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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 sys- tematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L cul- ture in a triply isotope-labeled ( 2 H/ 15 N/ 13 C) minimal medium. Thus the material is suitable for NMR spectro- scopic studies. The expected a-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interac- tions of CCR5 with its ligands, RANTES and MIP-1b were assessed by surface plasmon resonance yielding KD values in the nanomolar range. Using size exclusion chromatog- raphy, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oli- gomer 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.

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.

High-resolution nuclear magnetic resonance (NMR) has emerged as one of the most versatile tools for the quantitative study of structure, kinetics, and thermodynamics of biomolecules and their interactions at atomic resolution. Traditionally, nuclear Overhauser enhancements (NOEs) and chemical shift perturbation methods are used to determine molecular geometries and to identify contact surfaces, but more recently, weak anisotropic orientation, anisotropic diffusion, and scalar couplings across hydrogen bonds provide additional information. Examples of such technologies are shown as applied to the quantitative characterization of function and thermodynamics of several biomacromolecules. In particular, (1) the structural and dynamical changes of the TipA multidrug resistance protein are followed upon antibiotic binding, (2) the trimer-monomer equilibrium and thermal unfolding of foldon, a small and very efficient trimerization domain of the T4 phagehead, is described in atomic detail, and (3) the changes of individual protein hydrogen bonds during thermal unfolding are quantitatively followed by scalar couplings across hydrogen bonds.

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.

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.

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.

We have recently shown that an energy penalty for the incorporation of residual tensorial constraints into molecular structure calculations can be formulated without the explicit knowledge of the Saupe orientation tensor (Moltke and Grzesiek, J. Biomol. NMR, 1999, 15, 77–82). Here we report the implementation of such an algorithm into the program X-PLOR. The new algorithm is easy to use and has good convergence properties. The algorithm is used for the structure refinement of the HIV-1 Nef protein using 252 dipolar coupling restraints. The approach is compared to the conventional penalty function with explicit knowledge of the orientation tensor's amplitude and rhombicity. No significant differences are found with respect to speed, Ramachandran core quality or coordinate precision.

A wealth of information has been gathered during the past decades that water molecules do play an important role in the structure, dynamics, and function of bacteriorhodopsin (bR) and purple membrane. Light-induced structural alterations in bR as detected by X-ray and neutron diffraction at low and high resolution are discussed in relationship to the mechanism of proton pumping. The analysis of high resolution intermediate structures revealed photon-induced rearrangements of water molecules and hydrogen bonds concomitant with conformational changes in the chromophore and the protein. These observations led to an understanding of key features of the pumping mechanism, especially the vectoriality and the different modes of proton translocation in the proton release and uptake domain of bR. In addition, water molecules influence the function of bR via equilibrium fluctuations, which must occur with adequate amplitude so that energy barriers between conformational states can be overcome.

The transport of protons across membranes is an important process in cellular bioenergetics. The light-driven proton pump bacteriorhodopsin is the best-characterized protein providing this function. Photon energy is absorbed by the chromophore retinal, covalently bound to Lys 216 via a protonated Schiff base. The light-induced all-trans to 13-cis isomerization of the retinal results in deprotonation of the Schiff base followed by alterations in protonatable groups within bacteriorhodopsin. The changed force field induces changes, even in the tertiary structure, which are necessary for proton pumping. The recent report of a high-resolution X-ray crystal structure for the late M intermediate of a mutant bacteriorhopsin (with Asp 96-->Asn) displays the structure of a proton pathway highly disturbed by the mutation. To observe an unperturbed proton pathway, we determined the structure of the late M intermediate of wild-type bacteriorhodopsin (2.25 A resolution). The cytoplasmic side of our M2 structure shows a water net that allows proton transfer from the proton donor group Asp 96 towards the Schiff base. An enlarged cavity system above Asp 96 is observed, which facilitates the de- and reprotonation of this group by fluctuating water molecules in the last part of the cycle.

The photon-driven proton translocator bacteriorhodopsin is considered to be the best understood membrane protein so far. It is nowadays regarded as a model system for photosynthesis, ion pumps and seven transmembrane receptors. The profound knowledge came from the applicability of a variety of modern biophysical techniques which have often been further developed with research on bacteriorhodopsin and have delivered major contributions also to other areas. Most prominent examples are electron crystallography, solid-state NMR spectroscopy and time-resolved vibrational spectroscopy. The recently introduced method of crystallising a membrane protein in the lipidic cubic phase led to high-resolution structures of ground state bacteriorhodopsin and some of the photocycle intermediates. This achievement in combination with spectroscopic results will strongly advance our understanding of the functional mechanism of bacteriorhodopsin on the atomic level. We present here the current knowledge on specific aspects of the structural and functional dynamics of the photoreaction of bacteriorhodopsin with a focus on techniques established in our institute.

The diffusive properties of biomacromolecules within the aqueous phase of polyacrylamide gels are described. High quality NMR spectra can be obtained under such conditions. As compared to water, a fivefold reduction in the translational diffusion constant, but only a 1.6-fold decrease (1.4-fold increase) in amide-15N T2 (T1) are observed for human ubiquitin within a 10% acrylamide gel. Weak alignment of the solute macromolecules can be achieved within such gels by vertical or radial compression or by the embedding of magnetically oriented purple membrane fragments. The methods are applied to deriveresidual dipolar couplings for human HIV-1 Nef and ubiquitin.

Bacteriorhodopsin is the one of the best-studied models of an ion pump. Five atomic models are now available, yet their comparison reveals differences of some loops connecting the seven transmembrane alpha-helices. In an attempt to resolve this enigma, topographs were recorded in aqueous solution with the atomic force microscope (AFM) to reveal the most native surface structure of bacteriorhodopsin molecules in the purple membrane. Individual peptide loops were observed with a lateral resolution of between 4.5 A and 5.8 A, and a vertical resolution of about 1 A. The AFM images demonstrate for the first time, that the shape, the position, and the flexibility of individual polypeptide loops depend on the packing arrangement of bacteriorhodopsin molecules in the lipid bilayer.

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.

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.

The entry of the human immunodeficiency virus 1 (HIV-1) into host cells requires the sequential interaction of the viral envelope glycoprotein 120 (gp120) with the host-cell factor CD4 and with either CCR5 (CC chemokine receptor 5) or CXCR4, both G-protein coupled receptors (GPCR). This leads to the fusion of viral and host cell membranes. The normal physiological role of CCR5, however, is the regulation of immune-cell trafficking upon activation by its endogenous ligands: macrophage inflammatory protein 1α (MIP-1α), MIP-1β and RANTES (Regulated on Activation, Normal T-cell Expressed and Secreted). Since both, viral gp120 and the chemokines bind to the extracellular parts of the receptor, binding of a natural ligand, e.g. RANTES, obstructs the interaction of CCR5 with the viral protein, thereby hindering HIV infection. This makes RANTES and other chemokines potential lead structures for novel anti-HIV agents.Here, we present advances on the study of the interactions of recombinantly-expressed CCR5 with different chemokine variants and the small-molecule inhibitor maraviroc. Experiments are performed by nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR), with CCR5 incorporated in either detergent micelles, lipid bilayers, or the synthetic model membrane system of nanodiscs. Mechanistic implications of these results are discussed.While a 3D structure of CCR5 is still lacking, these data have the potential to shed some new light on our understanding of HIV infection, as well as on the topic of cellular signalling through chemokines. As the quality of preparation of recombinant CCR5 samples improves, further milestones are expected towards structural models of the ligand-CCR5 complexes.

The integral membrane protein bacteriorhodopsin (bR) is an excellent example to show how information is obtained from different biophysical methods to understand the function of this protein on the basis of its structure and dynamics. There are only a few such proteins, which attracted the interest of a large number of research groups.

The existence of two different M-state structures in the photocycle of the bacteriorhodopsin mutant ASP38ARG was proved. At pH 6.7 (0 to −6°C) a spectroscopic M intermediate (M 1 ) that does not differ significantly in its tertiary structure from the light-adapted ground state accumulates under illumination. At pH>9 another state (M 2 ), characterized by additional pronounced changes in the Fourier transform infrared difference spectrum in the region of the amide I and II bands, accumulates. The M 2 intermediate trapped at pH 9.6 displays the same changes in the x-ray diffraction intensities under continuous illumination as previously described for x-ray experiments with the mutant ASP96ASN. These observations indicate that in this mutant the altered charge distribution at neutral pH controls the tertiary structural changes that seem to be necessary for proton translocation.

The tertiary structural changes occurring during the photocycle of bacteriorhodopsin (BR) are assigned by X-ray diffraction to distinct M states, M1 and M2. Purple membranes (PM) of the mutant Asp96Asn at 15, 57, 75 and 100% relative humidity (r.h.) were studied in a parallel X-ray diffraction and Fourier transform infrared (FTIR) spectroscopic investigation. Light-dependent conformational changes of BR-Asp96Asn are observed at high hydration levels (100 and 75% r.h.) but not in partially dehydrated samples (57 and 15% r.h.). The FTIR spectra of continuously illuminated samples at low and high hydration, despite some differences, are characteristic of the M intermediate. The changes in diffraction patterns of samples in the M2 state are of the same magnitude as those of wild-type samples trapped with GuaHCl in the M(G) state. Additional large changes in the amide bands of the FTIR spectra occur between M2 and M(G). This suggests, that the tertiary structural changes between M1 and M2 are responsible for the switch opening the cytoplasmic half-channel of BR for reprotonation to complete the catalytic cycle. These tertiary structural changes seem to be triggered by a charge redistribution which might be a common feature of retinal proteins also in signal transduction.

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. © 2012 Van den Bergh et al., SCOPUS: ar.j, info:eu-repo/semantics/published