Activated STING in a vascular and pulmonary syndrome.

BACKGROUND: The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation. METHODS: We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-ß, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls. RESULTS: We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mononuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibroblasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients' lymphocytes was reduced by JAK inhibitors. CONCLUSIONS: STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173. (Funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases; ClinicalTrials.gov number, NCT00059748.).

Squeezing protein shells: how continuum elastic models, molecular dynamics simulations, and experiments coalesce at the nanoscale.

The current rapid growth in the use of nanosized particles is fueled in part by our increased understanding of their physical properties and ability to manipulate them, which is essential for achieving optimal functionality. Here we report detailed quantitative measurements of the mechanical response of nanosized protein shells (viral capsids) to large-scale physical deformations and compare them with theoretical descriptions from continuum elastic modeling and molecular dynamics (MD). Specifically, we used nanoindentation by atomic force microscopy to investigate the complex elastic behavior of Hepatitis B virus capsids. These capsids are hollow, approximately 30 nm in diameter, and conform to icosahedral (5-3-2) symmetry. First we show that their indentation behavior, which is symmetry-axis-dependent, cannot be reproduced by a simple model based on Föppl-von Kármán thin-shell elasticity with the fivefold vertices acting as prestressed disclinations. However, we can properly describe the measured nonlinear elastic and orientation-dependent force response with a three-dimensional, topographically detailed, finite-element model. Next, we show that coarse-grained MD simulations also yield good agreement with our nanoindentation measurements, even without any fitting of force-field parameters in the MD model. This study demonstrates that the material properties of viral nanoparticles can be correctly described by both modeling approaches. At the same time, we show that even for large deformations, it suffices to approximate the mechanical behavior of nanosized viral shells with a continuum approach, and ignore specific molecular interactions. This experimental validation of continuum elastic theory provides an example of a situation in which rules of macroscopic physics can apply to nanoscale molecular assemblies.

HIV-1 rev depolymerizes microtubules to form stable bilayered rings.

We describe a novel interaction between HIV-1 Rev and microtubules (MTs) that results in the formation of bilayered rings that are 44-49 nm in external diameter, 3.4-4.2 MD (megadaltons) in mass, and have 28-, 30-, or 32-fold symmetry. Ring formation is not sensitive to taxol, colchicine, or microtubule-associated proteins, but requires Mg(2+) and is inhibited by maytansine. The interaction involves the NH(2)-terminal domain of Rev and the face of tubulin exposed on the exterior of the MTs. The NH(2)-terminal half of Rev has unexpected sequence similarity to the tubulin-binding portion of the catalytic/motor domains of the microtubule-destabilizing Kin I kinesins. We propose a model wherein binding of Rev dimers to MTs at their ends causes segments of two neighboring protofilaments to peel off and close into rings, circumferentially containing 14, 15, or 16 tubulin heterodimers, with Rev bound on the inside. Rev has a strong inhibitory effect on aster formation in Xenopus egg extracts, demonstrating that it can interact with tubulin in the presence of normal levels of cellular constituents. These results suggest that Rev may interact with MTs to induce their destabilization, a proposition consistent with the previously described disruption of MTs after HIV-1 infection.

A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G.

The high-resolution three-dimensional structure of a single immunoglobulin binding domain (B1, which comprises 56 residues including the NH2-terminal Met) of protein G from group G Streptococcus has been determined in solution by nuclear magnetic resonance spectroscopy on the basis of 1058 experimental restraints. The average atomic root-mean-square distribution about the mean coordinate positions is 0.27 angstrom (A) for the backbone atoms, 0.65 A for all atoms, and 0.39 A for atoms excluding disordered surface side chains. The structure has no disulfide bridges and is composed of a four-stranded beta sheet, on top of which lies a long helix. The central two strands (beta 1 and beta 4), comprising the NH2- and COOH-termini, are parallel, and the outer two strands (beta 2 and beta 3) are connected by the helix in a +3x crossover. This novel topology (-1, +3x, -1), coupled with an extensive hydrogen-bonding network and a tightly packed and buried hydrophobic core, is probably responsible for the extreme thermal stability of this small domain (reversible melting at 87 degrees C).

Kinetics of folding of the all-beta sheet protein interleukin-1 beta.

The folding of the all-beta sheet protein, interleukin-1 beta, was studied with nuclear magnetic resonance (NMR) spectroscopy, circular dichroism, and fluorescence. Ninety percent of the beta structure present in the native protein, as monitored by far-ultraviolet circular dichroism, was attained within 25 milliseconds, correlating with the first kinetic phase determined by tryptophan and 1-anilinonaphthalene-8-sulfonate fluorescence. In contrast, formation of stable native secondary structure, as measured by quenched-flow deuterium-hydrogen exchange experiments, began after only 1 second. Results from the NMR experiments indicated the formation of at least two intermediates with half-lives of 0.7 to 1.5 and 15 to 25 seconds. The final stabilization of the secondary structure, however, occurs on a time scale much greater than 25 seconds. These results differ from previous results on mixed alpha helix-beta sheet proteins in which both the alpha helices and beta sheets were stabilized very rapidly (less than 10 to 20 milliseconds).

Epitope diversity of hepatitis B virus capsids: quasi-equivalent variations in spike epitopes and binding of different antibodies to the same epitope.

To investigate the range of antigenic variation of HBV capsids, we have characterized the epitopes for two anti-capsid antibodies by cryo-electron microscopy and image reconstruction of Fab-labeled capsids to approximately 10A resolution followed by molecular modeling. Both antibodies engage residues on the protruding spikes but their epitopes and binding orientations differ. Steric interference effects limit maximum binding to approximately 50% average occupancy in each case. However, the occupancies of the two copies of a given epitope that are present on a single spike differ, reflecting subtle distinctions in structure and hence, binding affinity, arising from quasi-equivalence. The epitope for mAb88 is conformational but continuous, consisting of a loop-helix motif (residues 77-87) on one of the two polypeptide chains in the spike. In contrast, the epitope for mAb842, like most conformational epitopes, is discontinuous, consisting of a loop on one polypeptide chain (residues 74-78) combined with a loop-helix element (residues 78-83) on the other. The epitope of mAb842 is essentially identical with that previously mapped for mAb F11A4, although the binding orientations of the two monoclonal antibodies (mAbs) differ, as do their affinities measured by surface plasmon resonance. From the number of monoclonals (six) whose binding had to be characterized to give the first duplicate epitope, we estimate the total number of core antigen (cAg) epitopes to be of the order of 20. Given that different antibodies may share the same epitope, the potential number of distinct anti-cAg clones should be considerably higher. The observation that the large majority of cAg epitopes are conformational reflects the relative dimensions of a Fab (large) and the small size and close packing of the motifs that are exposed and accessible on the capsid surface.

Specific complex of human immunodeficiency virus type 1 rev and nucleolar B23 proteins: dissociation by the Rev response element.

The human immunodeficiency virus type 1 (HIV) Rev protein is thought to be involved in the export of unspliced or singly spliced viral mRNAs from the nucleus to the cytoplasm. This function is mediated by a sequence-specific interaction with a cis-acting RNA element, the Rev response element (RRE), present in these intron-containing RNAs. To identify possible host proteins involved in Rev function, we fractionated nuclear cell extracts with a Rev affinity column. A single, tightly associated Rev-binding protein was identified; this protein is the mammalian nucleolar protein B23. The interaction between HIV Rev and B23 is very specific, as it was observed in complex cell extracts. The complex is also very stable toward dissociation by high salt concentrations. Despite the stability of the Rev-B23 protein complex, the addition of RRE, but not control RNA, led to the displacement of B23 and the formation of a specific Rev-RRE complex. The mammalian nucleolar protein B23 or its amphibian counterpart No38 is believed to function as a shuttle receptor for the nuclear import of ribosomal proteins. B23 may also serve as a shuttle for the import of HIV Rev from the cytoplasm into the nucleus or nucleolus to allow further rounds of export of RRE-containing viral RNAs.

The solution structure of the N-terminal domain of hepatocyte growth factor reveals a potential heparin-binding site.

BACKGROUND: Hepatocyte growth factor (HGF) is a multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. The N-terminal (N) domain of HGF, containing a hairpin-loop region, is important for receptor binding and the potent biological activities of HGF. The N domain is also the primary binding site for heparin or heparan sulfate, which enhances, receptor/ligand oligomerization and modulates receptor-dependent mitogenesis. The rational design of artificial modulators of HGF signaling requires a detailed understanding of the structures of HGF and its receptor, as well as the role of heparin proteoglycan; this study represents the first step towards that goal. RESULTS: We report here a high-resolution structure of the N domain of HGF. This first structure of HGF reveals a novel folding topology with a distinct pattern of charge distribution and indicates a possible heparin-binding site. CONCLUSIONS: The hairpin-loop region of the N domain plays a major role in stabilizing the structure and contributes to a putative heparin-binding site, which explains why it is required for biological functions. These results suggest several basic and/or polar residues that may be important for use in further mutational studies of heparin binding.

Evidence that high mannose glycopeptides are able to functionally interact with recombinant tumor necrosis factor and recombinant interleukin 1.

Both recombinant tumor necrosis factor (rTNF) and recombinant interleukin 1 (rIL-1) are able to mediate vascular collapse and death in a previously described murine model, using galactosamine to enhance the toxicity of these cytokines. Unexpectedly, both acid-treated tumor necrosis factor (TNF) and a site-specifically mutagenized form of interleukin 1 (IL-1) (His-30----Arg-30), which fails to bind to the IL-1 receptor, retain full in vivo toxicity in this model of TNF- and IL-1-mediated shock. Previous studies have shown that rTNF and rIL-1 exhibit two functionally distinct binding regions. Both cytokines bind to their respective cell surface receptors and they also express lectin like binding specificity (Muchmore and Decker, J. Biol. Chem., 261: 13404-13407, 1986; Muchmore and Decker, J. Immunol., 138: 2541-2546, 1987) for defined oligosaccharides. The specificity of these two types of interactions is quite different. Cell surface receptors for IL-1 and TNF demonstrate essentially no cross-reactivity, whereas, in the case of carbohydrate binding, competition studies reveal an almost identical carbohydrate specificity for the structure Man5(6)GlcNAc2-Asn. Man5(6)GlcNAc2-Asn binding is either unaffected or actually enhanced by either acid treatment of rTNF or mutation at His-30 for rIL-1. Both deoxymannojirimycin and swainsonine, inhibitors of glycoprotein processing, raise intracellular levels of Man5-9GlcNAc2 and enhance the in vitro biological activity of both rTNF and rIL-1. Conversely, castanosperimine, a glucosidase I inhibitor which blocks the synthesis of mature high mannose structures, inhibits the biological activity of IL-1. These observations support the hypothesis that some effects of IL-1 and TNF may involve interaction with high mannose-substituted glycoproteins.

A recombinant Chinese hamster ovary cell line containing a 300-fold amplified tetramer of the hepatitis B genome together with a double selection marker expresses high levels of viral protein.

A new series of double-selection plasmids containing recombinant genes expressing the neomycin phosphotransferase (NEO) of transposon Tn5 and mouse dihydrofolate reductase (DHFR) in mammalian cells is described. Activity of the recombinant DHFR gene varied more than 50-fold, depending on the location of the simian virus 40 72 base-pair repeat or enhancer, which is part of the promoter of the NEO unit. A NEO-DHFR module with the enhancer located at the 3' end of the DHFR gene was inserted into a plasmid containing four tandem head-to-tail copies of the hepatitis B virus (HBV) genome and the new plasmid was used to transform DHFR- Chinese hamster ovary cells. In one of the cell lines obtained, an unrearranged copy of the HBV tetramer could be amplified 300-fold by increasing selective pressure with methotrexate, resulting in a proportional increase of the synthesis of HBV surface antigen. Four different mRNAs detected in the amplified cell line probably encode HBV core protein, pre-S and surface antigens, and the X protein. As a result of the DNA amplification, synthesis of HBV proteins is no longer restricted to resting cells. Integrated plasmid sequences appear to be stable during the amplification process.

Low resolution structure of interleukin-1 beta in solution derived from 1H-15N heteronuclear three-dimensional nuclear magnetic resonance spectroscopy.

A low resolution solution structure of the cytokine interleukin-1 beta, a 153 residue protein of molecular weight 17,400, has been determined on the basis of 446 nuclear Overhauser effect (NOE) derived approximate interproton distance restraints involving solely NH, C alpha H and C beta H protons, supplemented by 90 distance restraints for 45 hydrogen bonds, and 79 phi torsion angle restraints. With the exception of 27 C alpha H-C alpha H NOEs, all the NOEs were assigned from a three-dimensional 1H-1H NOE 15N-1H heteronuclear multiple quantum coherence (HMQC) spectrum. The torsion angle restraints were obtained from accurate 3JHN alpha coupling constants measured from a HMQC-J spectrum, while the hydrogen bonds were derived from a qualitative analysis of the NOE, coupling constant and amide exchange data. A total of 20 simulated annealing (SA) structures was computed using the hybrid distance geometry-dynamical simulated annealing method. The solution structure of IL-1 beta comprises 12 beta-strands arranged in three pseudo-symmetrical topological units (each consisting of 5 anti-parallel beta-strands), joined by turns, short loops and long loops. The core of the structure, which is made up of the 12 beta-strands, together with the turns joining strands I and II, strands VIII and IX and strands X and XI, is well determined with a backbone atomic root-mean-square (r.m.s.) distribution about the mean co-ordinate positions of 1.2(+/- 0.1) A. The loop conformations, on the other hand, are poorly determined by the current data. A comparison of the core of the low resolution solution structure of IL-1 beta with that of the X-ray structure indicates that they are similar, with a backbone atomic r.m.s. difference of only 1.5 A between the co-ordinates of the restrained minimized mean of the SA structures and the X-ray structure.

Determination of the positions of bound water molecules in the solution structure of reduced human thioredoxin by heteronuclear three-dimensional nuclear magnetic resonance spectroscopy.

The presence of bound water molecules in the solution structure of reduced human thioredoxin has been investigated using three-dimensional 1H rotating frame Overhauser 1H-15N multiple quantum coherence spectroscopy. It is demonstrated that the backbone amide protons of Lys21, Lys39, Lys82, Gly83 and Asn102, as well as the side-chain amide group of Asn102, are in close proximity to bound water molecules. Examination of the high-resolution solution structure of reduced human thioredoxin reveals that these results are best accounted for by four bound water molecules. Subsequent simulated annealing calculations carried out on the basis of interproton distance and hydrogen bonding restraints to the bound water molecules, supplemented by the original set of experimental restraints used in the calculation of the three-dimensional structure of human thioredoxin, permit a more precise localization of the bound water positions. Potential hydrogen bonds to these water molecules are described and a comparison is made to corresponding bound water molecules in the crystal structure of oxidized Escherichia coli thioredoxin.

Hepatitis B virus capsid: localization of the putative immunodominant loop (residues 78 to 83) on the capsid surface, and implications for the distinction between c and e-antigens.

Hepatitis B virus capsid protein comprises a 149 residue "assembly" domain that polymerizes into icosahedral particles, and a 34 residue RNA-binding "protamine" domain. Recently, the capsid structure has been studied to resolutions below 10 A by cryo-electron microscopy, revealing much of its alpha-helical substructure and that it appears to have a novel fold for a capsid protein; however, the resolution is still too low for chain-tracing by conventional criteria. Aiming to establish a fiducial marker to aid in the process of chain-tracing, we have used cryo-microscopy to pinpoint the binding site of a monoclonal antibody that recognizes the peptide from residues 78 to 83. This epitope resides on the outer rim of the 30 A long spikes that protrude from the capsid shell. These spikes are four-helix bundles formed by the pairing of helix-turn-helix motifs from two subunits; by means of a tilting experiment, we have determined that this bundle is right-handed. Variants of the same protein present two clinically important and non-crossreactive antigens: core antigen (HBcAg), which appears early in infection as assembled capsids; and the sentinel e-antigen (HBeAg), a non-particulate form. Knowledge of the binding site of our anti-HBcAg antibody bears on the molecular basis of the distinction between the two antigens, which appears to reflect conformational differences between the assembled and unassembled states of the capsid protein dimer, in addition to epitope masking in capsids.

Secondary structure of the ribonuclease H domain of the human immunodeficiency virus reverse transcriptase in solution using three-dimensional double and triple resonance heteronuclear magnetic resonance spectroscopy.

The solution structure of the ribonuclease H domain of HIV-1 reverse transcriptase has been investigated by three-dimensional double and triple resonance heteronuclear magnetic resonance spectroscopy. The domain studied has 138 residues and comprises residues 427 to 560 of the 66 kDa reverse transcriptase with an additional four residues at the N terminus. Initial studies on the wild-type protein were hindered by severe differential line broadening, presumably due to conformational averaging. Mutation of the single tryptophan residue located in a loop at position 113 (position 535 in the reverse transcriptase sequence) to an alanine resulted in much improved spectral properties with no apparent change in structure. 1H, 15N and 13C backbone resonances were assigned sequentially using a range of three-dimensional double and triple resonance heteronuclear experiments on samples of uniformly (greater than 95%) 15N and 15N/13C-labeled protein, and the secondary structure was elucidated from a qualitative analysis of data derived from three-dimensional 15N- and 13C-edited nuclear Overhauser enhancement spectra. The secondary structure comprises three alpha-helices and five strands arranged in a mixed parallel/antiparallel beta-sheet with a +1, +1, -3x, -1x topology. The C-terminal region from residue 114 onwards appears to be conformationally disordered in solution as evidenced by an almost complete absence of sequential and medium range nuclear Overhauser effects.

Determination of the secondary structure and global topology of the 44 kDa ectodomain of gp41 of the simian immunodeficiency virus by multidimensional nuclear magnetic resonance spectroscopy.

The gp41 protein of the human (HIV) and simian (SIV) immunodeficiency viruses is part of the envelope glycoprotein complex gp41/gp120 which plays an essential role in viral infection. We present a multidimensional NMR study on the trimeric 44 kDa soluble ectodomain of SIV gp41 (e-gp41), comprising residues 27 to 149. Despite the large molecular weight and very limited spectral dispersion, complete backbone 1H, 13C, 13CO and 15N assignments have been made using a combination of triple resonance experiments on uniformly 13C/15N and 2H/13C/15N-labeled samples. The secondary structure of SIV e-gp41, derived on the basis of 13C chemical shifts, NH exchange rates, medium range nuclear Overhauser enhancements (NOE), and 3JHNalpha coupling constants, consists of a 49 residue helix at the N terminus (residues 29 to 77) and a 40 residue helix at the C terminus (residues 108 to 147), connected by a 30 residue loop which does not display any of the characteristics of regular secondary structure. The cross-peak intensities of the loop region in scalar correlation experiments suggests that it is more mobile than the core helical regions. The presence, however, of numerous long range NOEs, both intra and inter-subunit, within the loop indicates that it adopts a well-defined structure in which the loops from the three subunits interact with each other. Based on a number of long range intra and inter-subunit NOEs, a topological model is presented for the symmetric SIV e-gp41 trimer in which the N-terminal helices are packed within the protein interior in a parallel trimeric coiled-coil arrangement, while the C-terminal helices are located on the protein exterior, oriented antiparallel to the N-terminal helices.

Filamentous hemagglutinin of Bordetella pertussis. A bacterial adhesin formed as a 50-nm monomeric rigid rod based on a 19-residue repeat motif rich in beta strands and turns.

The filamentous hemagglutinin (FHA) of Bordetella pertussis is an adhesin that binds the bacteria to cells of the respiratory epithelium in whooping-cough infections. Mature FHA is a 220 kDa secretory protein that is highly immunogenic and has been included in acellular vaccines. We have investigated its structure by combining electron microscopy and circular dichroism spectroscopy (CD) with computational analysis of its amino acid sequence. The FHA molecule is 50 nm in length and has the shape of a horseshoe nail: it has a globular head that appears to consist of two domains; a 35 nm-long shaft that averages 4 nm in width, but tapers slightly from the head end; and a small, flexible, tail. Mass measurements by scanning transmission electron microscopy establish that FHA is a monomer. Its sequence contains two regions of tandem 19-residue pseudo-repeats: the first, of 38 cycles, starts at residue 344; the second, of 13 cycles, starts at residue 1440. The repeat motifs are predicted to consist of short beta-strands separated by beta-turns, and secondary structure measurements by CD support this prediction. We propose a hairpin model for FHA in which the head is composed of the terminal domains; the shaft consists mainly of the repeat regions conformed as amphipathic, hyper-elongated beta-sheets, with their hydrophobic faces apposed; and the tail is composed of the intervening sequence. Further support for the model was obtained by immuno-labeling electron microscopy. The 19-residue repeats of FHA have features in common with the leucine-rich repeats (LRRs) that are present in many eukaryotic proteins, including some adhesion factors. The model is also compared with the two other classes of filamentous proteins that are rich in beta-structure, i.e. viral adhesins and two beta-helical secretory proteins. Our proposed structure implies how the functionally important adhesion sites and epitopes of FHA are distributed: its tripeptide (RGD) integrin-binding site is assigned to the tail; the putative hemagglutination site forms part of the head; and two classes of immunodominant epitopes are assigned to opposite ends of the molecule. Possible mechanisms are discussed for two modes of FHA-mediated adhesion.

Monomer-trimer equilibrium of the ectodomain of SIV gp41: insight into the mechanism of peptide inhibition of HIV infection.

The monomer-trimer equilibrium of the ectodomain of SIV gp41 (residues 27-149, e-gp41) has been characterized by analytical ultracentrifugation, circular dichroism (CD), and NMR spectroscopy. Based on analytical ultracentrifugation experiments performed at different rotor speeds and protein concentrations, the equilibrium association constant for the SIV e-gp41 trimer is 3.1 x 10(11) M(-2). The presence of intermolecular nuclear Overhauser effects in a mixture of 12C and 13C-labeled e-gp41 prepared under nondenaturing conditions unambiguously demonstrates that there is a dynamic equilibrium between the monomer and trimer. The CD spectra taken as a function of SIV e-gp41 concentration suggest that the helical content of the monomeric state does not change significantly relative to that of the trimeric state. The relevance of the monomer-trimer equilibrium is discussed with respect to gp41 function and the inhibitory properties of gp41 peptides.

Is human thioredoxin monomeric or dimeric?

We have examined the molecular weight and rotational correlation time of human thioredoxin by analytical ultracentrifugation and NMR spectroscopy, respectively. Two variants of human thioredoxin were studied, namely human thioredoxin identical in amino acid sequence to the one whose NMR structure we previously determined (C62A, C69A, C73A, M74T) and human thioredoxin (C62A, C69A, C73A, M74) containing the wild-type amino acid methionine at position 74. In both cases, the experimental data indicate that the predominant species is monomeric and we find no evidence for the existence of a well-defined dimeric form as was observed in the recently reported crystal structure (Weichsel et al., 1996) of human thioredoxin and the C73S mutant.

Refined solution structure and backbone dynamics of HIV-1 Nef.

The tendency of HIV-1 Nef to form aggregates in solution, particularly at pH values below 8, together with its large fraction of highly mobile residues seriously complicated determination of its three-dimensional structure, both for heteronuclear solution NMR (Grzesiek et al., 1996a, Nat Struct Biol 3:340-345) and for X-ray crystallography (Lee et al., 1996, Cell 85:931-942). Methods used to determine the Nef structure by NMR at pH 8 and 0.6 mM concentration are presented, together with a detailed description of Nef's secondary and tertiary structure. The described techniques have general applicability for the NMR structure determination of proteins that are aggregating and/or have limited stability at low pH values. Extensive chemical shift assignments are reported for backbone and side chain 1H, 13C, and 15N resonances of the HIV-1 Nef deletion mutants NEF delta 2-39, NEF delta 2-39, delta 159-173, and of NEF delta 2-39, delta 159-173 in complex with the SH3 domain of the Hck tyrosine protein kinase. Besides a type II polyproline helix, Nef's structure consists of three alpha-helices, a 3(10) helix, and a five-stranded anti-parallel beta-sheet. The analysis of 15N relaxation parameters of the backbone amide sites reveals that all the secondary structure elements are non-mobile on the picosecond to nanosecond and on the millisecond time scale. A large number of slowly exchanging amide protons provides evidence for the stability of the Nef core even on the time scale of hours. Significant internal motions on the ps to ns time scale are detected for residues 60 to 71 and for residues 149 to 180, which form solvent-exposed loops. The residues of the HIV-1 protease cleavage site (W57/L58) do not exhibit large amplitude motions on the sub-nanosecond time scale, and their side chains insert themselves into a hydrophobic crevice formed between the C-terminus of helix 1 and the N-terminus of helix 2. A refined structure has been determined based on additional constraints for side-chain and backbone dihedral angles derived from a large number of three-bond J-coupling and ROE data.

The extracellular domain of immunodeficiency virus gp41 protein: expression in Escherichia coli, purification, and crystallization.

The env gene of SIV and HIV-1 encodes a single glycoprotein gp 160, which is processed to give a noncovalent complex of the soluble glycoprotein gp120 and the transmembrane glycoprotein gp41. The extracellular region (ectodomain), minus the N-terminal fusion peptide, of gp41 from HIV-1 (residues 27-154) and SIV (residues 27-149) have been expressed in Escherichia coli. These insoluble proteins were solubilized and subjected to a simple purification and folding scheme, which results in high yields of soluble protein. Purified proteins have a trimeric subunit composition and high alpha-helical content, consistent with the predicted coil-coil structure. SIV gp41 containing a double cysteine mutation was crystallized. The crystals are suitable for X-ray structure determination and, preliminary analysis, together with additional biochemical evidence, indicates that the gp41 trimer is arranged as a parallel bundle with threefold symmetry.