Mutations in lambda repressor''s amino-terminal domain: implications for protein stability and DNA binding.

The DNA binding properties of 52 different single-amino acid substitutions in lambda repressor's amino-terminal domain have been characterized. Seven proteins bearing mutations that change solvent-exposed side chains have been purified. The amino-terminal domains of these mutant repressors are folded and are comparable to the wild-type amino-terminal domain in thermal stability. In contrast, a purified mutant repressor bearing a substitution in a buried side chain contains an amino-terminal domain with decreased thermal stability. We argue that mutations that alter solvent-exposed wild-type side chains define residues that form the operator DNA binding surface of lambda repressor whereas completely or partially buried mutations exert their effect by decreasing protein stability.     

The solution structure of the Raf-1 cysteine-rich domain: a novel ras and phospholipid binding site.

The Raf-1 protein kinase is the best-characterized downstream effector of activated Ras. Interaction with Ras leads to Raf-1 activation and results in transduction of cell growth and differentiation signals. The details of Raf-1 activation are unclear, but our characterization of a second Ras-binding site in the cysteine-rich domain (CRD) and the involvement of both Ras-binding sites in effective Raf-1-mediated transformation provides insight into the molecular aspects and consequences of Ras-Raf interactions. The Raf-1 CRD is a member of an emerging family of domains, many of which are found within signal transducing proteins. Several contain binding sites for diacylglycerol (or phorbol esters) and phosphatidylserine and are believed to play a role in membrane translocation and enzyme activation. The CRD from Raf-1 does not bind diacylglycerol but interacts with Ras and phosphatidylserine. To investigate the ligand-binding specificities associated with CRDs, we have determined the solution structure of the Raf-1 CRD using heteronuclear multidimensional NMR. We show that there are differences between this structure and the structures of two related domains from protein kinase C (PKC). The differences are confined to regions of the CRDs involved in binding phorbol ester in the PKC domains. Since phosphatidylserine is a common ligand, we expect its binding site to be located in regions where the structures of the Raf-1 and PKC domains are similar. The structure of the Raf-1 CRD represents an example of this family of domains that does not bind diacylglycerol and provides a framework for investigating its interactions with other molecules.     

Three-dimensional structure of the amino-terminal domain of syntaxin 6, a SNAP-25 C homolog

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are required for intracellular membrane fusion, and are differentially localized throughout the cell. SNAREs on vesicle and target membranes contain "SNARE motifs" which interact to form a four-helix bundle that contributes to the fusion of two membranes. SNARE motif sequences fall into four classes, homologous to the neuronal proteins syntaxin 1a, VAMP 2, and the N- and C-terminal SNARE motifs of SNAP-25 (S25N and S25C), and it is thought that one member from each class interacts to form a SNARE complex. Many SNAREs also feature N-terminal domains believed to function in regulating SNARE complex assembly or other aspects of vesicle transport. Syntaxin 6 is a SNARE found primarily in endosomal transport vesicles and whose SNARE motif shows significant homology to both syntaxin 1a and S25C. The crystal structure of the syntaxin 6 N-terminal domain reveals strong structural similarity with the N-terminal domains of syntaxin family members syntaxin 1a, Sso1p, and Vam3p, despite a very low level of sequence similarity. The syntaxin 6 SNARE motif can substitute for S25C in in vitro binding experiments, supporting the classification of syntaxin 6 as an S25C family member. Secondary structure prediction of SNARE proteins shows that the N-terminal domains of many syntaxin, S25N, and S25C family members are likely to be similar to one another, but are distinct from those of VAMP family members, indicating that syntaxin, S25N, and S25C SNAREs may have shared a common ancestor.     

Receptor-binding domain of SARS-Cov spike protein: Soluble expression in purification and functional characterization

AIM: To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus (SARS-Cov), and to analyze its receptor binding ability.METHODS: Three fusion tags (glutathione S-transferase,GST; thioredoxin, Trx; maltose-binding protein, MBP),which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli( BL21( DE3 ) and Rosetta-gamiB(DE3) strains). The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay.RESULTS: RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 (DE3) and Rosetta-gamiB (DE3) under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21(DE3), and the protein was purified by AKTA Prime Chromatography system. The ELISA data anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD's binding ability to ACE2 (angiotensin-converting enzyme 2)positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and respectively.CONCLUSION: In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in E. coli BL21(DE3); data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E. coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.     

Proposed mechanism of bacteriophage lambda induction: acquisition of binding sites for lambda repressor by DNA of the host.

Interference with the in vitro binding of lambda phage repressor to lambda operator DNA was observed when Escherichia coli DNA containing the following lesions was present in the reaction mixture: (a) DNA with single-strand breaks from pancreatic DNase (nicked DNA); (B) DNA isolated from thymine-straved cells; (c) DNA from ultraviolet-treated cells; (d) DNA of mitomycin-treated cells; and (e) DNA from a temperature-sensitive ligase mutant after 1 hr at 42 degrees. Normal E. coli DNA did not interfere. Binding of lambda cIing-minus repressor to operator DNA was not affected by E. coli DNA with lesions. DNAs from cells treated with increasing doses of mitomycin were proportionately more effective in competition for repressor, suggesting increasing binding sites per unit of DNA. A general model of virus induction is proposed, based on binding affinity of ultraviolet-sensitive repressors for single-strand breaks in the host DNA. The model is extended also to the presumptive repressor of cell division.     

Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

The autocrine/paracrine peptide signaling molecules such as growth factors have many promising biologic activities for clinical applications. However, one cannot expect specific therapeutic effects of the factors administered by ordinary drug delivery systems as they have limited target specificity and short half-lives in vivo. To overcome the difficulties in using growth factors as therapeutic agents, we have produced fusion proteins consisting of growth factor moieties and a collagen-binding domain (CBD) derived from Clostridium histolyticum collagenase. The fusion proteins carrying the epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) at the N terminal of CBD (CBEGF/CBFGF) tightly bound to insoluble collagen and stimulated the growth of BALB/c 3T3 fibroblasts as much as the unfused counterparts. CBEGF, when injected subcutaneously into nude mice, remained at the sites of injection for up to 10 days, whereas EGF was not detectable 24 h after injection. Although CBEGF did not exert a growth-promoting effect in vivo, CBFGF, but not bFGF, strongly stimulated the DNA synthesis in stromal cells at 5 days and 7 days after injection. These results indicate that CBD may be used as an anchoring unit to produce fusion proteins nondiffusible and long-lasting in vivo.     

Structural and functional characterization of the pore-forming domain of pinholin S2168

Pinholin S²¹68 triggers the lytic cycle of bacteriophage φ21 in infected Escherichia coli. Activated transmembrane dimers oligomerize into small holes and uncouple the proton gradient. Transmembrane domain 1 (TMD1) regulates this activity, while TMD2 is postulated to form the actual “pinholes.” Focusing on the TMD2 fragment, we used synchrotron radiation-based circular dichroism to confirm its α-helical conformation and transmembrane alignment. Solid-state ¹⁵N-NMR in oriented DMPC bilayers yielded a helix tilt angle of τ = 14°, a high order parameter (S_mol = 0.9), and revealed the azimuthal angle. The resulting rotational orientation places an extended glycine zipper motif (G₄₀xxxS₄₄xxxG₄₈) together with a patch of H-bonding residues (T₅₁, T₅₄, N₅₅) sideways along TMD2, available for helix–helix interactions. Using fluorescence vesicle leakage assays, we demonstrate that TMD2 forms stable holes with an estimated diameter of 2 nm, as long as the glycine zipper motif remains intact. Based on our experimental data, we suggest structural models for the oligomeric pinhole (right-handed heptameric TMD2 bundle), for the active dimer (right-handed Gly-zipped TMD2/TMD2 dimer), and for the full-length pinholin protein before being triggered (Gly-zipped TMD2/TMD1-TMD1/TMD2 dimer in a line).

Upon infecting a host, bacteriophages release their newly produced offspring into the environment by lysis of the host cell. The infection cycle of double-stranded DNA bacteriophages that infect gram-negative bacteria is regulated by small viral membrane proteins, so-called holins (1–3). These holins accumulate in an inactive form in the hosts’ cytoplasmic membrane during phage morphogenesis (3). At an allele-specific time, the holins start to form big membrane lesions. Through these holes the muralytic endolysins escape from the cytoplasm and start to degrade the peptidoglycane layer (4, 5). Host lysis is then completed by the Rz–Rz1 spanin complex that disintegrates the outer membrane (6). In addition to this canonical holin endolysin system, a second class of holins, referred to as pinholins, has been described (3, 7). This class is represented by pinholin S²¹68 of lambdoid phage φ21 (7, 8). In contrast to canonical holins, for example those encoded by phage λ, the holes formed by pinholins are much smaller (“pinholes”) and do not allow the passage of proteins (7, 9).The pinholin S²¹68 protein is encoded by gene S²¹ of the lysis cassette, which possesses a dual start motif to allow expression of an antipinholin as well. This antipinholin protein has three additional N-terminal amino acids (M₁-K₂-S₃) and serves as a specific inhibitor of pinholin (Fig. 1A) (8, 10). The (anti)pinholin protein structure consists of two transmembrane domains (TMDs) and an unstructured C terminus (8). It is postulated that TMD1 possesses regulatory functions, while TMD2 is responsible for the actual pinhole formation (8, 11–13). During phage morphogenesis, (anti)pinholin initially accumulates as inactive heterodimers in the cytoplasmic membrane. For pinhole formation, TMD1 has to flip out of the membrane to allow TMD2–TMD2 interactions that build up the pinhole (Fig. 1B) (8, 14). The presence of antipinholin reduces the pinholin activity, because the additional charge on the N terminus delays the flipping of TMD1 (8, 11). Previous cross-linking and modeling studies by Pang et al. (9) have suggested that the pinholes are composed of seven pinholin S²¹68 monomers. These heptamers are supposed to form small holes with an inner diameter of around 1.5 nm, which lead to a collapse of the proton gradient across the membrane (9).Open in a separate windowFig. 1.Pinholin S²¹68. (A) Primary structure of the (anti)pinholin. The dual start motif of gene S²¹ of phage φ21 allows the expression of both antipinholin S²¹71 and pinholin S²¹68. The postulated TMDs are indicated, and our synthetic TMD2 fragment (W₃₆-E₆₂) is underlined. (B) In its inactive state, both helices of pinholin S²¹68 are supposed to be aligned in a transmembrane state (Left). Above a certain threshold concentration, TMD1 is supposed to flip out of the membrane into a surface bound state (Right) (14). This triggering allows further self-assembly through TMD2–TMD2 interactions and leads to the formation of a small pinhole pore by a putative heptamer (9, 13).The machinery also contains associated endolysins carrying an N-terminal secretory signal, called the signal-anchor release (SAR) domain. In their membrane-tethered state, the SAR-endolysins are enzymatically inactive, which prevents premature lysis (15, 16). This inactive state is highly dependent on the presence of an intact membrane potential (7, 17). After disruption of this potential by the formation of pinholes, the SAR-endolysins are released from the membrane, and become refolded and activated during this process (7, 16).Drew et al. (18) demonstrated recently that the wild-type protein is predominately α-helical (∼83%) in DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine; di-14:0-PC) vesicles. Ahammad et al. (14) from the same group used electron paramagnetic resonance (EPR) experiments on synthetic reconstituted pinholin to show that TMD1 is partly externalized from the lipid, whereas TMD2 is stably membrane inserted. In the present study, we aimed to determine the detailed membrane orientation of the postulated pore-forming TMD2 fragment and examine its putative role as the basic homo-/hetero-/oligomeric assembly unit. We used synchrotron radiation-based circular dichroism spectroscopy (SRCD) for quantitative secondary structure determination, and oriented CD (SROCD) for qualitative orientational analysis. Accurate parameters on the helix alignment were obtained by solid-state NMR spectroscopy (ssNMR) on nonperturbing selective ¹⁵N-isotope labels, which yielded a detailed picture of the TMD2 as it is positioned in the membrane. Using a vesicle leakage assay based on ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt)/DPX (p-xylene-bis-pyridinium bromide), we proved that TMD2 is indeed the pore-forming domain and could estimate the size of the pinhole from the leakage of fluorescein-isothiocyanate-dextrans (FITC-dextrans, FDs). Based on these experimental data, we have constructed putative models for the pinholin in all three stages of its functional cycle.     

Expression of the functional extracellular domain of human thyrotropin receptor using a vaccinia virus system: its purification and analysis of autoantibody binding

We produced substantial amount of the extracellular domain of the human TSH receptor (TSHRE) that has a tag of six histidines at C-terminus as a soluble form in the human cell line HeLa using a vaccinia virus system. By sequential nickel-chelating and lentil lectin column chromatography, TSHRE was purified to about 70% purity, with the recovery of around 0.1-0.2 mg TSHRE/L culture (5 x 10(8) cells/liter culture). The purified TSHRE interacted with TSH as well as Graves' autoantibodies to TSHR. However, the affinity of TSHRE for TSH was much lower than that of intact TSHR. The IC50 value for inhibition of TSH-dependent cAMP synthesis by TSHRE was about 10(-8) mol/L. Most importantly, the purified TSHRE inhibited the binding of the IgG of Graves' patients to thyroid membrane. About 1 microg/mL (2 x 10(-8) mol/L) TSHRE neutralized most of the autoantibody activity of patients' sera tested in the TSH binding inhibitory immunoglobulin (TBII) assay. Moreover, this protein neutralized thyroid stimulatory antibody-induced cAMP synthesis with an IC50 of 1 x 10(-9) mol/L and completely at 0.5-1 microg/mL (1-2 x 10(-8) mol/L). In the simple enzyme-linked immunosorbent assay, the TSHRE immobilized on the wells coated with nickel showed significantly higher binding with the IgGs from Graves' patients than in those from normal individuals. This autoantibody-reactive TSHRE will be useful for further studies on the diagnosis, pathogenesis, and the development of therapy of Graves' disease.     

Molecular and functional characterization of sex hormone binding globulin in zebrafish

SHBG (sex hormone binding globulin) transports androgens and estrogens in the blood of vertebrates including fish. Orthologs of SHBG in fish are poorly defined, and we have now obtained a zebrafish SHBG cDNA and characterized the zebrafish SHBG gene and protein through molecular biological, biochemical, and informatics approaches. Amino-terminal analysis of zebrafish SHBG indicated that its deduced precursor sequence includes a 25-residue secretion polypeptide and exhibits 22-27% homology with mammalian SHBG sequences and 41% with a deduced fugufish SHBG sequence. The 356-residue mature zebrafish SHBG (39,243 Da) sequence comprises a tandem repeat of laminin G-like domains typical of SHBG sequences; contains three N-glycosylation sites; and exists as a 105,000 +/- 8700 Da homodimer. Zebrafish SHBG exhibits a high affinity and specificity for sex steroids. An RT-PCR indicated that SHBG mRNA first appears in zebrafish larva, and SHBG mRNA was localized within the liver and gut at this stage of development by whole-mount in situ hybridization. In adult fish, SHBG mRNA was found in liver, testis, and gut. In the liver, immunoreactive SHBG was present in hepatocytes and concentrated in intrahepatic bile duct cells, whereas in the testis it was confined to cells surrounding the seminiferous tubule cysts. In the intestine, immunoreactive SHBG was present in the stroma and epithelial cells of the villous projections and the surrounding muscle. The production and presence of SHBG in the gut of developing and adult zebrafish suggests a novel role for this protein in regulating sex steroid action at this site.     

Deubiquitinating function of ataxin-3: insights from the solution structure of the Josephin domain

Spinocerebellar ataxia type 3 is a human neurodegenerative disease resulting from polyglutamine tract expansion. The affected protein, ataxin-3, which contains an N-terminal Josephin domain followed by tandem ubiquitin (Ub)-interacting motifs (UIMs) and a polyglutamine stretch, has been implicated in the function of the Ub proteasome system. NMR-based structural analysis has now revealed that the Josephin domain binds Ub and has a papain-like fold that is reminiscent of that of other deubiquitinases, despite primary sequence divergence but consistent with its deubiqutinating activity. Mutation of the catalytic Cys enhances the stability of a complex between ataxin-3 and polyubiquitinated proteins. This effect depends on the integrity of the UIM region, suggesting that the UIMs are bound to the substrate polyubiquitin during catalysis. We propose that ataxin-3 functions as a polyubiquitin chain-editing enzyme.     

Secondary structure of Src homology 2 domain of c-Abl by heteronuclear NMR spectroscopy in solution.

The Src homology 2 (SH2) domain is a recognition motif thought to mediate the association of the cytoplasmic proteins involved in signal transduction by binding to phosphotyrosyl-containing sequences in proteins. Assignments of nearly all 1H and 15N resonances of the SH2 domain from the c-Abl protein-tyrosine kinase have been obtained from homonuclear and heteronuclear NMR experiments. The secondary structure has been elucidated from the pattern of nuclear Overhauser effects, from vicinal coupling constants, and from observation of slowly exchanging amino hydrogens. The secondary structure contains two alpha-helices and eight beta-strands, six of which are arranged in two contiguous, antiparallel beta-sheets. Residues believed to be involved in phosphotyrosyl ligand binding are on a face of one beta-sheet. The alignment of homologous sequences on the basis of secondary structure suggests a conserved global fold in a family of SH2 domains.     

Solution structure of the Zbeta domain of human DNA-dependent activator of IFN-regulatory factors and its binding modes to B- and Z-DNAs

The DNA-dependent activator of IFN-regulatory factors (DAI), also known as DLM-1/ZBP1, initiates an innate immune response by binding to foreign DNAs in the cytosol. For full activation of the immune response, three DNA binding domains at the N terminus are required: two Z-DNA binding domains (ZBDs), Zα and Zβ, and an adjacent putative B-DNA binding domain. The crystal structure of the Zβ domain of human DAI (hZβ(DAI)) in complex with Z-DNA revealed structural features distinct from other known Z-DNA binding proteins, and it was classified as a group II ZBD. To gain structural insights into the DNA binding mechanism of hZβ(DAI), the solution structure of the free hZβ(DAI) was solved, and its bindings to B- and Z-DNAs were analyzed by NMR spectroscopy. Compared to the Z-DNA-bound structure, the conformation of free hZβ(DAI) has notable alterations in the α3 recognition helix, the "wing," and Y145, which are critical in Z-DNA recognition. Unlike some other Zα domains, hZβ(DAI) appears to have conformational flexibility, and structural adaptation is required for Z-DNA binding. Chemical-shift perturbation experiments revealed that hZβ(DAI) also binds weakly to B-DNA via a different binding mode. The C-terminal domain of DAI is reported to undergo a conformational change on B-DNA binding; thus, it is possible that these changes are correlated. During the innate immune response, hZβ(DAI) is likely to play an active role in binding to DNAs in both B and Z conformations in the recognition of foreign DNAs.     

Separate sites for binding and nicking of bacteriophage lambda DNA by terminase.

The cohesive end site (cos) is the site of action of bacteriophage lambda terminase, the enzyme that introduces staggered nicks to generate the 12-base cohesive ends of mature lambda DNA. Deletion mutations that remove the lambda cohesive end sequence have been isolated after in vitro mutagenesis. The deletions were obtained by digesting the DNA of a cos duplication phage with S1 nuclease to remove the cohesive ends and adjacent base pairs, followed by blunt end ligation and DNA packaging into phage particles. cos2 is the result of a 22-base-pair deletion that exactly removes the segment of rotational symmetry that includes the cohesive end sequence. The cos2 mutation abolishes nicking by terminase but does not affect terminase binding. We conclude that cos contains two sites that interact with terminase: cosN, the nicking site; and cosB, a binding site for terminase.     

Human lambda light-chain constant region gene CMor lambda: the primary structure of lambda VI Bence Jones protein Mor.

Serologic, structural, and genetic analyses have shown that the constant (C) region of human kappa light chains is encoded by a single gene, whereas that of lambda chains is encoded by multiple genes. We have determined the complete C region amino acid sequence of two monoclonal lambda VI light chains, Bence Jones proteins Sut and Mor. The C region of lambda chains Sut and Mor consists of 105 residues, as is characteristic for human lambda light chains, of which 102 are identical in sequence. Protein Sut has the C region sequence associated with the C lambda isotype Mcg-, Kern-, Oz+ and represents a product of the C lambda 3 (Kern-, Oz+) gene. Protein Mor has a C region sequence associated with Mcg-, Kern-, and Oz- proteins but differs from protein Sut by the presence of three amino acid interchanges at positions 168, 176, and 194. These substitutions distinguish protein Mor from lambda chains encoded by the C lambda 1 (Mcg+), C lambda 2 (Kern-, Oz-), and C lambda 3 (Kern-, Oz+) genes and provide further evidence for polymorphism of the human C lambda genome. The gene encoding the C region sequence of lambda chain Mor is designated CMor lambda.     

Complete androgen insensitivity syndrome caused by a novel mutation in the ligand-binding domain of the androgen receptor: functional characterization

Mutations in the X-linked androgen receptor (AR) gene cause the androgen insensitivity syndrome by impairing androgen-dependent male sexual differentiation to varying degrees. Complete androgen insensitivity (CAIS) yields an external female phenotype, whereas affected cases of partial androgen insensitivity have various ambiguities of the genitalia. Here we describe a 46,XY phenotypically female patient with all of the characteristics of CAIS, i.e. primary amenorrhea, no axillary or pubic hair, female external genitalia, no uterus, and undescended testes. Defects in testosterone and dihydrotestosterone synthesis were excluded. The molecular basis of the disease was clarified by means of direct sequencing of PCR-amplified exonic fragments of the AR gene. An A to C transition in exon 4 of the AR gene led to a novel missense His(689)Pro mutation in the ligand-binding domain of the AR protein. Functional studies demonstrated that the mutated AR is unable to efficiently bind its natural ligand dihydrotestosterone and to trans-activate known androgen response elements. Analysis of the structural consequences of the His(689)Pro substitution suggests that this mutation is likely to perturb the conformation of the second helix of the AR ligand-binding domain, which contains residues critical for androgen binding.