolate reductase.

Use of augmented Lagrangians in the calculation of molecular conformations by distance geometry.

Distance geometry is a technique widely used to find atomic coordinates that agree with given upper and lower bounds on the interatomic distances. It is successful because it chooses at random some relatively good "trial coordinates" that take into account the whole molecule and all constraints at once. Customarily, these trial coordinates must be refined by minimizing a penalty function until the structure agrees with the original bounds. Here we present an alternative to minimizing the penalty function, which has the advantage of more precisely satisfying the bounds, showing more clearly when the bounds are mutually contradictory, and simultaneously optimizing an objective function subject to precise satisfaction of the bounds.