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Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in Escherichia coli, had unprecedentedly high binding affinities (Kd ∼10−12 m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.

Hepatitis B virus (HBV) is the leading cause of liver disease worldwide. While an adequate vaccine is available, current treatment options are limited, not highly effective, and associated with adverse effects, encouraging the development of alternative therapeutics. The HBV core gene encodes two different proteins: core, which forms the viral nucleocapsid, and pre-core, which serves as an immune modulator with multiple points of action. The two proteins mostly have the same sequence, although they differ at their N and C termini and in their dimeric arrangements. Previously, we engineered two human-framework antibody fragments (Fab/scFv) with nano- to picomolar affinities for both proteins. Here, by means of X-ray crystallography, analytical ultracentrifugation, and electron microscopy, we demonstrate that the antibodies have non-overlapping epitopes and effectively block biologically important assemblies of both proteins. These properties, together with the anticipated high tolerability and long half-lives of the antibodies, make them promising therapeutics.

Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP) is a principal component of the sarcomere. The three prevalent isoforms of ZASP in skeletal muscle are generated by alternative splicing of exons 9 and 10. The long isoforms, either having (ZASP-L) or lacking exon 10 (ZASP-LΔex10), include an N-terminal PDZ domain, an actin-binding region (ABR) with a conserved motif (ZM), and three C-terminal LIM domains. The short isoform (ZASP-S) lacks the LIM domains. Mutations, A147T and A165V, within the ZM of ZASP-LΔex10 cause myofibrillar myopathy, but the mechanism is unknown. We have prepared these proteins, their ABR, and the respective mutant variants in recombinant form, characterized them biophysically, and analyzed their actin-binding properties by surface plasmon resonance and electron microscopy. All the proteins were physically homogeneous and monomeric and had circular dichroic spectra consistent with partially folded conformations. Comparison of the NMR HSQC spectra of ZASP-S and the PDZ domain showed that the ABR is unstructured. ZASP-S and its mutant variants and ZASP-LΔex10 all bound to immobilized G-actin with high affinity (K(d) ≈ 10(−8) to 10(−9) M). Constructs of the isolated actin-binding region missing exon 10 (ABRΔ10) bound with lower affinity (K(d) ≈ 10(−7) M), but those retaining exon 10 (ABR+10) did so only weakly (K(d) ≈ 10(−5) M). ZASP-S, and the ABRΔ10, also induced F-actin and array formation, even in conditions of low ionic strength and in the absence of KCl and Mg(2+) ions. Interestingly, the ZM mutations A147T and A165V did not affect any of the results described above.

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-β, 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.)

The human lens proteins beta-crystallins are subdivided into acidic (betaA1-betaA4) and basic (betaB1-betaB3) subunit groups. These structural proteins exist at extremely high concentrations and associate into oligomers under physiological conditions. Crystallin acidic-basic pairs tend to form strong heteromolecular associations. The long N-terminal extensions of beta-crystallins may influence both homo- and heteromolecular interactions. However, identification of the critical regions of the extensions mediating protein associations has not been previously addressed. This was studied by comparing the self-association and heteromolecular associations of wild-type recombinant betaA3- and betaB1-crystallins and their N-terminally truncated counterparts (betaA3DeltaN30 and betaB1DeltaN56) using several biophysical techniques, including analytical ultracentrifugation and fluorescence spectroscopy. Removal of the N-terminal extension of betaA3 had no effect on dimerization or heteromolecular tetramer formation with betaB1. In contrast, the level of self-association of betaB1DeltaN56 increased, resulting in homotetramer formation, and heteromolecular association with betaA3 was blocked. Limited proteolysis of betaB1 produced betaB1DeltaN47, which is similar to intact protein formed dimers but in contrast showed enhanced heteromolecular tetramer formation with betaA3. The tryptic digestion was physiologically significant, corresponding to protease processing sites observed in vivo. Molecular modeling of the N-terminal betaB1 extension indicates structural features that position a mobile loop in the vicinity of these processing sites. The loop is derived from residues 48-56 which appear to be critical for mediating protein interactions with betaA3-crystallin.

MAP30 is a 30 kDa single-stranded, type-I ribosome inactivating protein (RIP) possessing anti-tumor and anti-HIV activities. It binds both ribosomal RNA and the HIV-1 long-terminal repeat DNA. To understand the structural basis for MAP30 activities, we undertook the study of MAP30 by solution NMR spectroscopy. We report nearly complete 1H, 13C, and 15N chemical shift assignments of its 263 amino acids. Based upon an analysis of secondary 13C chemical shifts, 3J(HNHA) coupling constants, hydrogen exchange data, and nuclear Overhauser effect patterns, we find that the secondary structure and beta-sheet topology of MAP30 are very similar to those of the ricin A chain, a subunit of the well-known type-II RIP, even though two proteins display distinct activities. We therefore suggest that MAP30 and ricin A chain share a similar three-dimensional fold, and that the reported functional differences between two proteins arise primarily from differences in local three-dimensional structure and other structural properties such as surface electrostatic potentials.

We present the solution structure of MAP30, a plant protein with anti-HIV and anti-tumor activities. Structural analysis and subsequent biochemical assays lead to several novel discoveries. First, MAP30 acts like a DNA glycosylase/apurinic (ap) lyase, an additional activity distinct from its known RNA N-glycosidase activity toward the 28S rRNA. Glycosylase/ap lyase activity explains MAP30's apparent inhibition of the HIV-1 integrase, MAP30's ability to irreversibly relax supercoiled DNA, and may be an alternative cytotoxic pathway that contributes to MAP30's anti-HIV/anti-tumor activities. Second, two distinct, but contiguous, subsites are responsible for MAP30's glycosylase/ap lyase activity. Third, Mn2+ and Zn2+ interact with negatively charged surfaces next to the catalytic sites, facilitating DNA substrate binding instead of directly participating in catalysis.

Matrix metalloproteinases (MMPs) function in the remodeling of the extracellular matrix that is integral for many normal and pathological processes. The tissue inhibitor of metalloproteinases family, including tissue inhibitor of metalloproteinases-2 (TIMP-2), regulates the activity of these multifunctional metalloproteinases. TIMP family members are proteinase inhibitors that contain six conserved disulfide bonds, one involving an amino-terminal cysteine residue that is critical for MMP inhibitor activity. TIMP-2 has been expressed in Escherichia coli, folded from insoluble protein, and functionally characterized. The wild type protein inhibited gelatinase A (MMP-2), whereas a variant with an alanine appended to the amino terminus (Ala+TIMP-2) was inactive. Removal of amino-terminal alanine by exopeptidase digestion restored protease inhibitor activity. This confirms the mechanistic importance of the amino-terminal amino group in the metalloproteinase inhibitory activity, as originally suggested from the x-ray structure of a complex of MMP-3 with TIMP-1 and a complex of TIMP-2 with MT-1-MMP. The Ala+TIMP-2 variant exhibited conformational, pro-MMP-2 complex formation and fibroblast growth modulating properties of the wild type protein. These findings demonstrate that Ala+TIMP-2 is an excellent biochemical tool for examining the specific role of MMP inhibition in the multiple functions ascribed to TIMPs.

GRL007 and GRL008, two structurally related nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) containing 3( R ),3a( S ),6a( R )- bis -tetrahydrofuranylurethane ( bis -THF) as the P2 moiety and a sulfonamide isostere consisting of benzene carboxylic acid and benzene carboxamide as the P2′ moiety, respectively, were evaluated for their antiviral activity and interactions with wild-type protease (PR WT ). Both GRL007 ( K i of 12.7 pM with PR WT ) and GRL008 ( K i of 8.9 pM) inhibited PR WT with high potency in vitro . X-ray crystallographic analysis of PR WT in complex with GRL007 or GRL008 showed that the bis -THF moiety of both compounds has three direct polar contacts with the backbone amide nitrogen atoms of Asp29 and Asp30 of PR WT . The P2′ moiety of both compounds showed one direct contact with the backbone of Asp30′ and a bridging polar contact with Gly48′ through a water molecule. Cell-based antiviral assays showed that GRL007 was inactive (50% effective concentration [EC 50 ] of >1 μM) while GRL008 was highly active (EC 50 of 0.04 μM) against wild-type HIV-1. High-performance liquid chromatography (HPLC)/mass spectrometry-based cellular uptake assays showed 8.1- and 84-fold higher intracellular concentrations of GRL008 than GRL007 in human MT-2 and MT-4 cell extracts, respectively. Thus, GRL007, in spite of its favorable enzyme-inhibitory activity and protease binding profile, exhibited a lack of antiviral activity in cell-based assays, most likely due to its compromised cellular uptake associated with its P2′ benzene carboxylic acid moiety. The anti-HIV-1 potency, favorable toxicity, and binding profile of GRL008 suggest that further optimization of the P2′ moiety may improve its antiretroviral features.

High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm; however, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates by low speed centrifugation. Following preextaction (or washing) protein is extracted from washed pellets using guanidine·HCl. The solubilized and unfolded protein is either directly folded as described in UNIT 6.1 or further purified by gel filtration in the presence of guanidine·HCl as described here. A support protocol describes the removal of guanidine·HCl from column fractions so they can be monitored by SDS-PAGE.

Pigment epithelium-derived factor (PEDF) is a neurotrophic protein present in low amounts in conditioned medium of cultured fetal human retinal pigment epithelial cells. Recently, the PEDF cDNA has been cloned from a fetal human cDNA library, and its derived amino acid sequence identified it as a member of the serine protease inhibitor (serpin) supergene family (Steele, F. R., Chader, G. J., Johnson, L. V., and Tombran-Tink, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1526-1530). We have prepared recombinant expression constructs from the fetal human PEDF cDNA and obtained milligram amounts of biologically active PEDF from Escherichia coli. The full-length open reading frame (Met1-Pro418) and a truncated form (Asp44-Pro418) were used in our constructs. Induction from a vector containing the truncated PEDF version, named pEV-BH, produced a protein (BH) of expected size (M(r) 42,800) associated with inclusion bodies, which contained 25-40% of expressed protein. After solubilization, BH was highly purified by gel filtration and cation exchange chromatography. The NH2-terminal sequence of the purified protein matched that of the pEV-BH construct. We have conducted neurite outgrowth assays in a human retinoblastoma Y-79 cell culture system. Recombinant PEDF (BH) demonstrated neurotrophic activity, as reported for the native PEDF. Thus, unfolded and refolded in vitro BH retained a potent biological activity. In parallel experiments, protease inhibition assays were performed. Recombinant PEDF did not have an effect on trypsin, chymotrypsin, elastase, cathepsin G, endoproteinase Lys-C, endoproteinase Glu-C, or subtilisin activity, suggesting that inhibition of known serine proteases is not the biochemical pathway for the PEDF neutrophic activity.

Mediator is a multisubunit coactivator required for initiation by RNA polymerase II. The Mediator tail subdomain, containing Med15/Gal11, is a target of the activator Gcn4 in vivo, critical for recruitment of native Mediator or the Mediator tail subdomain present in sin4Delta cells. Although several Gal11 segments were previously shown to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We show that interaction of Gcn4 with the Mediator tail in vitro and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo involve additive contributions from three different segments in the N terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we establish that B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrate that Gcn4 binds to the Gal11 KIX domain directly and, by NMR chemical shift analysis combined with mutational studies, we identify the likely binding site for Gcn4 on the KIX surface. Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator in vivo.

Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion bodies. To obtain the native (i.e., correctly folded) and hence active form of the protein from such aggregates, four steps are usually followed: (1) the cells are lysed and the are aggregates, (2) the cell wall and outer membrane components of the aggregates are removed, (3) the aggregates are solubilized (or extracted) with strong protein denaturants, and (4) the solubilized, denatured proteins are folded with concomitant oxidation of reduced cysteine residues into the correct disulfide bonds to obtain the native protein. This unit features three different approaches to the final step of protein folding and purification. In the first, guanidine HCl is used as the denaturant, after which the solubilized protein is folded (before purification) in an "oxido-shuffling" buffer system to increase the rate of protein oxidation. In the second, acetic acid is used to solubilize the protein which is then partially purified by gel filtration before folding, and then the protein is folded and oxidized by simple dialyzed against water. A Support Protocol is included for rapidly determining the amount of folded protein that contains the correct disulfide linkage pattern. Finally, folding and purification of a fusion protein is described using metal-chelate affinity chromatography.

The integrase protein of human immunodeficiency virus type 1 carries out a set of polynucleotidyl transfer reactions that result in the covalent attachment of the retroviral cDNA to host DNA. We have analyzed the activities of a set of deletion derivatives of the integrase protein. The analysis reveals that a central domain of only 137 amino acids is sufficient in vitro to catalyze a subset of the reactions carried out by the complete protein. This polypeptide contains an amino acid sequence motif, Asp-Xaa39-58-Asp-Xaa35-Glu (DX39-58DX35E, where X and the subscript indicate the intervening amino acids between the invariant acidic residues), that is found in the integrases of retroviruses and retrotransposons and also the transposase proteins of some bacterial transposable elements. We also find that the integrase protein can bind Zn2+, and the histidine and cysteine residues of another conserved motif (HX3-7HX23-32CX2C) are required for efficient Zn2+ binding. The activities displayed by deletion mutants suggest to us possible functions for the various parts of integrase.