Structural basis and dynamics of multidrug recognition in a minimal bacterial multidrug resistance system.

TipA is a transcriptional regulator found in diverse bacteria. It constitutes a minimal autoregulated multidrug resistance system against numerous thiopeptide antibiotics. Here we report the structures of its drug-binding domain TipAS in complexes with promothiocin A and nosiheptide, and a model of the thiostrepton complex. Drug binding induces a large transition from a partially unfolded to a globin-like structure. The structures rationalize the mechanism of promiscuous, yet specific, drug recognition: (i) a four-ring motif present in all known TipA-inducing antibiotics is recognized specifically by conserved TipAS amino acids; and (ii) the variable part of the antibiotic is accommodated within a flexible cleft that rigidifies upon drug binding. Remarkably, the identified four-ring motif is also the major interacting part of the antibiotic with the ribosome. Hence the TipA multidrug resistance mechanism is directed against the same chemical motif that inhibits protein synthesis. The observed identity of chemical motifs responsible for antibiotic function and resistance may be a general principle and could help to better define new leads for antibiotics.

Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor.

The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled ((2)H/(15)N/(13)C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected α-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1ß were assessed by surface plasmon resonance yielding K(D) values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineering.

Monocytes contribute to differential immune pressure on R5 versus X4 HIV through the adipocytokine visfatin/NAMPT.

BACKGROUND: The immune system exerts a diversifying selection pressure on HIV through cellular, humoral and innate mechanisms. This pressure drives viral evolution throughout infection. A better understanding of the natural immune pressure on the virus during infection is warranted, given the clinical interest in eliciting and sustaining an immune response to HIV which can help to control the infection. We undertook to evaluate the potential of the novel HIV-induced, monocyte-derived factor visfatin to modulate viral infection, as part of the innate immune pressure on viral populations. RESULTS: We show that visfatin is capable of selectively inhibiting infection by R5 HIV strains in macrophages and resting PBMC in vitro, while at the same time remaining indifferent to or even favouring infection by X4 strains. Furthermore, visfatin exerts a direct effect on the relative fitness of R5 versus X4 infections in a viral competition setup. Direct interaction of visfatin with the CCR5 receptor is proposed as a putative mechanism for this differential effect. Possible in vivo relevance of visfatin induction is illustrated by its association with the dominance of CXCR4-using HIV in the plasma. CONCLUSIONS: As an innate factor produced by monocytes, visfatin is capable of inhibiting infections by R5 but not X4 strains, reflecting a potential selective pressure against R5 viruses.

Site-specific measurement of slow motions in proteins.

We demonstrate that a quantitative measure of slow molecular motions in solid proteins can be accessed by measuring site-specific (15)N rotating-frame relaxation rates at high magic-angle-spinning frequencies.

Measurement of site-specific 13C spin-lattice relaxation in a crystalline protein.

We demonstrate that it is possible to record site-specific spin-lattice relaxation rates for the majority of (13)C sites in uniformly (13)C and (15)N labeled solid proteins as a result of the slowing down of proton-driven spin diffusion at sample spinning frequencies > or = 60 kHz, thus providing a series of new experimental probes for characterizing molecular dynamics in solid proteins.

From biomolecular structure to functional understanding: new NMR developments narrow the gap.

Biomolecular structure provides the framework for dynamics and function. However, the information of more than 50000 protein and nucleic acid structures solved today has not yet yielded enough insight such that function could be predicted from structure or that structures and dynamics could be predicted to high resolution from sequence alone. Further experimental information is urgently needed to guide the process of relating sequence, structure, dynamics, and ultimately function. Recent progress in solution NMR methods encompasses better detection of dynamics and molecular interactions, a more detailed characterization of folding transitions, increases of the molecular weight limit and sensitivity, strongly enhanced computational approaches, and the in-cell detection of molecules. These developments are highly complementary and narrow the gap between structure and functional understanding. This article discusses recent advances and applications.

Correlation of protein structure and dynamics to scalar couplings across hydrogen bonds.

NMR-observable scalar couplings across hydrogen bonds in nucleic acids and proteins present a quantitative measure for the geometry and--by the implicit experimental time averaging--dynamics of hydrogen bonds. We have carried out in-depth molecular dynamics (MD) simulations with various force fields on three proteins: ubiquitin, the GB1 domain of protein G, and the SMN Tudor domain, for which experimental h3JNC' scalar couplings of backbone hydrogen bonds and various high-resolution X-ray structures are available. Theoretical average values for h3JNC' were calculated from the snapshots of these MD simulations either by density functional theory or by a geometric parametrization (Barfield, M. J. Am. Chem. Soc. 2002, 124, 4158-4168). No significant difference was found between the two methods. The results indicate that time-averaging using explicit water solvation in the MD simulations improves significantly the agreement between experimental and theoretical values for the lower resolution structures ubiquitin (1.8 A), Tudor domain (1.8 A), and protein G (2.1 A). Only marginal improvement is found for the high-resolution structure (1.1 A) of protein G. Hence, experimental h3JNC' values are compatible with a static, high-resolution structural model. The MD averaging of the low-resolution structures moves the averages of the rHO distance and the H...O=C angle theta closer to their respective values in the high-resolution structures, thereby improving the agreement using experimental h3JNC' data. In contrast, MD averaging with implicit water models deteriorates the agreement with experiment for all proteins. The differing behavior can be explained by an artifactual lengthening of H-bonds caused by the implicit water models.

Improved detection of long-range residual dipolar couplings in weakly aligned samples by Lee-Goldburg decoupling of homonuclear dipolar truncation.

Homonuclear (1)H residual dipolar couplings (RDCs) truncate the evolution of transverse (1)H magnetization of weakly aligned molecules in high-resolution NMR experiments. This leads to losses in sensitivity or resolution in experiments that require extended (1)H evolution times. Lee-Goldburg decoupling schemes have been shown to remove the effects of homonuclear dipolar couplings, while preserving chemical shift evolution in a number of solid-state NMR applications. Here, it is shown that the Lee-Goldburg sequence can be effectively incorporated into INEPT- or HMQC-type transfer schemes in liquid state weak alignment experiments in order to increase the efficiency of the magnetization transfer. The method is applied to the sensitive detection of (1)H(N)-(13)C long-range RDCs in a three-dimensional HCN experiment. As compared to a conventional HCN experiment, an average sensitivity increase by a factor of 2.4 is obtained for a sample of weakly aligned protein G. This makes it possible to detect 170 long-range (1)H(N)-(13)C RDCs for distances up to 4.9 angstroms.

Structural basis for antibiotic recognition by the TipA class of multidrug-resistance transcriptional regulators.

The TipAL protein, a bacterial transcriptional regulator of the MerR family, is activated by numerous cyclic thiopeptide antibiotics. Its C-terminal drug-binding domain, TipAS, defines a subfamily of broadly distributed bacterial proteins including Mta, a central regulator of multidrug resistance in Bacillus subtilis. The structure of apo TipAS, solved by solution NMR [Brookhaven Protein Data Bank entry 1NY9], is composed of a globin-like alpha-helical fold with a deep surface cleft and an unfolded N-terminal region. Antibiotics bind within the cleft at a position that is close to the corresponding heme pocket in myo- and hemoglobin, and induce folding of the N-terminus. Thus the classical globin fold is well adapted not only for accommodating its canonical cofactors, heme and other tetrapyrroles, but also for the recognition of a variety of antibiotics where ligand binding leads to transcriptional activation and drug resistance.

Calcium-dependent homoassociation of E-cadherin by NMR spectroscopy: changes in mobility, conformation and mapping of contact regions.

Cadherins are calcium-dependent cell surface proteins that mediate homophilic cellular adhesion. The calcium-induced oligomerization of the N-terminal two domains of epithelial cadherin (ECAD12) was followed by NMR spectroscopy in solution over a large range of protein (10 microM-5 mM) and calcium (0-5 mM) concentrations. Several spectrally distinct states could be distinguished that correspond to a calcium-free monomeric form, a calcium-bound monomeric form, and to calcium-bound higher oligomeric forms. Chemical shift changes between these different states define calcium-binding residues as well as oligomerization contacts. Information about the relative orientation and mobility of the ECAD12 domains in the various states was obtained from weak alignment and 15N relaxation experiments. The data indicate that the calcium-free ECAD12 monomer adopts a flexible, kinked conformation that occludes the dimer interface observed in the ECAD12 crystal structure. In contrast, the calcium-bound monomer is already in a straight, non-flexible conformation where this interface is accessible. This mechanism provides a rational for the calcium-induced adhesiveness. Oligomerization induces chemical shift changes in an area of domain CAD1 that is centered at residue Trp-2. These shift changes extend to almost the entire surface of domain CAD1 at high (5 mM) protein concentrations. Smaller additional clusters of shift perturbations are observed around residue A80 in CAD1 and K160 in CAD2. According to weak alignment and relaxation data, the symmetry of a predominantly dimeric solution aggregate at 0.6 mM ECAD12 differs from the approximate C2-symmetry of the crystalline dimer.