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.     

Dissociation of the CD4 and CXCR4 binding properties of human immunodeficiency virus type 1 gp120 by deletion of the first putative alpha-helical conserved structure

To evaluate conserved structures of the surface gp120 subunit (SU) of the human immunodeficiency virus type 1 (HIV-1) envelope in gp120-cell interactions, we designed and produced an HIV-1 IIIB (HXB2R) gp120 carrying a deletion of amino acids E61 to S85. This sequence corresponds to a highly conserved predicted amphipathic alpha-helical structure located in the gp120 C1 region. The resultant soluble mutant with a deleted alpha helix 1 (gp120 DeltaalphaHX1) exhibited a strong interaction with CXCR4, although CD4 binding was undetectable. The former interaction was specific since it inhibited the binding of the anti-CXCR4 monoclonal antibody (12G5), as well as SDF1alpha, the natural ligand of CXCR4. Additionally, the mutant gp120 was able to bind to CXCR4(+)/CD4(-) cells but not to CXCR4(-)/CD4(-) cells. Although efficiently expressed on cell surface, HIV envelope harboring the deleted gp120 DeltaalphaHX1 associated with wild-type transmembrane gp41 was unable to induce cell-to-cell fusion with HeLa CD4(+) cells. Nevertheless, the soluble gp120 DeltaalphaHX1 efficiently inhibited a single round of HIV-1 LAI infection in HeLa P4 cells, with a 50% inhibitory concentration of 100 nM. Our data demonstrate that interaction with the CXCR4 coreceptor was maintained in a SUgp120 HIV envelope lacking alphaHX1. Moreover, in the absence of CD4 binding, the interaction of gp120 DeltaalphaHX1 with CXCR4 was sufficient to inhibit HIV-1 infection.     

Human immunodeficiency virus type 1 and 2 envelope glycoproteins oligomerize through conserved sequences

Hetero-oligomerization between human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (Env) truncation mutants and epitope-tagged gp160 is dependent on the presence of gp41 transmembrane protein (TM) amino acids 552 to 589, a putative amphipathic alpha-helical sequence. HIV-2 Env truncation mutants containing this sequence were also able to form cross-type hetero-oligomers with HIV-1 Env. HIV-2/HIV-1 hetero-oligomerization was, however, more sensitive to disruption by mutagenesis or increased temperature. The conservation of the Env oligomerization function of the HIV-1 and HIV-2 alpha-helical sequences suggests that retroviral TM alpha-helical motifs may have a universal role in oligomerization.     

Antigenic implications of human immunodeficiency virus type 1 envelope quaternary structure: oligomer-specific and -sensitive monoclonal antibodies

A majority of monoclonal antibodies (mAbs) raised against soluble oligomeric human immunodeficiency virus type 1 isolate IIIB (HIV-1IIIB) envelope (env) glycoprotein reacted with conformational epitopes within the gp120 or gp41 subunits. Of 35 mAbs directed against gp41, 21 preferentially reacted with oligomeric env. A subset of these mAbs reacted only with env oligomers (oligomer-specific mAbs). In contrast, only 1 of 27 mAbs directed against the gp120 subunit reacted more strongly with env oligomers than with monomers, and none were oligomer-specific. However, 50% of anti-gp120 mAbs preferentially recognized monomeric env, suggesting that some epitopes in gp120 are partially masked or altered by intersubunit contacts in the native env oligomer. Two mAbs to oligomer-dependent epitopes in gp41 neutralized HIV-1IIIB and HIV-1SF2, and binding of these mAbs to env was blocked by preincubation with HIV-1-positive human serum. Thus, immunization with soluble, oligomeric env elicits antibodies to conserved, conformational epitopes including a newly defined class of neutralizing antibodies that bind to oligomer-specific epitopes in gp41, and may also minimize the production of antibodies that preferentially react with monomeric env protein.     

Increased incorporation of chimeric human immunodeficiency virus type 1 gp120 proteins into Pr55gag virus-like particles by an Epstein-Barr virus gp220/350-derived transmembrane domain

Noninfectious Pr55gag virus-like particles containing high quantities of oligomeric human immunodeficiency virus type 1 (HIV-1) envelope (Env) proteins represent potential candidate immunogens for a vaccine against HIV-1 infection. Thus, chimeric env genes were constructed encoding the HIV-1 exterior glycoprotein gp120 which was covalently linked at different C-terminal positions to a transmembrane domain (TM) from the Epstein-Barr virus (EBV) major Env glycoprotein gp220/ 350. All chimeric Env-TM polypeptides as well as the wild-type HIV Env proteins were equally produced and incorporated at the outer surface of insect cells using the baculovirus expression system. In the presence of coexpressed HIV Pr55gag polyproteins significantly decreased amounts of wild-type Env proteins were presented at the cell surface, whereas the membrane incorporation of the Env-TM chimeras was not affected. Biochemical and immunoelectron microscopical analysis of particles that were efficiently released from these cells displayed the incorporation of both wild-type Env and chimeric Env-TM proteins on the surface of VLPs. However, the quantities of particle-associated chimeric Env-TM proteins exceeded those of incorporated wild-type Env proteins by a factor of 5-10. Chemical cross-linking and subsequent polyacrylamide gel electrophoresis of VLP-entrapped Env proteins revealed that the chimeric Env-TM proteins form homodimers and a higher-order oligomer, similar to that observed for wild-type Env proteins. Thus, the results of this study clearly demonstrate that the replacement of the gp41 transmembrane protein of gp160 by a heterologous, EBV gp220/350-derived membrane anchor provides an effective strategy to incorporate high quantities of oligomeric HIV gp120 proteins on the surface of Pr55gag virus-like particles.     

Membrane fusion induced by the HIV type 1 fusion peptide: modulation by factors affecting glycoprotein 41 activity and potential anti-HIV compounds

Peptides representing a sequence of 23 amino acid residues at the N terminus of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 bind and subsequently induce fusion of large unilamellar vesicles (LUV), an activity presumably related to gp41 function in viral infection. These in vitro effects can be modulated by several factors that are known to affect HIV-1 infectivity and gp41-mediated virus-cell fusion. Peptide-induced membrane fusion but not peptide binding can be inhibited by two factors known to block gp41 activity: a polar amino acid substitution V --> E in position 2 and the presence of the N-terminal hexapeptide of gp41 in addition to the parent sequence. Whereas inclusion of the alternative gp120 receptor galactosylceramide in membranes has virtually no effect, membrane cholesterol stimulates fusion activity. In view of its putative physiological relevance, we have used the fusion activity of the peptides as a tool to evaluate the inhibitory effect of antivirals that might target this sequence. We describe three dissimilar effects: Amphotericin B inhibits in a cholesterol-independent way peptide-induced fusion but not binding, human serum albumin inhibits binding and consequently fusion, and dextran sulfate (M(r) 5000) does not affect either binding or fusion.     

Comparative characterization of a wild type and transmembrane domain-deleted fatty acid amide hydrolase: identification of the transmembrane domain as a site for oligomerization

Fatty acid amide hydrolase (FAAH) is an integral membrane protein responsible for the hydrolysis of a number of primary and secondary fatty acid amides, including the neuromodulatory compounds anandamide and oleamide. Analysis of FAAH's primary sequence reveals the presence of a single predicted transmembrane domain at the extreme N-terminus of the enzyme. A mutant form of the rat FAAH protein lacking this N-terminal transmembrane domain (DeltaTM-FAAH) was generated and, like wild type FAAH (WT-FAAH), was found to be tightly associated with membranes when expressed in COS-7 cells. Recombinant forms of WT- and DeltaTM-FAAH expressed and purified from Escherichia coli exhibited essentially identical enzymatic properties which were also similar to those of the native enzyme from rat liver. Analysis of the oligomerization states of WT- and DeltaTM-FAAH by chemical cross-linking, sedimentation velocity analytical ultracentrifugation, and size exclusion chromatography indicated that both enzymes were oligomeric when membrane-bound and after solubilization. However, WT-FAAH consistently behaved as a larger oligomer than DeltaTM-FAAH. Additionally, SDS-PAGE analysis of the recombinant proteins identified the presence of SDS-resistant oligomers for WT-FAAH, but not for DeltaTM-FAAH. Self-association through FAAH's transmembrane domain was further demonstrated by a FAAH transmembrane domain-GST fusion protein which formed SDS-resistant dimers and large oligomeric assemblies in solution.     

Impairment of excitatory amino acid transport in astroglial cells infected with the human immunodeficiency virus type 1

Perturbation of astrocyte functions by HIV-1 infection may contribute to the pathogenesis of AIDS dementia complex (ADC). The present study investigated the possibility that astroglial transport of glutamate and aspartate, the major excitatory amino acids (EAAs) in the mammalian central nervous system (CNS), is altered by HIV-1 infection. Human U251 glioma cells were infected with the brain isolate SF162 of HIV-1. HIV-1 persisted in glial cells over several months. This nonproductive infection of glial cells was characterized by persistent expression of Nef over the time of the infection, and the transient presence of structural viral proteins, including the viral transmembrane glycoprotein gp41, which was detected during the initial 2 weeks following HIV-1 infection. The presence of gp41 in acutely HIV-1-infected glial cells coincided with a 36% decrease in D-[3H]aspartate uptake, owing to a reduction in the maximal transport capacity (vmax) for D-aspartate. The expression of typical astrocytic glutamate transporters EAAT1 and EAAT2 in U251 glioma cells was not altered by HIV-1 infection. To determine whether viral protein gp120, gp41, or Nef was involved in the impairment of EAA transport in acutely HIV-1-infected glial cells, effects of lentiviral lytic peptide type 1 (LLP-1) (corresponding to the carboxy terminus of gp41), recombinant SF2 gp120, and recombinant LAI Nef on D-[3H]aspartate uptake and the release of glutamate in glial cells were investigated. Only LLP-1 reduced D-[3H]aspartate uptake and facilitated the release of glutamate from glial cells in a concentration-dependent manner. These results suggest that the carboxy terminus of gp41 impairs EAA transport in glial cells, which may contribute to excitotoxic damage to neurons in HIV-1 infection of the CNS.     

A leucine zipper-like sequence from the cytoplasmic tail of the HIV-1 envelope glycoprotein binds and perturbs lipid bilayers

HIV-1 transmembrane envelope glycoprotein (gp41) has an unusually long cytoplasmic domain that has secondary associations with the inner leaflet of the membrane. Two highly amphiphatic alpha-helices in the cytoplasmic domain of gp41 have previously been shown to interact with lipid bilayers. We have detected a highly conserved leucine zipper-like sequence between the two alpha-helices. A peptide corresponding to this segment (residues 789-815, LLP-3) aggregates in aqueous solution, but spontaneously inserts into phospholipid membranes and dissociates into alpha-helical monomers. The peptide perturbs the bilayer structure resulting in the formation of micelles and other non-bilayer structures. Tryptophan fluorescence quenching experiments using brominated phospholipids revealed that the peptide penetrates deeply into the hydrophobic milieu of the membrane bilayer. The peptide interacts equally with zwitterionic and negatively-charged phospholipid membranes and is protected from proteolytic digestion in its membrane-bound state. Polarized attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy showed that the LLP-3 alpha-helix axis is about 70 degrees from the normal to the membrane plane. The ATR-FTIR CH2-stretching dichroic ratio increases when the peptide is incorporated into pure phospholipid membranes, further indicating that the peptide can deeply penetrate and perturb the bilayer structure. Integrating these data with what is already known about the membrane-associating features of adjacent segments, we propose a revised structural model in which a large portion of the cytoplasmic tail of the HIV-1 envelope glycoprotein is associated with the membrane.     

Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells

CD4-expressing T cells in lymphoid organs are infected by the primary strains of HIV and represent one of the main sources of virus replication. Gene therapy strategies are being developed that allow the transfer of exogenous genes into CD4(+) T lymphocytes whose expression might prevent viral infection or replication. Insights into the mechanisms that govern virus entry into the target cells can be exploited for this purpose. Major determinants of the tropism of infection are the CD4 molecules on the surface of the target cells and the viral envelope glycoproteins at the viral surface. The best characterized and most widely used gene transfer vectors are derived from Moloney murine leukemia virus (MuLV). To generate MuLV-based retroviral gene transfer vector particles with specificity of infection for CD4-expressing cells, we attempted to produce viral pseudotypes, consisting of MuLV capsid particles and the surface (SU) and transmembrane (TM) envelope glycoproteins gp120-SU and gp41-TM of HIV type 1 (HIV-1). Full-length HIV-1 envelope glycoproteins were expressed in the MuLV env-negative packaging cell line TELCeB6. Formation of infectious pseudotype particles was not observed. However, using a truncated variant of the transmembrane protein, lacking sequences of the carboxyl-terminal cytoplasmic domain, pseudotyped retroviruses were generated. Removal of the carboxyl-terminal domain of the transmembrane envelope protein of HIV-1 was therefore absolutely required for the generation of the viral pseudotypes. The virus was shown to infect CD4-expressing cell lines, and infection was prevented by antisera specific for gp120-SU. This retroviral vector should prove useful for the study of HIV infection events mediated by HIV-1 envelope glycoproteins, and for the targeting of CD4(+) cells during gene therapy of AIDS.     

Induction of antibodies against epitopes inaccessible on the HIV type 1 envelope oligomer by immunization with recombinant monomeric glycoprotein 120

An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected to elicit antibodies preferentially neutralizing mutant variants of HIV-BRU lacking the N306 glycan. Therefore, two guinea pigs were immunized with monomeric wild-type HIV-BRU gp120 possessing the N306 glycan and immune sera were tested for neutralization against target viruses HIV-BRU, -A308, and -A308T321. HIV-A308 and HIV-A308T321 lack the N306 glycan; HIV-A308T321 contains an additional mutation at the tip of V3 rendering it resistant to MAb binding at this epitope. Both immune sera preferentially neutralized the two mutant virus variants lacking the N306 glycan, with a 10- to 20-fold increase in neutralization titer compared with the wild-type HIV-BRU. Thus, immunization with monomeric HIV-BRU gp120 elicited antibodies preferentially neutralizing HIV variants lacking the N306 glycan. In addition to antibodies directed against the tip of V3, other antibodies directed against epitopes shielded by the N306 glycan on the envelope oligomer were elicited by the immunization, as demonstrated by the ability of the immune sera to neutralize HIV-A308T321. One such epitope was overlapping the NEA-9284 epitope located at the amino-terminal flank of the V3 loop. Our results demonstrate that monomeric gp120 contains immunogenic structures inaccessible on the envelope oligomer. The limited ability of recombinant gp120 vaccines to induce neutralizing antibodies against primary isolates may thus not exclusively reflect genetic variation.     

The C-terminal region of the stalk domain of ubiquitous human kinesin heavy chain contains the binding site for kinesin light chain

The motor protein kinesin is a heterotetramer composed of two heavy chains of approximately 120 kDa and two light chains of approximately 65 kDa protein. Kinesin motor activity is dependent on the presence of ATP and microtubules. The kinesin light chain-binding site in human kinesin heavy chain was determined by reconstituting in vitro a complex of recombinant heavy and light chains. The proteins expressed in bacteria included oligohistidine-tagged fragments of human ubiquitous kinesin heavy chain, spanning most of the stalk and all of the tail domain (amino acids 555-963); and untagged, essentially full-length human kinesin light chain (4-569) along with N-terminal (4-363) and C-terminal (364-569) light chain fragments. Heavy chain fragments were attached to Ni2+-charged beads and incubated with untagged light chain fragments. Analysis of eluted complexes by SDS-PAGE and immunoblotting mapped the light chain-binding site in heavy chain to amino acids 771-813, a region close to the C-terminal end of the heavy chain stalk domain. In addition, only the full-length and N-terminal kinesin light chain fragments bound to this heavy chain region. Within this heavy chain region are four highly conserved contiguous heptad repeats (775-802) which are predicted to form a tight alpha-helical coiled-coil interaction with the heptad repeat-containing N-terminus of the light chain, in particular region 106-152 of human light chain. This predicted hydrophobic, alpha-helical coiled-coil interaction is supported by both circular dichroism spectroscopy of the recombinant kinesin heavy chain fragment 771-963, which displays an alpha-helical content of 70%, and the resistance of the heavy/light chain interaction to high salt (0.5 M).     

Effect of major deletions in the V1 and V2 loops of a macrophage-tropic HIV type 1 isolate on viral envelope structure, cell entry, and replication

Two HIV-1 envelope mutant proteins were generated by introducing deletions in the first and second hypervariable gp120 regions (V1 and V2 loops, respectively) of a macrophage-tropic primary HIV-1 isolate, SF162, to study the effect of the deleted sequences on envelope structure, viral entry, and replication potentials. The first mutant lacked 17 amino acids of the V1 loop and the latter 30 amino acids of the V2 loop. A comparison of the immunochemical structure of the wild-type and mutant monomeric and virion-associated gp120 molecules revealed that the V1 and V2 loop deletions differentially altered the structure of the V3 loop, the CD4-binding site, and epitopes within conserved regions of gp120. Regardless of differences in structure, both mutated envelope proteins supported viral replication into peripheral blood mononuclear cells to levels comparable to those of the wild-type SF162 virus. However, they decreased the viral replication potential in macrophages, even though they did not alter the coreceptor usage of the viruses. These studies support and extend previous observations that a complex structural interaction between the V1, V2, and V3 loops and elements of the CD4-binding site of gp120 controls entry of virus into cells. The present studies, however, suggest that the effect of the V1 and V2 loops in viral entry is cell dependent.     

Induction of CD4+ T cell depletion in mice doubly transgenic for HIV gp120 and human CD4

It has been suggested that loss of uninfected T cells in HIV infection occurs because of lymphocyte activation resulting in cell death by apoptosis. To address the question of whether cross-linking of CD4/HIV gp120 complexes by antibodies were sufficient to induce T cell depletion in vivo, we developed an animal model of continuous interaction between human CD4 (hCD4), gp120 and anti-gp120 antibodies in the absence of other viral factors. Double-transgenic mice have been generated in which T cells express on their membrane hCD4 and secrete HIV gp120. Although these mice have hCD4/gp120 complexes present on the surface of T cells, they do not show gross immunological abnormalities, and they are able to produce anti-gp120 antibodies following immunization with denaturated gp120. However, double-transgenic mice with antibodies to gp120, when immunized with tetanus toxoid, mount an IgG response that is significantly lower than that of double-transgenic mice without antibodies to gp120. Furthermore, the presence of anti-gp120 antibodies leads to CD4+ T cell depletion and immunodeficiency in the absence of HIV infection. Thus, the antibody response to gp120 can lead to CD4+ T cell attrition in vivo.     

The YXXL sequences of a transmembrane protein of bovine leukemia virus are required for viral entry and incorporation of viral envelope protein into virions

The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.     

Oxidative stress and interleukins in seminal plasma during leukocytospermia

Various roles for the viral receptor, CD4, have been proposed in facilitating human immunodeficiency virus type 1 (HIV-1) entry, including virion binding to the target cell and the induction of conformational changes in the viral envelope glycoproteins required for the membrane fusion reaction. Here, we compare the structural requirements in the CDR2-like loop of CD4 domain 1, the major contact site of the gp120 envelope glycoprotein, for gp120 binding and virus entry. For every CD4 mutant examined, the level of cell surface expression and the gp120 binding affinity were sufficient to explain the relative ability to function as a viral receptor. The decrease in relative infectibility associated with decreased gp120 binding affinity was more pronounced at lower cell surface CD4 concentrations. These results imply that both receptor density and affinity determine the efficiency of HIV-1 entry and that specific structures in the CD4 residues examined are probably not required for HIV-1 entry functions other than gp120 binding.     

Resistance to a drug blocking human immunodeficiency virus type 1 entry (RPR103611) is conferred by mutations in gp41

A triterpene derived from betulinic acid (RPR103611) blocks human immunodeficiency virus type 1 (HIV-1) infection and fusion of CD4+ cells with cells expressing HIV-1 envelope proteins (gp120 and gp41), suggesting an effect on virus entry. This compound did not block infection by a subtype D HIV-1 strain (NDK) or cell-cell fusion mediated by the NDK envelope proteins. The genetic basis of drug resistance was therefore addressed by testing envelope chimeras derived from NDK and a drug-sensitive HIV-1 strain (LAI, subtype B). A drug-resistant phenotype was observed for all chimeras bearing the ectodomain of NDK gp41, while the origins of gp120 and of the membrane anchor and cytoplasmic domains of gp41 had no apparent role. The envelope gene of a LAI variant, fully resistant to the antiviral effect of RPR103611, was cloned and sequenced. Its product differed from the parental sequence at two positions in gp41, with changes of arginine 22 to alanine (R22A) and isoleucine 84 to serine (I84S), the gp120 being identical. In the context of LAI gp41, the I84S substitution was sufficient for drug resistance. Therefore, in two different systems, differences in gp41 were associated with sensitivity or resistance to RPR103611. Modifications of gp41 can affect the quaternary structure of gp120 and gp41 and the accessibility of gp120 to antiviral agents such as neutralizing antibodies. However, a direct effect of RPR103611 on a gp41 target must also be envisioned, in agreement with the blocking of apparently late steps of HIV-1 entry. This compound could be a valuable tool for structure-function studies of gp41.     

Women among a population at a psychiatric service in Senegal (psychosocial aspects)

Exposure of porin from Escherichia coli to glutardialdehyde followed by SDS-gel electrophoresis in 3% polyacrylamide yielded three bands that were identified in order of decreasing mobility as monomers and two and three cross-linked polypeptide chains. The distribution of protein among the three species for different extents of reaction showed a remarkably good agreement with corresponding values predicted from cross linking theory for an oligomer composed of three identical subunits arranged according to a 3-fold rotation axis. Electrophoresis performed in 7.5% polyacrylamide yielded four bands that were assigned to polypeptide chain monomers, dimers, and two types of trimers carrying two and three intersubunit cross-links. Our findings provide evidence that the central premise of cross-linking theory, viz. that the intersubunit cross-links formed upon exposure of a protein to a bifunctional reagent be governed by the symmetry of the molecule, is valid. Careful interpretation of cross-linking experiments thus proves an effective method to assess the oligomeric structure of a protein and reveal the symmetry underlying the spatial arrangement of the subunits within the molecule.     

Intrastrand cross-linked actin between Gln-41 and Cys-374. III. Inhibition of motion and force generation with myosin

Structural and functional properties of intrastrand, ANP (N-(4-azido-2-nitrophenyl)-putrescine) cross-linked actin filaments, between Gln-41 and Cys-374 on adjacent monomers, were examined for several preparations of such actin. Extensively cross-linked F-actin (with 12% un-cross-linked monomers) lost at 60 degrees C the ability to activate myosin ATPase at a 100-fold slower rate and unfolded in CD melting experiments at a temperature higher by 11 degrees C than the un-cross-linked actin. Electron microscopy and image reconstruction of these filaments did not reveal any gross changes in F-actin structure but showed a change in the orientation of subdomain 2 and a decrease in interstrand connectivity. Rigor and weak (in the presence of ATP) myosin subfragment (S1) binding and acto-S1 ATPase did not show major changes upon 50% and 90% ANP cross-linking of F-actin; the Kd and Km values were little affected by the cross-linking, and the Vmax decreased by 50% for the extensively cross-linked actin. The cross-linking of actin (50%) decreased the mean speed and the number of sliding filaments in the in vitro motility assays by approximately 35% while the relative force, as measured by using external load in these assays, was inhibited by approximately 25%. The mean speed of actin filaments decreased with the increase in their cross-linking and approached 0 for the 90% cross-linked actin. Also examined were actin filaments reassembled from cross-linked and purified ANP cross-linked dimers, trimers, and oligomers. All of these filaments had the same acto-S1 ATPase and rigor S1 binding properties but different behavior in the in vitro motility assays. Filaments made of cross-linked dimers moved at approximately 50% of the speed of the un-cross-linked actin. The movement of filaments made of cross-linked trimers was inhibited more severely, and the oligomer-made filaments did not move at all. These results show the uncoupling between force generation and other events in actomyosin interactions and emphasize the role of actin filament structure and dynamics in the contractile process.     

The hepatic interleukin-6 receptor. Studies on its structure and regulation by phorbol 12-myristate 13-acetate-dexamethasone

Recombinant human 125I-interleukin-6 (IL-6) was cross-linked with the homobifunctional reagent disuccinimidyl suberate to human hepatoma cells (HepG2). Three recombinant human 125I-IL-6-containing complexes of apparent molecular masses of 100, 120, and 200 kDa were immunoprecipitated with specific antibodies to human IL-6 or to the 80-kDa IL-6 receptor subunit. We show by immunoprecipitation, peptide mapping, and by the use of a cleavable heterobifunctional cross-linker (Denny-Jaffe reagent) that different polypeptides are involved in the formation of the 100- and 120-kDa IL-6-containing complexes. The molecular compositions of the 100- and 120-kDa cross-linked complexes were identified. The 100-kDa complex consisted of one ligand and one IL-6 receptor subunit, glycoprotein 80 (gp80), whereas the 120-kDa complex was found to be composed of one ligand and a polypeptide which was immunoprecipitable with the monoclonal antibody AM64 directed against gp130. Exposure of HepG2 cells to phorbol 12-myristate 13-acetate (PMA) or PMA-dexamethasone led to an increase in the 80-kDa IL-6 receptor mRNA and functional receptor protein. Whereas treatment of HepG2 cells with PMA led to an increase in the formation of gp80.gp130.IL-6 complexes determined by cross-linking, no corresponding increase in high affinity binding sites was found. The existence of a third IL-6 receptor subunit present in limiting amounts on HepG2 cells is proposed to explain this discrepancy. Evidence is presented that the 80-kDa IL-6 receptor up-regulation by PMA-dexamethasone is caused by the depletion of protein kinase C since the protein kinase C inhibitor staurosporine mimics the effect of PMA-dexamethasone.