Peptide Inhibitors of Dengue-Virus Entry Target a Late-Stage Fusion Intermediate

The mechanism of membrane fusion by “class II” viral fusion proteins follows a pathway that involves large-scale domain rearrangements of the envelope glycoprotein (E) and a transition from dimers to trimers. The rearrangement is believed to proceed by an outward rotation of the E ectodomain after loss of the dimer interface, followed by a reassociation into extended trimers. The ∼55-aa-residue, membrane proximal “stem” can then zip up along domain II, bringing together the transmembrane segments of the C-terminus and the fusion loops at the tip of domain II. We find that peptides derived from the stem of dengue-virus E bind stem-less E trimer, which models a conformational intermediate. In vitro assays demonstrate that these peptides specifically block viral fusion. The peptides inhibit infectivity with potency proportional to their affinity for the conformational intermediate, even when free peptide is removed from a preincubated inoculum before infecting cells. We conclude that peptides bind virions before attachment and are carried with virions into endosomes, the compartment in which acidification initiates fusion. Binding depends on particle dynamics, as there is no inhibition of infectivity if preincubation and separation are at 4°C rather than 37°C. We propose a two-step model for the mechanism of fusion inhibition. Targeting a viral entry pathway can be an effective way to block infection. Our data, which support and extend proposed mechanisms for how the E conformational change promotes membrane fusion, suggest strategies for inhibiting flavivirus entry.     

Molecular Diagnostics of Fine Needle Aspiration for the Presurgical Screening of Thyroid Nodules

“The incidence of thyroid cancer, the most common endocrine malignancy, is rising. The two most common types of thyroid cancer are papillary and follicular” thyroid carcinomas. “Fine-needle aspiration (FNA) of thyroid nodules” can permit to detect many genetic mutations and other molecular alterations, including RAS and BRAF point mutations, PAX8/peroxisome proliferator-activated receptor (PPAR)γ and “RET/PTC rearrangements, occurring in thyroid papillary and follicular carcinomas” (more than 70% of cases), which can be used successfully to improve the diagnosis “and the management of patients with thyroid nodules”. The most extensive experience has been accumulated with “the diagnostic use of BRAF mutation”, which is highly specific for malignancy. “Testing FNA samples for a panel of mutations” that typically includes RAS, BRAF, PAX8/PPARγ and RET/PTC could permit to achieve the biggest diagnostic impact. “The accuracy of cancer diagnosis in thyroid nodules could be improved significantly using these and other emerging molecular markers”.     

Immunoglobulin G subclasses of anti-thyroid peroxidase autoantibodies in human autoimmune thyroid diseases

A distribution of immunoglobulin G (IgG) subclass of anti-thyroid peroxidase (TPO) autoantibodies was studied to know whether anti-TPO autoantibodies are closely implicated in the pathogenesis of human autoimmune thyroid diseases. As a result of analyzing 14 patients' sera, 7 with Graves' disease and 7 with Hashimoto's thyroiditis, anti-TPO autoantibodies were found to consist of mainly IgG1 subclass. Percentages of both IgG1 and IgG2 subclasses in IgG class of autoantibodies corresponded to those in the normal serum composition, whereas IgG3 subclass was scarcely contained in anti-TPO autoantibodies and IgG4 subclass markedly increased. It was thought that anti-TPO autoantibodies had a capability to lyse thyroid follicular cells by the mechanism of antibody-dependent complement-mediated cytolysis, because IgG1 and IgG2 subclasses of antibodies can fix complement and TPO locates in apical membrane surface of thyroid follicular cells. Comparing Graves' disease with Hashimoto's thyroiditis, mean percentages of both IgG1 and IgG2 subclasses of 2 groups were statistically different. Namely, sera of patients with Graves' disease had higher and lower mean percentages of IgG1 and IgG2 subclasses of autoantibodies, respectively, than those with Hashimoto's thyroiditis, though no plausible explanation for these differences can be offered at the present time.     

The Crystal Structures of Yeast Get3 Suggest a Mechanism for Tail-Anchored Protein Membrane Insertion

Tail-anchored (TA) proteins represent a unique class of membrane proteins that contain a single C-terminal transmembrane helix. The post-translational insertion of the yeast TA proteins into the ER membrane requires the Golgi ER trafficking (GET) complex which contains Get1, Get2 and Get3. Get3 is an ATPase that recognizes and binds the C-terminal transmembrane domain (TMD) of the TA proteins. We have determined the crystal structures of Get3 from two yeast species, S. cerevisiae and D. hansenii, respectively. These high resolution crystal structures show that Get3 contains a nucleotide-binding domain and a “finger” domain for binding the TA protein TMD. A large hydrophobic groove on the finger domain of S. cerevisiae Get3 structure might represent the binding site for TMD of TA proteins. A hydrophobic helix from a symmetry-related Get3 molecule sits in the TMD-binding groove and mimics the TA binding scenario. Interestingly, the crystal structures of the Get3 dimers from S. cerevisiae and D. hansenii exhibit distinct conformations. The S. cerevisiae Get3 dimer structure does not contain nucleotides and maintains an “open” conformation, while the D. hansenii Get3 dimer structure binds ADP and stays in a “closed” conformation. We propose that the conformational changes to switch the Get3 between the open and closed conformations may facilitate the membrane insertions for TA proteins.     

Human thyroid peroxidase: mapping of autoantibodies, conformational epitopes to the enzyme surface

The enzyme, thyroid peroxidase (TPO), is a dominant antigen in thyroid autoimmune diseases. Autoantibodies recognised two major dominant conformational epitopes termed A and B. The epitopes have been defined by mAbs, but the amino acid residues which constitute these determinants remain unknown. Using a model of TPO, built from the structure of myeloperoxidase (MPO), we have synthesised peptides corresponding to exposed loops and generated rabbit antibodies to the peptides. Antisera to peptide sequence 599-617 (peptide 14) representing a highly protrusive loop on the TPO, showed the highest inhibition in 65 sera from patients positive with anti-TPO antibodies. The inhibition was by 15-80% (mean 41%), and no other antibody showed any inhibition. Binding of hFabs to the B determinant on TPO was inhibited by anti-peptide 14 antibodies more then 85%, but not Fabs to the A determinant. In conclusion, the peptide 14 defines a sequence taking part in building up the B major conformational epitope. None of generated anti-peptide antibodies alone inhibited the binding of human Fabs to the A epitope, however a combination of four anti-peptide antibodies (P1, P12, P14 and P18) inhibits Fabs binding to the A determinant by more then 60% and autoantibodies binding from 65% to 94%. Combination of antibodies reacting with peptides outside the surface defined by those four antipeptide antibodies did not give any inhibition of Fabs to TPO. The inhibition of Fabs and auto Abs to TPO by this combination of anti-peptide Abs is the result of steric hindrance as none of these Abs individually inhibited auto Abs' or Fabs' binding to TPO. The four peptides define an area on the enzyme surface where the A and B major conformational epitopes are localised.     

Mapping of a linear autoantigenic epitope within the human thyroid peroxidase using recombinant DNA techniques.

Autoantibodies directed against the thyroid peroxidase (TPO), the thyroid microsomal antigen, are widely used to diagnose human autoimmune thyroid disease. A cloned 3.088 kb cDNA coding for the entire mature human TPO was isolated from a cDNA library derived from a pathological thyroid gland of a Graves' disease patient and used further to generate a so-called TPO epitope cDNA library in order to map linear autoantigenic epitopes involving a recombinant molecular biology approach. The TPO epitope cDNA library consisting of randomly fragmented cDNA sequences inserted in the expression vector pGEX-2T was expressed in Escherichia coli and screened with characterized anti-TPO autoantisera from Hashimoto's disease patients. All the sera were positively tested with a purified thyroid microsomal antigen fraction (TMA/TPO). Only about 1% of examined autoantisera were able to recognize bacterial expressed recombinant TPO representing sequential antigenic determinants. A corresponding autoantigenic epitope with 61 amino acids in length was located at the C-terminus of human TPO.     

Characteristics of monoclonal antibodies against human thyroid peroxidase for use in immunoaffinity chromatography and immunoassays

Two types of monoclonal antibodies (MABs) against human thyroid peroxidase (TPO) have been obtained, which interact with spatially separated conformational epitopes of the antigen (Ka values are in the range 10(8)-10(9) M(-1)). The binding site of MAB F8 is in the immunodominant region of the TPO molecule, in the vicinity of the autoantigenic determinants, whereas the epitope specific for MAB A1 lies outside this location. Both MABs retain the ability to form immune complexes after solid-phase immobilization and chemical modification with a biotin derivative. The above properties suggest that MABs A1 and F8 may be used in immunoaffinity chromatography (isolation and purification of TPO from natural sources) and immunoassays for determinations of TPO (in biological fluids) and TPO autoantibodies (in human blood serum).     

Characteristics of monoclonal antibodies against human thyroid peroxidase for use in immunoaffinity chromatography and immunoassays

Two types of monoclonal antibodies (MABs) against human thyroid peroxidase (TPO) have been obtained, which interact with spatially separated conformational epitopes of the antigen (K _a values are in the range 10⁸–10⁹ M^?1). The binding site of MAB F8 is in the immunodominant region of the TPO molecule, in the vicinity of the autoantigenic determinants, whereas the epitope specific for MAB A1 lies outside this location. Both MABs retain the ability to form immune complexes after solid-phase immobilization and chemical modification with a biotin derivative. The above properties suggest that MABs A1 and F8 may be used in immunoaffinity chromatography (isolation and purification of TPO from natural sources) and immunoassays for determinations of TPO (in biological fluids) and TPO autoantibodies (in human blood serum).     

Thyroperoxidase, an auto-antigen with a mosaic structure made of nuclear and mitochondrial gene modules.

A lambda gt11 cDNA library was constructed from a normal human thyroid and screened with a rabbit anti-porcine thyroperoxidase antibody. A series of thyroperoxidase (TPO) clones were obtained which allowed determination of the complete primary structure of the protein. The library was also screened with serum from a patient with Hashimoto's thyroiditis, an autoimmune disease characterized by the presence in the serum of high titers of autoantibodies directed against the 'microsomal antigen' (McAg). Comparison of the cDNA sequences from TPO clones and McAg clones provides definite proof that the McAg is TPO. A short segment of TPO was characterized as bearing a major epitope involved in autoimmunity. The primary structure of TPO was 42% homologous to myeloperoxidase (MPO). It contains, in addition, a C-terminal extension with a membrane anchor region contiguous to two domains encoded by modules belonging to the EGF and C4b gene families. The existence in TPO of still another domain presenting a significant homology with a putative heme-binding region of cytochrome C oxidase polypeptide I raises the possibility that a mitochondrial gene module has contributed a piece to the evolution of a typical nuclear mosaic gene.     

Human thyroid peroxidase: mapping of autoantibodies,conformational epitopes to the enzyme surface

Abstract

The enzyme, thyroid peroxidase (TPO), is a dominant antigen in thyroid autoimmune diseases. Autoantibodies recognised two major dominant conformational epitopes termed A and B. The epitopes have been defined by mAbs, but the amino acid residues which constitute these determinants remain unknown. Using a model of TPO, built from the structure of myeloperoxidase (MPO), we have synthesised peptides corresponding to exposed loops and generated rabbit antibodies to the peptides.

Antisera to peptide sequence 599–617 (peptide 14) representing a highly protrusive loop on the TPO, showed the highest inhibition in 65 sera from patients positive with anti-TPO antibodies. The inhibition was by 15–80% (mean 41%), and no other antibody showed any inhibition. Binding of hFabs to the B determinant on TPO was inhibited by anti-peptide 14 antibodies more then 85%, but not Fabs to the A determinant. In conclusion, the peptide 14 defines a sequence taking part in building up the B major conformational epitope.

None of generated anti-peptide antibodies alone inhibited the binding of human Fabs to the A epitope, however a combination of four anti-peptide antibodies (P1, P12, P14 and P18) inhibits Fabs binding to the A determinant by more then 60% and autoantibodies binding from 65% to 94%. Combination of antibodies reacting with peptides outside the surface defined by those four anti-peptide antibodies did not give any inhibition of Fabs to TPO.

The inhibition of Fabs and auto Abs to TPO by this combination of anti-peptide Abs is the result of steric hindrance as none of these Abs individually inhibited auto Abs' or Fabs' binding to TPO.

The four peptides define an area on the enzyme surface where the A and B major conformational epitopes are localised.     

Protein radical formation on thyroid peroxidase during turnover as detected by immuno-spin trapping

Thyroid peroxidase (TPO) is a 933 amino acid residue, heme-containing, integral membrane glycoprotein that catalyzes two steps in the maturation of the thyroid hormone precursor. As with other peroxidases, these reactions require hydrogen peroxide and initial enzyme oxidation. Previous researchers studied the oxidative state of the TPO heme moiety using spectrophotometric and catalytic analyses. We use a novel antiserum to 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to detect radical-derived DMPO spin-trapped TPO. Our work reveals that TPO generates radical adducts in the presence of H2O2, but that the generation of these adducts can be suppressed by the addition of substrates and inhibitors. Chemical alteration of the tyrosine residues of TPO greatly reduces the generation of TPO-DMPO adducts. Iodide strongly suppresses the H2O2-generated production of TPO radical adducts and protects the enzyme from loss of enzyme activity. Because the normal catalytic mechanism of TPO involves the production of radical species, TPO is potentially more susceptible to oxidative damage than most enzymes which do not require H2O2 as a substrate. We hypothesize that oxidatively damaged TPO may trigger the production of anti-TPO autoantibodies, resulting in the development of autoimmune thyroid disorders. Evidence that correlates iodine deficiencies with development of thyroid autoimmune disorders supports this conjecture.     

Search for the autoantibody immunodominant region on thyroid peroxidase: epitopic footprinting with a human monoclonal autoantibody locates a facet on the native antigen containing a highly conformational epitope

Autoantibodies to thyroid peroxidase (TPO) are the hallmark of the humoral autoimmune response in human autoimmune thyroiditis (Hashimoto's thyroiditis). The majority of TPO autoantibodies in individual patients' sera interact with a restricted immunodominant region on TPO. Although this region can be mapped, previous studies have failed to localize its position on the TPO molecule. We, therefore, used a footprinting approach that can localize a highly conformational, discontinuous epitope on a very large molecule. Extensive biotinylation ( approximately 15 biotins/molecule protein) of lysine residues on the surface of purified, native TPO resulted in loss of multiple tryptic cleavage sites, as determined by analysis of tryptic polypeptide fragments on reverse-phase HPLC. TPO was then complexed with a monoclonal human autoantibody Fab (TR1.9) before biotinylation. After dissociation from TR1.9, TPO was recovered by gel filtration. A trypsin site, previously observed to be lost after TPO biotinylation, was restored when biotinylation was performed on the TPO-TR1.9 complex. The epitope-protected lysine (K) was present in a 30-aa TPO fragment that, by N-terminal sequencing, was found to be K713. Altered recognition by TR1.9 of a TPO-myeloperoxidase chimeric molecule involving this region supported the epitope protection data. In conclusion, we provide the first identification of an amino acid residue (K713) comprising part of an epitope within the TPO immunodominant region. This focal residue localizes the facet on the large, highly complex TPO molecule that contains the immunodominant region and provides the basis for rational guided mutagenesis studies to more fully characterize this region.     

Alternatively spliced form of human thyroperoxidase, TPOzanelli: activity, intracellular trafficking, and role in hormonogenesis

Thyroperoxidase (TPO), a type I transmembrane heme containing glycoprotein, catalyzes iodide organification and thyroid hormone synthesis. One of the two main alternatively spliced forms of this enzyme, TPOzanelli, which is present in Graves's disease thyroid tissue, has a cytoplasmic domain completely modified. In the first stage of this study, the results of RT-PCR experiments showed that the TPOzanelli mRNA is present in normal thyroid tissue. We then generated CHO cell lines expressing the wild-type TPO (TPO1) and the alternatively spliced form TPOzanelli. Upon investigating a panel of 12 mAbs directed against the extracellular domain of TPO1 and sera from patients with a high titer of TPO autoantibodies, we observed that (i) the three-dimensional structure of this domain is similar in both isoforms; (ii) the autoantibodies recognize TPOzanelli as well as TPO1. The results of pulse chase and cell surface biotinylation experiments showed that the TPOzanelli has a shorter half-life (7 versus 11 h) and is expressed at the cell surface in lesser amounts than TPO1 (7 versus 15%). The total enzymatic activity and cell surface activity were determined in CHO cells expressing TPO1 and TPOzanelli, and TPO1 and TPOzanelli were found to have similar levels of activity. It was established that approximately 20% of the TPO purified from a Graves' disease thyroid gland was precipitated by polyclonal antibodies directed against a specific part of the cytoplasmic tail of TPOzanelli. This confirmed that the protein corresponding to the mRNA is present in the thyroid tissue. All in all, these results indicate that TPOzanelli can be expected to play a role in thyroid hormone synthesis and in thyroid autoimmunity.     

Domain-elongation NMR spectroscopy yields new insights into RNA dynamics and adaptive recognition

By simplifying the interpretation of nuclear magnetic resonance spin relaxation and residual dipolar couplings data, recent developments involving the elongation of RNA helices are providing new atomic insights into the dynamical properties that allow RNA structures to change functionally and adaptively. Domain elongation, in concert with spin relaxation measurements, has allowed the detailed characterization of a hierarchical network of local and collective motional modes occurring at nanosecond timescale that mirror the structural rearrangements that take place following adaptive recognition. The combination of domain elongation with residual dipolar coupling measurements has allowed the experimental three-dimensional visualization of very large amplitude rigid-body helix motions in HIV-1 transactivation response element (TAR) that trace out a highly choreographed trajectory in which the helices twist and bend in a correlated manner. The dynamic trajectory allows unbound TAR to sample many of its ligand bound conformations, indicating that adaptive recognition occurs by “conformational selection” rather than “induced fit.” These studies suggest that intrinsic flexibility plays essential roles directing RNA conformational changes along specific pathways.     

Characterization of thyroid follicular cell apical plasma membrane peroxidase using monoclonal antibody

Thyroid peroxidase (TPO) located in the apical plasma membrane of follicular cells was investigated by means of a membrane-immunofluorescent technique. The epitope of TPO recognized by a murine monoclonal antibody (mAb 30.1.2) was identified on the apical membrane surface. Trypsinization removed TPO immunoreactivity and enzymatic activity after 60 min of incubation at 37 degrees C. The epitope reappeared on the apical membrane surface after short term culture for 120 min without the addition of TSH. With TSH the time required for reappearance was only 30 min. TPO activity was regenerated under both conditions. Since dibutyryl cyclic AMP could not accelerate the reappearance of the epitope, it was thought that TPO reappearance is mediated by other than the adenylate cyclase-cyclic AMP system.     

Human autoantibodies modulate the T cell epitope repertoire but fail to unmask a pathogenic cryptic epitope

Abs can tune the responses of Ag-specific T cells by influencing the nature of the epitope repertoire displayed by APCs. We explored the interaction between human self-reactive T cells and human monoclonal autoantibodies from combinatorial Ig-gene libraries derived from autoimmune thyroiditis patients and specific for the main autoantigen thyroid peroxidase (TPO). All human mAbs extensively influenced the T cell epitope repertoire recognized by different TPO-specific T cell clones. The action of the human mAbs was complex, because sometimes the same Ab suppressed or enhanced the epitopes recognized by the 10 different TPO-specific T cell clones. The human mAbs could modulate the epitope repertoire when TPO was added exogenously and when expressed constitutively on the surface of APCs. However, they could not unmask an immunodominant cryptic TPO epitope. In this study, we show that human autoantibodies influence the activity of self-reactive T cells and prove their relevance in concealing or exposing epitopes recognized by self-reactive T cells. However, our results further stress the biological significance of the immunodominant cryptic epitope we have defined and its potential importance in the evolution of autoimmunity.     

Distinct immunological and biochemical properties of thyroid peroxidase purified from human thyroid glands and recombinant protein produced in insect cells.

The biosynthesis of thyroid hormone from thyroglobulin is catalysed by thyroid peroxidase (TPO), an integral membrane protein. TPO is also a major autoantigen in autoimmune thyroid disease and autoantibodies to TPO are markers for disease activity. Large quantities of purified TPO are essential for elucidating its structure and understanding its role in disease activity. We describe the high yield purification of full-length recombinant human TPO from baculovirus infected insect cells and compare it to purified native TPO from human thyroid glands. In contrast to native human TPO, the human TPO produced in insect cells as a recombinant protein was insoluble and resistant to solubilisation in detergents. Reversible substitution of lysine residues with citraconic anhydride led to increased solubility of the recombinant TPO, allowing high-yield purification by monoclonal antibody chromatography. The purified enzyme preparation was shown to be TPO by its reactivity with monoclonal and polyclonal antibodies by enzyme linked immunosorbent assay and Western blotting. Both the human and recombinant purified TPO preparations also react with sera from patients with autoimmune thyroid disease, although the binding of conformational dependent autoantibodies was considerably lower to the recombinant TPO than to the native TPO. This suggests that the recombinant TPO may differ in some aspects of its tertiary structure. The purified recombinant TPO was devoid of enzyme activity, in contrast to the enzymatically active, purified human TPO preparations. Both preparations contained comparable amounts of haem (R(z)=0.269), but a shift in the Soret band of recombinant TPO (402 nm) from that of natural TPO (409 nm) indicates that the lack of enzymatic activity of the recombinant enzyme may be due to changes in the protein backbone surrounding the haem. Both the purified native and recombinant TPO, under non-denaturing conditions, show evidence of high molecular mass oligomers, although the latter preparation is prone to a greater degree of aggregation. In conclusion, our studies indicate that recombinant TPO generated in insect cells is conformationally distinct from the native TPO, is insoluble and enzymatically inactive, consistent with the difficulties associated with its purification and crystallisation.     

Unique gating properties of C. elegans ClC anion channel splice variants are determined by altered CBS domain conformation and the R-helix linker

All eukaryotic and some prokaryotic ClC anion transport proteins have extensive cytoplasmic C-termini containing two cystathionine-β-synthase (CBS) domains. CBS domain secondary structure is highly conserved and consists of two α-helices and three β-strands arranged as β1-α1-β2-β3-α2. ClC CBS domain mutations cause muscle and bone disease and alter ClC gating. However, the precise functional roles of CBS domains and the structural bases by which they regulate ClC function are poorly understood. CLH-3a and CLH-3b are C. elegans ClC anion channel splice variants with strikingly different biophysical properties. Splice variation occurs at cytoplasmic N- and C-termini and includes several amino acids that form α2 of the second CBS domain (CBS2). We demonstrate that interchanging α2 between CLH-3a and CLH-3b interchanges their gating properties. The “R-helix” of ClC proteins forms part of the ion-conducting pore and selectivity filter and is connected to the cytoplasmic C-terminus via a short stretch of cytoplasmic amino acids termed the “R-helix linker”. C-terminus conformation changes could cause R-helix structural rearrangements via this linker. X-ray structures of three ClC protein cytoplasmic C-termini suggest that α2 of CBS2 and the R-helix linker could be closely apposed and may therefore interact. We found that mutating apposing amino acids in α2 and the R-helix linker of CLH-3b was sufficient to give rise to CLH-3a-LIKE gating. We postulate that the R-helix linker interacts with CBS2 α2, and that this putative interaction provides a pathway by which cytoplasmic C-terminus conformational changes induce conformational changes in membrane domains that in turn modulate ClC function.Key words: ClC channel, chloride channel, homology model     

Conformational Plasticity of the Essential Membrane-associated Mannosyltransferase PimA from Mycobacteria

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of peripheral membrane-associated GTs for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here we used single molecule force spectroscopy techniques to study the mechanical and conformational properties of PimA. In our studies, we engineered a polyprotein containing PimA flanked by four copies of the well characterized I27 protein, which provides an unambiguous mechanical fingerprint. We found that PimA exhibits weak mechanical stability albeit displaying β-sheet topology expected to unfold at much higher forces. Notably, PimA unfolds following heterogeneous multiple step mechanical unfolding pathways at low force akin to molten globule states. Interestingly, the ab initio low resolution envelopes obtained from small angle x-ray scattering of the unliganded PimA and the PimA·GDP complexed forms clearly demonstrate that not only the “open” and “closed” conformations of the GT-B enzyme are largely present in solution, but in addition, PimA experiences remarkable flexibility that undoubtedly corresponds to the N-terminal “Rossmann fold” domain, which has been proved to participate in protein-membrane interactions. Based on these results and on our previous experimental data, we propose a model wherein the conformational transitions are important for the mannosyltransferase to interact with the donor and acceptor substrates/membrane.     

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates

We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the “reaching dimer” previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition.