Effect of oxidation and mutation on the conformational dynamics and fibril assembly of amyloidogenic peptides derived from apolipoprotein C-II.

The oxidation of methionine residues in proteins can inhibit the self-assembly of proteins to form amyloid fibrils. For human apolipoprotein (apo) C-II the oxidation of methionine at position 60 inhibits fibril formation by the mature protein and by the core peptides apoC-II(56-76) and apoC-II(60-70). To investigate the molecular nature of these effects, we carried out fully solvated, all-atom molecular dynamics simulations of the structural changes in apoC-II(56-76) associated with substitutions of oxidized methionine (Met ox) at position 60. The results with apoC-II(56-76) (Met ox) showed less flexibility in structure, leading to a perturbation of the hydrophobic core. Valine substitution at position 60 showed an increased tendency to explore a wide range of conformational space, whereas the behavior of the Gln substitution mutant was similar to the wild-type peptide. These simulations are consistent with kinetic measurements which showed that a Met60Gln substitution within apoC-II(56-76) had little effect on the rate of fibril formation whereas substitution of Met ox or Val at position 60 lead to significant inhibition of peptide fibril formation. The effects of amino acid modification and substitutions on the kinetics of peptide fibril formation differ from the effects observed with full-length apoC-II inferring that additional mechanisms are involved in fibril formation by mature apoC-II.

Conformation and orientation of penetratin in phospholipid membranes.

The binding, conformation and orientation of a hydrophilic vector peptide penetratin in lipid membranes and its state of self-association in solution were examined using circular dichroism (CD), analytical ultracentrifugation and fluorescence spectroscopy. In aqueous solution, penetratin exhibited a low helicity and sedimented as a monomer in the concentration range approximately 50-500 microM. The partitioning of penetratin into phospholipid vesicles was determined using tryptophan fluorescence anisotropy titrations. The apparent penetratin affinity for 20% phosphatidylserine/80% egg phosphatidylcholine vesicles was inversely related to the total peptide concentration implying repulsive peptide-peptide interactions on the lipid surface. The circular dichroism spectra of the peptide when bound to unaligned 20% phosphatidylserine/80% egg phosphatidylcholine vesicles and aligned hydrated phospholipid multilayers were attributed to the presence of both alpha-helical and beta-turn structures. The orientation of the secondary structural elements was determined using oriented circular dichroism spectroscopy. From the known circular dichroism tensor components of the alpha-helix, it can be concluded that the orientation of the helical structures is predominantly perpendicular to the membrane surface, while that of the beta-type carbonyls is parallel to the membrane surface. On the basis of our observations, we propose a novel model for penetratin translocation.

Biophysical characterization of interactions involving importin-alpha during nuclear import.

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-alpha/beta heterodimer. Importin-alpha contains the NLS binding site, whereas importin-beta mediates the translocation through the nuclear pore. We characterized the interactions involving importin-alpha during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-alpha is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-beta (stoichiometry, 1:1; K(D) = 1.1 x 10(-8) m) increases the affinity for NLSs; the importin-alpha/beta complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K(D) = 3.5 x 10(-8) m and 4.8 x 10(-8) m, respectively) comparable with those of a truncated importin-alpha lacking the autoinhibitory domain (T-antigen NLS, K(D) = 1.7 x 10(-8) m; nucleoplasmin NLS, K(D) = 1.4 x 10(-8) m). The autoinhibitory domain (as a separate peptide) binds the truncated importin-alpha, and the crystal structure of the complex resembles the structure of full-length importin-alpha. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-alpha and provide a quantitative description of the binding and regulatory steps during nuclear import.

Identification of a determinant of epidermal growth factor receptor ligand-binding specificity using a truncated, high-affinity form of the ectodomain.

Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (hEGF), mouse EGF (mEGF), and human transforming growth factor alpha (hTGF-alpha) with high affinity despite the significant differences in the amino acid sequences of the ligands and the receptors. In contrast, the chicken EGFR can discriminate between mEGF (and hEGF) and hTGF-alpha and binds the EGFs with approximately 100-fold lower affinity. The regions responsible for this poor binding are known to be Arg(45) in hEGF and the L2 domain in the chicken EGFR. In this study we have produced a truncated form of the hEGFR ectodomain comprising residues 1-501 (sEGFR501), which, unlike the full-length hEGFR ectodomain (residues 1-621, sEGFR621), binds hEGF and hTGF-alpha with high affinity (K(D) = 13-21 and 35-40 nM, respectively). sEGFR501 was a competitive inhibitor of EGF-stimulated mitogenesis, being almost 10-fold more effective than the full-length EGFR ectodomain and three times more potent than the neutralizing anti-EGFR monoclonal antibody Mab528. Analytical ultracentrifugation showed that the primary EGF binding sites on sEGFR501 were saturated at an equimolar ratio of ligand and receptor, leading to the formation of a 2:2 EGF:sEGFR501 dimer complex. We have used sEGFR501 to generate three mutants with single position substitutions at Glu(367), Gly(441), or Glu(472) to Lys, the residue found in the corresponding positions in the chicken EGFR. All three mutants bound hTGF-alpha and were recognized by Mab528. However, mutant Gly(441)Lys showed markedly reduced binding to hEGF, implicating Gly(441), in the L2 domain, as part of the binding site that recognizes Arg(45) of hEGF.

The molecular chaperone, alpha-crystallin, inhibits amyloid formation by apolipoprotein C-II.

Under lipid-free conditions, human apolipoprotein C-II (apoC-II) exists in an unfolded conformation that over several days forms amyloid ribbons. We examined the influence of the molecular chaperone, alpha-crystallin, on amyloid formation by apoC-II. Time-dependent changes in apoC-II turbidity (at 0.3 mg/ml) were suppressed potently by substoichiometric subunit concentrations of alpha-crystallin (1-10 microg/ml). alpha-Crystallin also inhibits time-dependent changes in the CD spectra, thioflavin T binding, and sedimentation coefficient of apoC-II. This contrasts with stoichiometric concentrations of alpha-crystallin required to suppress the amorphous aggregation of stressed proteins such as reduced alpha-lactalbumin. Two pieces of evidence suggest that alpha-crystallin directly interacts with amyloidogenic intermediates. First, sedimentation equilibrium and velocity experiments exclude high affinity interactions between alpha-crystallin and unstructured monomeric apoC-II. Second, the addition of alpha-crystallin does not lead to the accumulation of intermediate sized apoC-II species between monomer and large aggregates as indicated by gel filtration and sedimentation velocity experiments, suggesting that alpha-crystallin does not inhibit the relatively rapid fibril elongation upon nucleation. We propose that alpha-crystallin interacts stoichiometrically with partly structured amyloidogenic precursors, inhibiting amyloid formation at nucleation rather than the elongation phase. In doing so, alpha-crystallin forms transient complexes with apoC-II, in contrast to its chaperone behavior with stressed proteins.

Sedimentation analysis of novel DNA structures formed by homo-oligonucleotides.

Sedimentation velocity analysis has been used to examine the base-specific structural conformations and unusual hydrogen bonding patterns of model oligonucleotides. Homo-oligonucleotides composed of 8-28 residues of dA, dT, or dC nucleotides in 100 mM sodium phosphate, pH 7.4, at 20 degrees C behave as extended monomers. Comparison of experimentally determined sedimentation coefficients with theoretical values calculated for assumed helical structures show that dT and dC oligonucleotides are more compact than dA oligonucleotides. For dA oligonucleotides, the average width (1.7 nm), assuming a cylindrical model, is smaller than for control duplex DNA whereas the average rise per base (0.34 nm) is similar to that of B-DNA. For dC and dT oligonucleotides, there is an increase in the average widths (1.8 nm and 2.1 nm, respectively) whereas the average rise per base is smaller (0.28 nm and 0.23 nm, respectively). A significant shape change is observed for oligo dC(28) at lower temperatures (10 degrees C), corresponding to a fourfold decrease in axial ratio. Optical density, circular dichroism, and differential scanning calorimetry data confirm this shape change, attributable from nuclear magnetic resonance analysis to i-motif formation. Sedimentation equilibrium studies of oligo dG(8) and dG(16) reveal extensive self-association and the formation of G-quadruplexes. Continuous distribution analysis of sedimentation velocity data for oligo dG(16) identifies the presence of discrete dimers, tetramers, and dodecamers. These studies distinguish the conformational and colligative properties of the individual bases in DNA and their inherent capacity to promote specific folding pathways.

The interaction of human apolipoprotein C-I with sub-micellar phospholipid.

Mature human apolipoprotein C-I (apoC-I), comprising 57 amino acids, is the smallest member of the plasma apolipoprotein family. Amphipathic helical regions within apoC-I, common to this class of proteins, are mediators of lipid binding, a process that underlies the functional properties of apoC-I, including the capacity to activate the plasma enzyme LCAT, to disrupt apoE mediated receptor interactions and to inhibit cholesterol ester transfer protein. To examine apoC-I/phospholipid interactions, we have developed an expression system in Escherichia coli to obtain purified apoC-I with yields of approximately 4-5 mg per L of culture. The purified product has properties similar to plasma-derived apoC-I including self-association in the lipid-free state and induced alpha-helical content in the presence of egg-yolk phosphatidylcholine and dimyristoylglycerophosphocholine vesicles. We chose the short-chain phospholipid, dihexanoylglycerophosphocholine (Hex2Gro-PCho), to examine the interaction of apoC-I with submicellar phospholipid. Circular dichroism spectroscopy and cross-linking experiments show that apoC-I acquires helical content and remains self-associated at submicellar concentrations of Hex2Gro-PCho (4 mM). Sedimentation equilibrium studies of apoC-I at submicellar levels of Hex2Gro-PCho and analysis of the effects of apoC-I on the 1H NMR spectrum of Hex2Gro-PCho indicate micelle induction by apoC-I, and establish the capacity of apoC-I to assemble individual phospholipid molecules.

NMR structure of human apolipoprotein C-II in the presence of sodium dodecyl sulfate.

The structure and protein-detergent interactions of apolipoprotein C-II (apoC-II) in the presence of SDS micelles have been investigated using circular dichroism and heteronuclear NMR techniques applied to (15)N-labeled protein. Micellar SDS, a commonly used mimetic of the lipoprotein surface, inhibits the aggregation of apoC-II and induces a stable structure containing approximately 60% alpha-helix as determined by circular dichroism. NMR reveals the first 12 residues of apoC-II to be structurally heterogeneous and largely disordered, with the rest of the protein forming a predominantly helical structure. Three regions of helical conformation, residues 16-36, 50-56, and 63-77, are well-defined by NMR-derived constraints, with the intervening regions showing more loosely defined helical conformation. The structure of apoC-II is compared to that determined for other apolipoproteins in a similar environment. Our results shed light on the lipid interactions of apoC-II and its mechanism of lipoprotein lipase activation.

Sub-micellar phospholipid accelerates amyloid formation by apolipoprotein C-II.

Lipid-free human apolipoprotein C-II (apoC-II) forms amyloid fibrils with characteristic beta-structure. This conformation is distinct from the alpha-helical fold of lipid-bound apoC-II. We have investigated the effect of the short-chain phospholipid, dihexanoylphosphatidylcholine (DHPC) on amyloid formation by apoC-II. The alpha-helical content of apoC-II increases in the presence of micellar DHPC (16 mM) and amyloid formation is inhibited. However, at sub-micellar DHPC concentrations (below 8 mM) amyloid formation is accelerated 6 fold. These results suggest that individual phospholipid molecules in vivo may exert significant effects on amyloid folding pathways.

Self-association of human apolipoprotein E3 and E4 in the presence and absence of phospholipid.

Human apolipoprotein E (apoE) exists as three main isoforms, differing by single amino acid substitutions, with the apoE4 isoform strongly linked to the incidence of late onset Alzheimer's disease. We have expressed and purified apoE3 and apoE4 from Escherichia coli and compared their hydrodynamic properties by gel permeation liquid chromatography, capillary electrophoresis, circular dichroism, and sedimentation methods. Sedimentation velocity experiments, employing a new method for determining the size distribution of polydisperse macromolecules in solution (Schuck, P. (2000) Biophys. J. 78, 1606-1619), provide direct evidence for the heterogeneous solution structures of apoE3 and apoE4. In a lipid-free environment, apoE3 and apoE4 exist as a slow equilibrium mixture of monomer, tetramer, octamer, and a small proportion of higher oligomers. Both sedimentation velocity and equilibrium experiments indicate that apoE4 has a greater propensity to self-associate. We also demonstrate that apoE3 and apoE4 oligomers dissociate significantly in the presence of dihexanoylphosphatidylcholine micelles (20 mm) and to a lesser extent at submicellar concentrations (4 mm). The alpha-helical content for both isoforms was almost identical (50%) in the presence and absence of dihexanoylphosphatidylcholine. These results reveal that apoE oligomers undergo phospholipid-induced dissociation to folded monomers, suggesting the monomeric form prevails on the lipoprotein surface in vivo.

Human apolipoprotein C-II forms twisted amyloid ribbons and closed loops.

Human apolipoprotein C-II (apoC-II) self-associates in solution to form aggregates with the characteristics of amyloid including red-green birefringence in the presence of Congo Red under cross-polarized light, increased fluorescence in the presence of thioflavin T, and a fibrous structure when examined by electron microscopy. ApoC-II was expressed and purified from Escherichia coli and rapidly exchanged from 5 M guanidine hydrochloride into 100 mM sodium phosphate, pH 7.4, to a final concentration of 0.3 mg/mL. This apoC-II was initially soluble, eluting as low molecular weight species in gel filtration experiments using Sephadex G-50. Circular dichroism (CD) spectroscopy indicated predominantly unordered structure. Upon incubation for 24 h, apoC-II self-associated into high molecular weight aggregates as indicated by elution in the void volume of a Sephadex G-50 column, by rapid sedimentation in an analytical ultracentrifuge, and by increased light scattering. CD spectroscopy indicated an increase in beta-sheet content, while fluorescence emission spectroscopy of the single tryptophan revealed a blue shift and an increase in maximum intensity, suggesting repositioning of the tryptophan into a less polar environment. Electron microscopy of apoC-II aggregates revealed a novel looped-ribbon morphology (width 12 nm) and several isolated closed loops. Like all of the conserved plasma apolipoproteins, apoC-II contains amphipathic helical regions that account for the increase in alpha-helix content on lipid binding. The increase in beta-structure accompanying apoC-II fibril formation points to an alternative folding pathway and an in vitro system to explore the general tendency of apolipoproteins to form amyloid in vivo.

Stoichiometry, kinetic and binding analysis of the interaction between epidermal growth factor (EGF) and the extracellular domain of the EGF receptor.

The kinetics, binding equilibria and stoichiometry of the interaction between epidermal growth factor and the soluble extracellular domain of the epidermal growth factor receptor (sEGFR), produced in CHO cells using a bioreactor, have been studied by three methods: analytical ultracentrifugation, biosensor analysis using surface plasmon resonance detection (BIAcore 2000) and fluorescence anisotropy. These studies were performed with an sEGFR preparation purified in the absence of detergent using a mild two step chromatographic procedure employing anion exchange and size exclusion HPLC. The fluorescence anisotropy and analytical ultracentrifugation data indicated a 1:1 molar binding ratio between EGF and the sEGFR. Analytical ultracentrifugation further indicated that the complex comprised 2EGF:2sEGFR, consistent with the model proposed recently by Lemmon et al. (1997). Global analysis of the BIAcore binding data showed that a simple Langmuirian interaction does not adequately describe the EGF:sEGFR interaction and that more complex interaction mechanisms are operative. Furthermore, analysis of solution binding data using either fluorescence anisotropy or the biosensor, to determine directly the concentration of free sEGFR in solution competition experiments, yielded Scatchard plots which were biphasic and Hill coefficients of less than unity. Taken together our data indicate that in solution there are two sEGFR populations; one which binds EGF with a KD of 2-20 nM and the other with a KD of 400-550 nM.

Peptide self-association in aqueous trifluoroethanol monitored by pulsed field gradient NMR diffusion measurements.

Defining the self-association state of a molecule in solution can be an important step in NMR-based structure determination. This is particularly true of peptides, where there can be a relatively small number of long-range interactions and misinterpretation of an intermolecular NOE as an intramolecular contact can have a dramatic influence on the final calculated structure. In this paper, we have investigated the use of translational self-diffusion coefficient measurements to detect self-association in aqueous trifluoroethanol of three peptides which are analogues of the C-terminal region of human neuropeptide Y. Experimentally measured diffusion coefficients were extrapolated to D0, the limiting value as the peptide concentration approaches zero, and then converted to D(20,w), the diffusion coefficient after correction for temperature and the viscosity of the solvent. A decrease in D(20,w) of about 16% was found for all three peptides in aqueous TFE (30% by volume) compared with water, which is in reasonable agreement with the expected decrease upon dimerisation, the presence of which was indicated by sedimentation equilibrium measurements. Apparent molecular masses of these peptides in both solutions were also calculated from their diffusion coefficients and similar results were obtained. Several potential internal standards, including acetone, acetonitrile, dimethylsulfoxide and dioxane, were assessed as monitors of solution viscosity over a range of trifluoroethanol concentrations. Compared with independent measurements of viscosity, acetonitrile was the most accurate standard among these four. The practical limitations of a quantitative assessment of peptide self-association from translational diffusion coefficients measured by PFGNMR, including the calculation of apparent molecular mass, are also discussed.

Apolipoprotein C-II39-62 activates lipoprotein lipase by direct lipid-independent binding.

Apolipoprotein C-II (apoC-II) is an exchangeable plasma apolipoprotein and an endogenous activator of lipoprotein lipase (LpL). Genetic deficiencies of apoC-II and overexpression of apoC-II in transgenic mice are both associated with severe hyperlipidemia, indicating a complex role for apoC-II in the regulation of blood lipid levels. ApoC-II exerts no effect on the activity of LpL for soluble substrates, suggesting that activation occurs via the formation of a lipid-bound complex. We have synthesized a peptide corresponding to amino acid residues 39-62 of mature human apoC-II. This peptide does not bind to model lipid surfaces but retains the ability to activate LpL. Conjugation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) to the N-terminal alpha-amino group of apoC-II39-62 facilitated determination of the affinity of the peptide for LpL using fluorescence anisotropy measurements. The dissociation constant describing this interaction was 0.23 microM, and was unchanged when LpL was lipid-bound. Competitive binding studies showed that apoC-II39-62 and full-length apoC-II exhibited the same affinity for LpL in aqueous solution, whereas the affinity for full-length apoC-II was increased at least 1 order of magnitude in the presence of lipid. We suggest that while the binding of apoC-II to the lipid surface promotes the formation of a high-affinity complex of apoC-II and LpL, activation occurs via direct helix-helix interactions between apoC-II39-62 and the loop covering the active site of LpL.

Helical structure and self-association in a 13 residue neuropeptide Y Y2 receptor agonist: relationship to biological activity.

The solution structure and self-association behaviour of a 13 residue peptide analogue of the C-terminal region of human neuropeptide Y (NPY) have been investigated. NMR analysis of Ac[Leu(28,31)]NPY(24-36), a potent Y2 receptor agonist, shows that it is unstructured in aqueous solution at 5-20 degrees C, but forms a well-defined helix (encompassing residues 25-35) in 40% trifluoroethanol/water at 20 degrees C. Sedimentation experiments show that, in contrast to many peptides in aqueous trifluoroethanol, Ac[Leu(28,31)]NPY(24-36) associates to form a trimer or, more likely, a tetramer in 40% trifluoroethanol, even though it is monomeric in water. This is consistent with the observation of inter-molecular nuclear Overhauser enhancements in trifluoroethanol. Possible models of the associated form that are consistent with the NMR data are described. The relevance of the helical structure observed in trifluoroethanol to the structure of this peptide bound to the NPY Y2 receptor is discussed.

Mass spectrometry to characterize the binding of a peptide to a lipid surface.

The binding of an amphipathic alpha-helical peptide to small unilamellar lipid vesicles has been examined using chemical derivitization and mass spectrometry. The peptide is derived from the sequence of human apolipoprotein C-II (apoC-II), the protein activator of lipoprotein lipase (LpL). ApoC-II(19-39) forms approximately 60% alpha-helix upon binding to model egg yolk phosphatidylcholine small unilamellar vesicles. Measurement of the affinity of the peptide for lipid by spectrophotometric methods is complicated by the contribution of scattered light to optical signals. Instead, we characterize the binding event using the differential labeling of lysine residues by the lipid- and aqueous-phase cross-linkers, disuccinimidyl suberate (DSS) and bis(sulfosuccinimidyl) suberate (BS(3)), respectively. In aqueous solution, the three lysine residues of the peptide are accessible to both cross-linkers. In the presence of lipid, the C-terminal lysine residue becomes inaccessible to the lipid-phase cross-linker DSS, but remains accessible to the aqueous-phase cross-linker, BS(3). We use mass spectrometry to characterize this binding event and to derive a dissociation constant for the interaction (K(d) = 5 microM). We also provide evidence for the formation of dimeric cross-linked peptide when high densities of peptide are bound to the lipid surface.

Helix-helix association of a lipid-bound amphipathic alpha-helix derived from apolipoprotein C-II.

The interaction of a peptide derived from the sequence of apolipoprotein C-II (apoC-II) with a model lipid surface has been investigated by fluorescence spectroscopy. ApoC-II19-39, labeled at the N-terminus with 7-nitrobenz-2-oxa-1,3-diazole (NBD), bound to small unilamellar vesicles of phosphatidylcholine with a dissociation constant of 6 microM. The lipid-bound NBD-labeled peptide exhibited a red-edge excitation shift in its emission maximum and anisotropy, consistent with insertion of the probe into the motionally restricted, polar environment provided by the bilayer interface. The small Stokes shift of the NBD fluorophore permits electronic energy homotransfer between peptides on the lipid surface and results in depolarization of the NBD emission. At high surface densities of lipid-bound peptide, the anisotropy of the NBD probe was 33% lower than in corresponding samples in which electronic energy homotransfer was prevented by the addition of an unlabeled peptide. The efficiency of energy transfer between probes was not consistent with a random distribution of peptides on the lipid surface, indicating instead the self-association of lipid-bound apoC-II19-39. We propose that the role of this sequence in apoC-II is not only to mediate binding of protein to a lipid surface, but also to stabilize the lipoprotein complexes by associating with other amphipathic helices within apoC-II and with other apolipoproteins.

Physicochemical characterization of an antagonistic human interleukin-6 dimer.

A noncovalently bound dimeric form of recombinant human IL-6 interleukin-6 (IL-6D) was shown to be an antagonist for IL-6 activity, in a STAT3 tyrosine phosphorylation assay using HepG2 cells, under conditions where it does not dissociate into monomeric IL-6 (IL-6M). The fluorescence from Trp157, the single tryptophan residue in the primary sequence of IL-6, is altered in IL-6D, where the wavelength maximum is blue-shifted by 3 nm and the emission intensity is reduced by 30%. These data suggest that Trp157 is close to, but not buried by, the dimer interface. Both IL-6D and IL-6M are compact molecules, as determined by sedimentation velocity analysis, and contain essentially identical levels of secondary and tertiary structure, as determined by far- and near-UV CD, respectively. IL-6D and IL-6M show the same susceptibility to limited proteolytic attack, and exhibit identical far-UV CD-monitored urea-denaturation profiles with the midpoint of denaturation occurring at 6.0 +/- 0.1 M urea. However, IL-6D was found to dissociate prior to the complete unfolding of the protein, with a midpoint of dissociation of 3 M urea, suggesting that dissociation and dimerization occur when the protein is in a partially unfolded state. Based on these results, we suggest that IL-6D is a metastable domain-swapped dimer, comprising two monomeric units where identical helices from each protein chain are swapped through the loop regions at the "top" of the protein (i.e., the region of the protein most distal from the N- and C-termini). Such an arrangement would account for the antagonistic activity of IL-6D. In this model, receptor binding site I, which comprises residues in the A/B loop and the C-terminus of the protein, is free to bind the IL-6 receptor. However, site III, which includes Trp157 and residues in the C/D loop and N-terminal end of helix D, and perhaps site II, which comprises residues in the A and C helices, are no longer able to bind the signal transducing component of the IL-6 receptor complex, gp130.

Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein.

We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type 1 (HTLV-1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C-terminal alpha-helix to the predicted N-terminal alpha-helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X-ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N-terminal alpha-helical sequences.