Three-dimensional solution structure of the 44 kDa ectodomain of SIV gp41.

The solution structure of the ectodomain of simian immunodeficiency virus (SIV) gp41 (e-gp41), consisting of residues 27-149, has been determined by multidimensional heteronuclear NMR spectroscopy. SIV e-gp41 is a symmetric 44 kDa trimer with each subunit consisting of antiparallel N-terminal (residues 30-80) and C-terminal (residues 107-147) helices connected by a 26 residue loop (residues 81-106). The N-terminal helices of each subunit form a parallel coiled-coil structure in the interior of the complex which is surrounded by the C-terminal helices located on the exterior of the complex. The loop region is ordered and displays numerous intermolecular and non-sequential intramolecular contacts. The helical core of SIV e-gp41 is similar to recent X-ray structures of truncated constructs of the helical core of HIV-1 e-gp41. The present structure establishes unambiguously the connectivity of the N- and C-terminal helices in the trimer, and characterizes the conformation of the intervening loop, which has been implicated by mutagenesis and antibody epitope mapping to play a key role in gp120 association. In conjunction with previous studies, the solution structure of the SIV e-gp41 ectodomain provides insight into the binding site of gp120 and the mechanism of cell fusion. The present structure of SIV e-gp41 represents one of the largest protein structures determined by NMR to date.     

SIV gp41 binds to membranes both in the monomeric and trimeric states: consequences for the neuropathology and inhibition of HIV infection

The viral envelope glycoprotein gp41 mediates membrane fusion in HIV/SIV infection. gp41 ectodomain (e-gp41, residues 27-149), which was shown to interact with phospholipid membranes, exists in an equilibrium between the monomeric and trimeric states. Here, we analyzed, by intrinsic Trp fluorescence and resonance energy transfer, whether SIV e-gp41-membrane interaction depends on the gp41 oligomeric state. We found that both gp41 monomers and trimers bind membranes, with the monomers' full binding being reached at substantially lower lipid to protein ratios. Furthermore, the different characteristics of the Trp fluorescence of monomers and trimers enabled us to detect binding of each form at concentrations at which both species were present. CD spectroscopy revealed that the secondary structure of gp41 monomers does not change upon membrane binding, suggesting that membrane-bound monomeric-gp41 is a possible target for DP-178, a potent peptide inhibitor of HIV infection. The consequences of the interaction between monomeric and trimeric gp41 with membranes in HIV/SIV infection, its inhibition, and its associated neuropathologies are discussed.     

Design, expression, and immunogenicity of a soluble HIV trimeric envelope fragment adopting a prefusion gp41 configuration

The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) is comprised of non-covalently associated gp120/gp41 subunits that form trimeric spikes on the virion surface. Upon binding to host cells, Env undergoes a series of structural transitions, leading to gp41 rearrangement necessary for fusion of viral and host membranes. Until now, the prefusion state of gp41 ectodomain (e-gp41) has eluded molecular and structural analysis, and thus assessment of the potential of such an e-gp41 conformer to elicit neutralizing antibodies has not been possible. Considering the importance of gp120 amino (C1) and carboxyl (C5) segments in the association with e-gp41, we hypothesize that these regions are sufficient to maintain e-gp41 in a prefusion state. Based on the available gp120 atomic structure, we designed several truncated gp140 variants by including the C1 and C5 regions of gp120 in a gp41 ectodomain fragment. After iterative cycles of protein design, expression and characterization, we obtained a variant truncated at Lys(665) that stably folds as an elongated trimer under physiologic conditions. Several independent biochemical/biophysical analyses strongly suggest that this mini-Env adopts a prefusion e-gp41 configuration that is strikingly distinct from the postfusion trimer-of-hairpin structure. Interestingly, this prefusion mini-Env, lacking the fragment containing the 2F5/4E10 neutralizing monoclonal antibody binding sites, displays no detectable HIV-neutralizing epitopes when employed as an immunogen in rabbits. The result of this immunogenicity study has important implications for HIV-1 vaccine design efforts. Moreover, this engineered mini-Env protein should facilitate three-dimensional structural studies of the prefusion e-gp41 and serve to guide future attempts at pharmacologic and immunologic intervention of HIV-1.     

Lineage-Specific Differences between Human and Simian Immunodeficiency Virus Regulation of gp120 Trimer Association and CD4 Binding

Metastable conformations of the gp120 and gp41 envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) must be maintained in the unliganded state of the envelope glycoprotein trimer. Binding of gp120 to the primary receptor, CD4, triggers the transition to an open conformation of the trimer, promoting interaction with the CCR5 chemokine receptor and ultimately leading to gp41-mediated virus-cell membrane fusion and entry. Topological layers in the gp120 inner domain contribute to gp120-trimer association in the unliganded state and to CD4 binding. Here we describe similarities and differences between HIV-1 and SIVmac gp120. In both viruses, the gp120 N/C termini and the inner domain β-sandwich and layer 2 support the noncovalent association of gp120 with the envelope glycoprotein trimer. Layer 1 of the SIVmac gp120 inner domain contributes more to trimer association than the corresponding region of HIV-1 gp120. On the other hand, layer 1 plays an important role in stabilizing the CD4-bound conformation of HIV-1 but not SIVmac gp120 and thus contributes to HIV-1 binding to CD4. In SIVmac, CD4 binding is instead enhanced by tryptophan 375, which fills the Phe 43 cavity of gp120. Activation of SIVmac by soluble CD4 is dependent on tryptophan 375 and on layer 1 residues that determine a tight association of gp120 with the trimer. Distinct biological requirements for CD4 usage have resulted in lineage-specific differences in the HIV-1 and SIV gp120 structures that modulate trimer association and CD4 binding.     

Biophysical characterization of gp41 aggregates suggests a model for the molecular mechanism of HIV-associated neurological damage and dementia

In human immunodeficiency virus (HIV)-infected individuals, the level of the HIV envelope protein gp41 in brain tissue is correlated with neurological damage and dementia. In this paper we show by biochemical methods and electron microscopy that the extracellular ectodomain of purified HIV and simian immunodeficiency virus gp41 (e-gp41) forms a mixture of soluble high molecular weight aggregate and native trimer at physiological pH. The e-gp41 aggregate is shown to be largely alpha-helical and relatively stable to denaturants. The high molecular weight form of e-gp41 is variable in size ranging from 7 to 70 trimers, which associate by interactions at the interior of the aggregate involving the loop that connects the N- and C-terminal helices of the e-gp41 core. The trimers are predominantly arranged with their long axes oriented radially, and the width of the high molecular weight aggregate corresponds to the length of two e-gp41 trimers (approximately 200 A). Using both light and electron microscopy combined with immunohistochemistry we show that HIV gp41 accumulates as an extracellular aggregate in the brains of HIV-infected patients diagnosed with dementia. We postulate that the high molecular weight aggregates of e-gp41 are responsible for HIV-associated neurological damage and dementia, consistent with known mechanisms of encephalopathy.     

On the interaction between gp41 and membranes: the immunodominant loop stabilizes gp41 helical hairpin conformation

gp41 is the protein responsible for the process of membrane fusion that allows primate lentiviruses (HIV and SIV) to enter into their host cells. gp41 ectodomain contains an N-terminal and a C-terminal heptad repeat region (NHR and CHR) connected by an immunodominant loop. In the absence of membranes, the NHR and CHR segments fold into a protease-resistant core with a trimeric helical hairpin structure. However, when the immunodominant loop is not present (either in a complex formed by HIV-1 gp41-derived NHR and CHR peptides or by mild treatment with protease of recombinant constructs of HIV-1 gp41 ectodomain, which also lack the N-terminal fusion peptide and the C-terminal Trp-rich region) membrane binding induces a conformational change in the gp41 core structure. Here, we further investigated whether covalently linking the NHR and CHR segments by the immunodominant loop affects this conformational change. Specifically, we analyzed a construct corresponding to a fragment of SIVmac239 gp41ectodomain (residues 27-149, named e-gp41) by means of surface plasmon resonance, Trp and rhodamine fluorescence, ATR-FTIR spectroscopy, and differential scanning calorimetry. Our results suggest that the presence of the loop stabilizes the trimeric helical hairpin both when e-gp41 is in aqueous solution and when it is bound to the membrane surface. Bearing in mind possible differences between HIV-1 and SIV gp41, and considering that the gp41 ectodomain constructs analyzed to date lack the N-terminal fusion peptide and the C-terminal Trp-rich region, we discuss our observations in relation to the mechanism of virus-induced membrane fusion.     

Three-dimensional structures of soluble CD4-bound states of trimeric simian immunodeficiency virus envelope glycoproteins determined by using cryo-electron tomography

The trimeric envelope glycoprotein (Env) spikes displayed on the surfaces of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) virions are composed of three heterodimers of the viral glycoproteins gp120 and gp41. Although binding of gp120 to cell surface CD4 and a chemokine receptor is known to elicit conformational changes in gp120 and gp41, changes in quaternary structure of the trimer have only recently been elucidated. For the HIV-1 BaL isolate, CD4 attachment results in a striking rearrangement of the trimer from a "closed" to an "open" conformation. The effect of CD4 on SIV trimers, however, has not been described. Using cryo-electron tomography, we have now determined molecular architectures of the soluble CD4 (sCD4)-bound states of SIV Env trimers for three different strains (SIVmneE11S, SIVmac239, and SIV CP-MAC). In marked contrast to HIV-1 BaL, SIVmneE11S and SIVmac239 Env showed only minor conformational changes following sCD4 binding. In SIV CP-MAC, where trimeric Env displays a constitutively "open" conformation similar to that seen for HIV-1 BaL Env in the sCD4-complexed state, we show that there are no significant further changes in conformation upon the binding of either sCD4 or 7D3 antibody. The density maps also show that 7D3 and 17b antibodies target epitopes on gp120 that are on opposites sides of the coreceptor binding site. These results provide new insights into the structural diversity of SIV Env and show that there are strain-dependent variations in the orientation of sCD4 bound to trimeric SIV Env.     

Expression, purification, and characterization of gp160e, the soluble, trimeric ectodomain of the simian immunodeficiency virus envelope glycoprotein, gp160

The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares approximately 25% sequence identity with gp160 from the human immunodeficiency virus, type I, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g. CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.     

Envelope Glycoprotein Cytoplasmic Domains from Diverse Lentiviruses Interact with the Prenylated Rab Acceptor

Lentivirus envelope glycoproteins have unusually long cytoplasmic domains compared to those of other retroviruses. To identify cellular binding partners of the simian immunodeficiency virus (SIV) envelope transmembrane protein (gp41) cytoplasmic domain (CD), we performed a yeast two-hybrid screen of a phytohemagglutinin-activated human T-cell cDNA library with the SIV gp41 CD. The majority of positive clones (50 of 54) encoded the prenylated Rab acceptor (PRA1). PRA1 is a 21-kDa protein associated with Golgi membranes that binds to prenylated Rab proteins in their GTP-bound state. While the cellular function of PRA1 is presently unknown, this protein appears to participate in intracellular vesicular trafficking, based on its cellular localization and ability to bind multiple members of the Rab protein family. Mammalian two-hybrid assays confirmed the interaction between the SIV gp41 CD and PRA1. Furthermore, gp41 sequences important for PRA1 binding were mapped to a central leucine-rich, amphipathic alpha-helix in the SIV gp41 cytoplasmic tail. Although the human immunodeficiency virus (HIV-1) gp41 CD failed to interact with PRA1 in the yeast two-hybrid system, its interaction with PRA1 was significantly better than that of the SIV gp41 CD in mammalian two-hybrid assays. Interestingly, PRA1 also interacted with the Env CDs of HIV-2, bovine immunodeficiency virus, equine infectious anemia virus, and feline immunodeficiency virus. Thus, PRA1 associates with envelope glycoproteins from widely divergent lentiviruses.     

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.     

Soluble mimetics of human immunodeficiency virus type 1 viral spikes produced by replacement of the native trimerization domain with a heterologous trimerization motif: characterization and ligand binding analysis

The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein, gp120, mediates binding to the viral receptors and, along with the transmembrane glycoprotein gp41, is a major target for neutralizing antibodies. We asked whether replacing the gp41 fusion/trimerization domain with a stable trimerization motif might lead to a more stable gp120 trimer that would be amenable to structural and immunologic analysis. To obtain stable gp120 trimers, a heterologous trimerization motif, GCN4, was appended to the C terminus of YU2gp120. Biochemical analysis indicated that the gp120-GCN4 trimers were superior to gp140 molecules in their initial homogeneity, and trilobed structures were observable by electron microscopy. Biophysical analysis of gp120-GCN4 trimers by isothermal titration calorimetry (ITC) and ultracentrifugation analyses indicated that most likely two molecules of soluble CD4 could bind to one gp120-GCN4 trimer. To further examine restricted CD4 stoichiometric binding to the gp120-GCN4 trimers, we generated a low-affinity CD4 binding trimer by introducing a D457V change in the CD4 binding site of each gp120 monomeric subunit. The mutant trimers could definitively bind only one soluble CD4 molecule, as determined by ITC and sedimentation equilibrium centrifugation. These data indicate that there are weak interactions between the gp120 monomeric subunits of the GCN4-stabilized trimers that can be detected by low-affinity ligand sensing. By similar analysis, we also determined that removal of the variable loops V1, V2, and V3 in the context of the gp120-GCN4 proteins allowed the binding of three CD4 molecules per trimer. Interestingly, both the gp120-GCN4 variants displayed a restricted stoichiometry for the CD4-induced antibody 17b of one antibody molecule binding per trimer. This restriction was not evident upon removal of the variable loops V1 and V2 loops, consistent with conformational constraints in the wild-type gp120 trimers and similar to those inherent in the functional Env spike. Thus, the gp120-GCN4 trimers demonstrate several properties that are consistent with some of those anticipated for gp120 in the context of the viral spike.     

HIV-1 gp41 and gp160 are hyperthermostable proteins in a mesophilic environment. Characterization of gp41 mutants.

HIV gp41(24-157) unfolds cooperatively over the pH range of 1.0-4.0 with T(m) values of > 100 degrees C. At pH 2.8, protein unfolding was 80% reversible and the DeltaH(vH)/DeltaH(cal) ratio of 3.7 is indicative of gp41 being trimeric. No evidence for a monomer-trimer equilibrium in the concentration range of 0.3-36 micro m was obtained by DSC and tryptophan fluorescence. Glycosylation of gp41 was found to have only a marginal impact on the thermal stability. Reduction of the disulfide bond or mutation of both cysteine residues had only a marginal impact on protein stability. There was no cooperative unfolding event in the DSC thermogram of gp160 in NaCl/P(i), pH 7.4, over a temperature range of 8-129 degrees C. When the pH was lowered to 5.5-3.4, a single unfolding event at around 120 degrees C was noted, and three unfolding events at 93.3, 106.4 and 111.8 degrees C were observed at pH 2.8. Differences between gp41 and gp160, and hyperthermostable proteins from thermophile organisms are discussed. A series of gp41 mutants containing single, double, triple or quadruple point mutations were analysed by DSC and CD. The impact of mutations on the protein structure, in the context of generating a gp41 based vaccine antigen that resembles a fusion intermediate state, is discussed. A gp41 mutant, in which three hydrophobic amino acids in the gp41 loop were replaced with charged residues, showed an increased solubility at neutral pH.     

The stoichiometry of trimeric SIV glycoprotein interaction with CD4 differs from that of anti-envelope antibody Fab fragments.

Human and simian immunodeficiency viruses infect host lymphoid cells by binding CD4 molecules via their gp160 envelope glycoproteins. Biochemical studies on recombinant SIVmac32H (pJ5) envelope ectodomain gp140 precursor protein show that the envelope is a trimer. Using size exclusion chromatography, quantitative amino acid analysis, analytical ultracentrifugation, and CD4-based competition assay, we demonstrate that the stoichiometry of CD4 receptor-oligomeric envelope interaction is 1:1. By contrast, Fab fragments of both neutralizing and non-neutralizing monoclonal antibodies bind at a 3:1 ratio. Thus, despite displaying equivalent CD4 binding sites on each of the three gp140 protomers within an uncleaved trimer, only one site binds the soluble 4-domain human CD4 extracellular segment. The anti-cooperativity and the faster k(off) of gp140 trimer:CD4 versus gp120 monomer:CD4 interaction suggest that CD4-induced conformational change is impeded in the intact envelope. The implications of these findings for immunity against human immunodeficiency virus and simian immunodeficiency virus are discussed.     

Expression, purification, and characterization of recombinant HIV gp140. The gp41 ectodomain of HIV or simian immunodeficiency virus is sufficient to maintain the retroviral envelope glycoprotein as a trimer

Efforts to understand the molecular basis of human immunodeficiency virus (HIV) envelope glycoprotein function have been hampered by the inability to generate sufficient quantities of homogeneous material. We now report on the high level expression, purification, and characterization of soluble HIV gp140 ectodomain proteins in Chinese hamster ovary-Lec3.2.8.1 cells. Gel filtration and analytical ultracentrifugation show that the uncleaved ADA strain-derived gp140 proteins are trimeric without further modification required to maintain oligomers. These spike proteins are native as judged by soluble CD4 (sCD4) (K(D) = 1-2 nm) and monoclonal antibody binding studies using surface plasmon resonance. CD4 ligation induces conformational change in the trimer, exposing the chemokine receptor binding site as assessed by 17b monoclonal antibody reactivity. Lack of anti-cooperativity in sCD4-ADA trimer interaction distinct from that observed with sCD4-SIV mac32H implies quaternary structural differences in ground states of their respective spike proteins.     

Cryo-electron tomographic structure of an immunodeficiency virus envelope complex in situ

The envelope glycoprotein (Env) complexes of the human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate viral entry and are a target for neutralizing antibodies. The receptor binding surfaces of Env are in large part sterically occluded or conformationally masked prior to receptor binding. Knowledge of the unliganded, trimeric Env structure is key for an understanding of viral entry and immune escape, and for the design of vaccines to elicit neutralizing antibodies. We have used cryo-electron tomography and averaging to obtain the structure of the SIV Env complex prior to fusion. Our result reveals novel details of Env organisation, including tight interaction between monomers in the gp41 trimer, associated with a three-lobed, membrane-distal gp120 trimer. A cavity exists at the gp41-gp120 trimer interface. Our model for the spike structure agrees with previously predicted interactions between gp41 monomers, and furthers our understanding of gp120 interactions within an intact spike.     

Design of a novel peptide inhibitor of HIV fusion that disrupts the internal trimeric coiled-coil of gp41

The pre-hairpin intermediate of gp41 from the human immunodeficiency virus (HIV) is the target for two classes of fusion inhibitors that bind to the C-terminal region or the trimeric coiled-coil of N-terminal helices, thereby preventing formation of the fusogenic trimer of hairpins. Using rational design, two 36-residue peptides, N36(Mut(e,g)) and N36(Mut(a,d)), were derived from the parent N36 peptide comprising the N-terminal helix of the gp41 ectodomain (residues 546-581 of HIV-1 envelope), characterized by analytical ultracentrifugation and CD, and assessed for their ability to inhibit HIV fusion using a quantitative vaccinia virus-based fusion assay. N36(Mut(e,g)) contains nine amino acid substitutions designed to disrupt interactions with the C-terminal region of gp41 while preserving contacts governing the formation of the trimeric coiled-coil. N36(Mut(a,d)) contains nine substitutions designed to block formation of the trimeric coiled-coil but retains residues that interact with the C-terminal region of gp41. N36(Mut(a,d)) is monomeric, is largely random coil, does not interact with the C34 peptide derived from the C-terminal region of gp41 (residues 628-661), and does not inhibit fusion. The trimeric coiled-coil structure is therefore a prerequisite for interaction with the C-terminal region of gp41. N36(Mut(e,g)) forms a monodisperse, helical trimer in solution, does not interact with C34, and yet inhibits fusion about 50-fold more effectively than the parent N36 peptide (IC(50) approximately 308 nm versus approximately 16 microm). These results indicate that N36(Mut(e,g)) acts by disrupting the homotrimeric coiled-coil of N-terminal helices in the pre-hairpin intermediate to form heterotrimers. Thus N36(Mut(e,g)) represents a novel third class of gp41-targeted HIV fusion inhibitor. A quantitative model describing the interaction of N36(Mut(e,g)) with the pre-hairpin intermediate is presented.     

Fast folding of the HIV-1 and SIV gp41 six-helix bundles

Human (HIV-1) and simian (SIV) immunodeficiency virus fusion with the host cell is promoted by the receptor-triggered refolding of the gp41 envelope protein into a stable trimer-of-hairpins structure that brings viral and cellular membranes into close proximity. The core of this hairpin structure is a six-helix bundle in which an inner homotrimeric coiled coil is buttressed by three antiparallel outer HR2 helices. We have used stopped-flow circular dichroism spectroscopy to characterize the unfolding and refolding kinetics of the six-helix bundle using the HIV-1 and SIV N34(L6)C28 polypeptides. In each case, the time-course of ellipticity changes in refolding experiments is well described by a simple two-state model involving the native trimer and the unfolded monomers. The unfolding free energy of the HIV-1 and SIV trimers and their urea dependence calculated from kinetic data are in very good agreement with data measured directly by isothermal unfolding experiments. Thus, formation of the gp41 six-helix bundle structure involves no detectable population of stable, partly folded intermediates. Folding of HIV-1 N34(L6)C28 is five orders of magnitudes faster than folding of its SIV counterpart in aqueous buffer: k(on),(HIV-1)=1.3 x 10(15)M(-2)s(-1) versus k(on),(SIV)=1.1 x 10(10)M(-2)s(-1). The unfolding rates are similar: k(off),(HIV-1)=1.1 x 10(-5)s(-1) versus k(off),(SIV=)5.7 x 10(-4)s(-1). Kinetic m-values indicate that the transition state for folding of the HIV-1 protein is significantly more compact than the transition state of the SIV protein. Replacement of a single SIV threonine by isoleucine corresponding to position 573 in the HIV-1 sequence significantly stabilizes the protein and renders the folding rate close to that of the HIV-1 protein yet without making the transition state of the mutant as compact as that of the HIV-1 protein. Therefore, the overall reduction of surface exposure in the high-energy transition state seems not to account for different folding rates. While the available biological evidence suggests that refolding of the gp41 protein is slow, our study implies that structural elements outside the trimer-of-hairpins limit the rate of HIV-1 fusion kinetics.     

Reversible and fast association equilibria of a molecular chaperone, gp57A, of bacteriophage T4

The association of a molecular chaperone, gp57A, of bacteriophage T4, which facilitates formation of the long and short tail fibers, was investigated by analytical ultracentrifugation, differential scanning microcalorimetry, and stopped-flow circular dichroism (CD) to establish the association scheme of the protein. Gp57A is an oligomeric alpha-helix protein with 79 amino acids. Analysis of the sedimentation velocity data by direct boundary modeling with Lamm equation solutions together with a more detailed boundary analysis incorporating association schemes led us to conclude that at least three oligomeric species of gp57A are in reversible and fast association equilibria and that a 3(mer)-6(mer)-12(mer) model described the data best. On the other hand, differential scanning microcalorimetry revealed a highly reversible two-step transition of dissociation/denaturation, both of which accompanied decrease in CD at 222 nm. The melting curve analysis revealed that it is consistent with a 6(mer)-3(mer)-1(mer) model. The refolding/association kinetics of gp57A measured by stopped-flow CD was consistent with the interpretation that the bimolecular reaction from trimer to hexamer was preceded by a fast alpha-helix formation in the dead-time. Trimer or hexamer is likely the functional oligomeric state of gp57A.     

Plant annexins form calcium-independent oligomers in solution

The oligomeric state in solution of four plant annexins, namely Anx23(Ca38), Anx24(Ca32), Anx(Gh1), and Anx(Gh2), was characterized by sedimentation equilibrium analysis and gel filtration. All proteins were expressed and purified as amino-terminal His(n) fusions. Sequencing of the Anx(Gh1) construct revealed distinct differences with the published sequence. Sedimentation equilibrium analysis of Anx23(Ca38), Anx24(Ca32), and Anx(Gh1) suggests monomer-trimer equilibria for each protein with association constants in the range of 0.9 x 10(10)-1.7 x 10(11) M(-2). All four proteins were subjected to analytical gel filtration under different buffer conditions. Observations from this experiment series agree quantitatively with the ultracentrifugation results, and strongly suggest calcium independence of the annexin oligomerization behavior; moreover, binding of calcium ions to the proteins seems to require disassembly of the oligomers. Anx(Gh2) showed a different elution profile than the other plant annexins; while having only a very small trimer content, this annexin seems to exist in a monomer-dimer equilibrium in solution.