Glycolipid membrane-binding domain of human erythrocyte acetylcholinesterase

The membrane-binding domain of human erythrocyte acetylcholinesterase is a small hydrophobic structure at the COOH-terminus of the enzyme subunits. Papain digestion cleaves a COOH-terminal dipeptide linked to the hydrophobic structure with the sequence His-Gly-ethanolamine-Z, where the ethanolamine is in amide linkage to the glycine and Z is a partially characterized glycolipid. This glycolipid includes a second residue of ethanolamine and a residue of glucosamine, both of which have free primary amino groups accessible to radiomethylation. The glycolipid also contains a carbohydrate residue or residues that bind to concanavalin A and nearly stoichiometric amounts of both palmitate and C22 unsaturated fatty acids. Similarities in this membrane-binding structure to those reported for trypanosome variant surface glycoproteins and Thy-1 glycoprotein suggest an important new category of posttranslational modifications involving the attachment of COOH-terminal glycolipid.     

Structural and membrane-binding properties of saposin D.

Saposin D is generated together with three similar proteins, saposins A, B and C, from a common precursor, called prosaposin, in acidic organelles such as late endosomes and lysosomes. Although saposin D has been reported to stimulate the enzymatic hydrolysis of sphingomyelin and ceramide, its physiological role has not yet been clearly established. In the present study we examined structural and membrane-binding properties of saposin D. At acidic pH, saposin D showed a great affinity for phospholipid membranes containing an anionic phospholipid such as phosphatidylserine or phosphatidic acid. The binding of saposin D caused destabilization of the lipid surface and, conversely, the association with the membrane markedly affected the fluorescence properties of saposin D. The presence of phosphatidylserine-containing vesicles greatly enhanced the intrinsic tyrosine fluorescence of saposin D, which contains tyrosines but not tryptophan residues. The structural properties of saposin D were investigated in detail using advanced MS analysis. It was found that the main form of saposin D consists of 80 amino acid residues and that the six cysteine residues are linked in the following order: Cys5-Cys78, Cys8-Cys72 and Cys36-Cys47. The disulfide pattern of saposin D is identical with that previously established for two other saposins, B and C, which also exhibit a strong affinity for lipids. The common disulfide structure probably has an important role in the interaction of these proteins with membranes. The analysis of the sugar moiety of saposin D revealed that the single N-glycosylation site present in the molecule is mainly modified by high-mannose-type structures varying from two to six hexose residues. Deglycosylation had no effect on the interaction of saposin D with phospholipid membranes, indicating that the glycosylation site is not related to the lipid-binding site. The association of saposin D with membranes was highly dependent on the composition of the bilayer. Neither ceramide nor sphingomyelin, sphingolipids whose hydrolysis is favoured by saposin D, promoted its binding, while the presence of an acidic phospholipid such as phosphatidylserine or phosphatidic acid greatly favoured the interaction of saposin D with vesicles at low pH. These results suggest that, in the acidic organelles where saposins are localized, anionic phospholipids may be determinants of the saposin D topology and, conversely, saposin D may affect the lipid organization of anionic phospholipid-containing membranes.     

Lipid-interacting properties of the N-terminal domain of human apolipoprotein C-III

The lipid-interacting properties of the N-terminal domain of human apolipoprotein C-III (apo C-III) were investigated. By molecular modeling, we predicted that the 6-20 fragment of apo C-III is obliquely orientated at the lipid/water interface owing to an asymmetric distribution of the hydrophobic residues when helical. This is characteristic of 'tilted peptides' originally discovered in viral fusion proteins and later in various proteins including some involved in lipoprotein metabolism. Since most tilted peptides were shown to induce liposome fusion in vitro, the fusogenic capacity of the 6-20 fragment of apo C-III was tested on unilamellar liposomes and compared with the well characterized SIV fusion peptide. Mutants were designed by molecular modeling to assess the role of the hydrophobicity gradient in the fusion. FTIR spectroscopy confirmed the predominantly helical conformation of the peptides in TFE solution and also in lipid-peptide complexes. Lipid-mixing experiments showed that the apo C-III (6-20) peptide is able to increase the fluorescence of a lipophilic fluorescent probe. The vesicle fusion was confirmed by core-mixing and leakage assays. The hydrophobicity gradient plays a key role in the fusion process because the mutant with no hydrophobic asymmetry but the same mean hydrophobicity as the wild type does not induce significant lipid fusion. The apo C-III (6-20) fragment is, however, less fusogenic than the SIV peptide, in agreement with their respective mean hydrophobicity. Since lipid fusion should not be the physiological function of the N-terminal domain of apo CIII, we suggest that its peculiar distribution of hydrophobic residues is important for the lipid-binding properties of apo C-III and should be involved in apolipoprotein and lipid exchanges crucial for triglyceride metabolism.     

Structural and functional characterization of the N-terminal domain of human Rad51D

Rad51D, one of five Rad51 paralogs, is required for homologous recombination and disruption of Holliday junctions with bloom syndrome protein (BLM) in vertebrates. The N-terminal domain of Rad51D is highly conserved in eukaryotic Rad51D orthologs and is essential for protein-protein interaction with XRCC2, but nothing is known about its individual structure or function. In this study, we determined the solution structure of the human Rad51D N-terminal domain (residues 1-83), which consists of four short helices flanked by long N- and C-terminal tails. Interestingly, the position of the N-terminal tail (residues 1-13) is fixed within the domain structure via several hydrophobic interactions between Leu4 and Thr27, Leu4 and Val28, and Val6 and Ile17. We show that the domain preferentially binds to ssDNA versus dsDNA and does not bind to a mobile Holliday junction by electrophoretic mobility shift assay. NMR titration and dynamics studies showed that human Rad51D-N interacts with ssDNA by positively charged and hydrophobic residues on its surface. The results suggest that the N-terminal domain of Rad51D is required for the ssDNA-specific binding function of human Rad51D and that the conserved N-terminal domains of other Rad51 paralogs may have distinguishable functions from each other in homologous recombination.     

Analysis of the properties of the N-terminal nucleotide-binding domain of human P-glycoprotein

Human P-glycoprotein, the MDR1 gene product, requires both Mg(2+)-ATP binding and hydrolysis to function as a drug transporter; however, the mechanism(s) defining these events is not understood. In the present study, we explored the nature of Mg(2+)-ATP binding in the N-terminal nucleotide-binding domain of human P-glycoprotein and identified the minimal functional unit required for specific ATP binding. Recombinant proteins encompassing amino acids within the region beginning at 348 and ending at 707 were expressed in Escherichia coli, purified from inclusion bodies under denaturing conditions, and renatured by rapid dilution. The ability of ATP to interact with these proteins was examined by use of the photoactive ATP analogue [alpha-(32)P]-8-azido-ATP. Photoaffinity labeling of recombinant proteins identified the region between amino acids 375 and 635 as the region necessary to obtain specific ATP-binding properties. Specific protein labeling was saturable, enhanced by Mg(2+), and inhibited by ATP. Recombinant proteins confined within the region beginning at amino acid 392 and ending at amino acid 590 demonstrated nonspecific [alpha-(32)P]-8-azido-ATP labeling. Nonspecific labeling was not enhanced by Mg(2+) and was inhibited only by high concentrations of ATP. Using a D555N mutated protein, we found that the conserved aspartate residue in the Walker B motif plays a role in magnesium-enhanced ATP-binding. Taken together, these data define the region of the N-terminal nucleotide-binding domain of P-glycoprotein that is required for specific ATP binding and suggest that magnesium may play a role in stabilizing the ATP-binding site.     

Structural study of the N-terminal domain of human MCM8/9 complex

1. Download : Download high-res image (201KB)
2. Download : Download full-size image

     

Structural analysis of the N-terminal domain of the human T-cell leukemia virus capsid protein

The N-terminal domain of the retroviral capsid (CA) protein is one of the least conserved regions encoded in the genome. Surprisingly, the three-dimensional structures of the CA from different genera exhibit alpha-helical structural features that are highly conserved. The N-terminal residues of the human immunodeficiency virus type 1 (HIV-1) and Rous sarcoma virus (RSV) capsid proteins form a beta-hairpin. To determine if this feature is conserved in the retroviral family, we cloned, expressed, purified, and solved the structure of a N-terminal 134 amino acid fragment (CA(134)) from the human T-cell leukemia virus type 1 (HTLV-I) using high resolution nuclear magnetic resonance (NMR) spectroscopy. The CA(134) fragment contains an N-terminal beta-hairpin and a central coiled-coil-like structure composed of six alpha-helices. The N-terminal Pro1 residue contacts Asp54 in the helical cluster through a salt bridge. Thus, the beta-hairpin is conserved and the helical cluster is structurally similar to other retroviral CA domains. However, although the same Asp residue defines the orientation of the hairpin in both the HTLV-1 and HIV-1 CA proteins, the HTLV-I hairpin is oriented away, rather than towards, the helical core. Significant differences were also detected in the spatial orientation and helical content of the long centrally located loop connecting the helices in the core. It has been proposed that the salt bridge allows the formation of a CA-CA interface that is important for the assembly of the conical cores that are characteristic of HIV-1. As HTLV-I forms spherical cores, the salt-bridge feature is apparently not conserved for this function although its role in determining the orientation of the beta-hairpin may be critical, along with the central loop. Comparison of three-dimensional structures is expected to elucidate the relationships between the retroviral capsid protein structure and its function.     

Structural analysis of the human galectin-9 N-terminal carbohydrate recognition domain reveals unexpected properties that differ from the mouse orthologue

Galectins are a family of β-galactoside-binding lectins that contain a conserved carbohydrate recognition domain (CRD). They exhibit high affinities for small β-galactosides as well as variable binding specificities for complex glycoconjugates. Structural and biochemical analyses of the mechanism governing specific carbohydrate recognition provide a useful template to elucidate the function of these proteins. Here we report the crystal structures of the human galectin-9 N-terminal CRD (NCRD) in the presence of lactose and Forssman pentasaccharide. Mouse galectin-9 NCRD, the structure of which was previously solved by our group, forms a non-canonical dimer in both the crystal state and in solution. Human galectin-9 NCRD, however, exists as a monomer in crystals, despite a high sequence identity to the mouse homologue. Comparative frontal affinity chromatography analysis of the mouse and human galectin-9 NCRDs revealed different carbohydrate binding specificities, with disparate affinities for complex glycoconjugates. Human galectin-9 NCRD exhibited a high affinity for Forssman pentasaccharide; the association constant for mouse galectin-9 NCRD was 100-fold less than that observed for the human protein. The combination of structural data with mutational studies demonstrated that non-conserved amino acid residues on the concave surface were important for determination of target specificities. The human galectin-9 NCRD exhibited greater inhibition of cell proliferation than the mouse NCRD. We discuss the biochemical and structural differences between highly homologous proteins from different species.     

Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase

The hydrophobic, membrane-binding domain of purified human erythrocyte acetylcholinesterase was labeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. The radiolabel was incorporated when the enzyme was prepared in detergent-free aggregates, in detergent micelles, or in phospholipid liposomes, but the highest percentage of labeling occurred in the detergent-free aggregates. Papain digestion of the enzyme released the hydrophobic domain, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate or gel exclusion chromatography demonstrated that the label was localized exclusively in the cleaved hydrophobic domain fragment. This fragment was purified in a three-step procedure. Digestion was conducted with papain attached to Sepharose CL-4B, and the supernatant was adsorbed to acridinium affinity resin to remove the hydrophilic enzyme fragment. The nonretained fragment associated with Triton X-100 micelles was then chromatographed on Sepharose CL-6B, and finally detergent was removed by chromatography on Sephadex LH-60 in an ethanol-formic acid solvent. The fragment exhibited an apparent molecular weight of 3100 on the Sephadex LH-60 column when compared with peptide standards. However, amino acid analysis of the purified fragment revealed only 1 mol each of histidine and glycine per mole of fragment in contrast to the 25-30 mole of amino acids expected on the basis of the molecular weight estimate. This result suggests a novel non-amino acid structure for the hydrophobic domain of human erythrocyte acetylcholinesterase.     

Supramolecular properties of the proline-rich gamma-Zein N-terminal domain

Zeins are maize storages proteins that accumulate inside large vesicles called protein bodies. gamma-Zein lines the inner face of the protein body membrane, and its N-terminal proline-rich repetitive domain with the sequence (VHLPPP)(8) appears to be necessary for the accumulation of the protein within the organelle. Synthetic (VHLPPP)(8) adopts an amphipathic polyproline II conformation. In a preliminary recent work we used atomic force microscopy to study the surface organization of the octamer and transmission electron microscopy to visualize aggregates of the peptide from aqueous solution. We previously envisioned two self-assembly models (i.e., the geometric and the micellar) that take into account the observed features. In the present work we studied in detail the self-assembly of the peptide in solution and found that the peptide is able to form cylindrical micelles. Fibrils formed on graphite are generated by assembly of solution micelles. Based on the results of these studies, we focused exclusively on the micellar model. To our knowledge we have characterized for the first time supramolecular aggregates of polyproline structures other than collagen. The spontaneous arrangement of (VHLPPP)(8) suggests a role for the N-terminal domain of gamma-zein in the process of the whole protein deposition in protein bodies.     

Structural analysis of the Sulfolobus solfataricus MCM protein N-terminal domain

The Mini-Chromosome Maintenance (MCM) proteins are candidates of replicative DNA helicase in eukarya and archaea. Here we report a 2.8 Å crystal structure of the N-terminal domain (residues 1–268) of the Sulfolobus solfataricus MCM (Sso MCM) protein. The structure reveals single-hexameric ring-like architecture, at variance from the protein of Methanothermobacter thermoautotrophicus (Mth). Moreover, the central channel in Sso MCM seems significantly narrower than the Mth counterpart, which appears to more favorably accommodate single-stranded DNA than double-stranded DNA, as supported by DNA-binding assays. Structural analysis also highlights the essential role played by the zinc-binding domain in the interaction with nucleic acids and allows us to speculate that the Sso MCM N-ter domain may function as a molecular clamp to grasp the single-stranded DNA passing through the central channel. On this basis possible DNA unwinding mechanisms are discussed.     

Insight into the location and dynamics of the annexin A2 N-terminal domain during Ca-induced membrane bridging

Annexin A2 (AnxA2) is a Ca²⁺- and phospholipid-binding protein involved in many cellular regulatory processes. Like other annexins, it is constituted by two domains: a conserved core, containing the Ca²⁺ binding sites, and a variable N-terminal segment, containing sites for interactions with other protein partners like S100A10 (p11). A wealth of data exists on the structure and dynamics of the core, but little is known about the N-terminal domain especially in the Ca²⁺-induced membrane-bridging process. To investigate this protein region in the monomeric AnxA2 and in the heterotetramer (AnxA2-p11)₂, the reactive Cys8 residue was specifically labelled with the fluorescent probe acrylodan and the interactions with membranes were studied by steady-state and time-resolved fluorescence. In membrane junctions formed by the (AnxA2-p11)₂ heterotetramer, the flexibility of the N-terminal domain increased as compared to the protein in solution. In “homotypic” membrane junctions formed by monomeric AnxA2, acrylodan moved to a more hydrophobic environment than in the protein in solution and the flexibility of the N-terminal domain also increased. In these junctions, this domain is probably not in close contact with the membrane surface, as suggested by the weak quenching of acrylodan observed with doxyl-PCs, but pairs of N-termini likely interact, as revealed by the excimer-forming probe pyrene-maleimide bound to Cys8. We present a model of monomeric AnxA2 N-terminal domain organization in “homotypic” bridged membranes in the presence of Ca²⁺.     

Structural analysis of the recognition mechanism of poly-N-acetyllactosamine by the human galectin-9 N-terminal carbohydrate recognition domain

Galectins are a family of beta-galactoside-specific lectins bearing a conserved carbohydrate recognition domain. Interactions between galectins and poly-N-acetyllactosamine sequences are critical in a variety of biological processes. Galectin-9, a member of the galectin family, has two carbohydrate recognition domains at both the N- and C-terminal regions. Here we report the crystal structure of the human galectin-9 N-terminal carbohydrate recognition domain in complex with N-acetyllactosamine dimers and trimers. These complex structures revealed that the galectin-9 N-terminal carbohydrate recognition domain can recognize internal N-acetyllactosamine units within poly-N-acetyllactosamine chains. Based on these complex structures, we propose two putative recognition modes for poly-N-acetyllactosamine binding by galectins.     

The N-terminal region of the human progesterone A-receptor. Structural analysis and the influence of the DNA binding domain

The role of the N-terminal region in nuclear receptor function was addressed by a biochemical and biophysical analysis of the progesterone receptor A-isoform lacking only the hormone binding domain (NT-A). Sedimentation studies demonstrate that NT-A is quantitatively monomeric, with a highly asymmetric shape. Contrary to dogma, the N-terminal region is structured as demonstrated by limited proteolysis. However, N-terminal structure is strongly stabilized by the DNA binding domain, possibly explaining the lack of structure seen in isolated activation domains. Upon DNA binding, NT-A undergoes N-terminal mediated assembly, suggestive of DNA-induced allostery, and consistent with changes in protease accessibility of sites outside the DNA binding domain. Microsequencing reveals that protease-accessible regions are limited to previously identified phosphorylation motifs and to functional domain boundaries.     

Role of the sterol carrier protein-2 N-terminal membrane binding domain in sterol transfer

Previous studies showed that the N-terminal 32 amino acids of sterol carrier protein-2 ((1-32)SCP(2)) comprise an amphipathic alpha-helix essential for SCP(2) binding to membranes [Huang et al. (1999) Biochemistry 38, 13231]. However, it is unclear whether membrane interaction of the (1-32)SCP(2) portion of SCP(2) is in itself sufficient to mediate intermembrane sterol transfer, possibly by altering membrane structure. In this study a fluorescent sterol exchange assay was used to resolve these issues and demonstrated that the SCP(2) N-terminal peptide (1-32)SCP(2) did not by itself enhance intermembrane sterol transfer but potentiated the ability of the SCP(2) protein to stimulate sterol transfer. Compared with SCP(2) acting alone, (1-32)SCP(2) potentiated the sterol transfer activity of SCP(2) by increasing the initial rate of sterol transfer by 2.9-fold and by decreasing the half-time of sterol transfer by 10-fold (from 11.6 to 1.2 min) without altering the size of the transferable fractions. The ability of a series of SCP(2) mutant N-terminal peptides to potentiate SCP(2)-mediated sterol transfer was directly correlated with membrane affinity of the respective peptide. N-Terminal peptide (1-32)SCP(2) did not potentiate intermembrane sterol transfer by binding sterol (dehydroergosterol), altering membrane fluidity (diphenylhexatriene) or membrane permeability (leakage assay). Instead, fluorescence lifetime measurements suggested that SCP(2) and (1-32)SCP(2) bound to membranes and thereby elicited a shift in membrane sterol microenvironment to become more polar. In summary, these data for the first time showed that while the N-terminal membrane binding domain of SCP(2) was itself inactive in mediating intermembrane sterol transfer, it nevertheless potentiated the ability of SCP(2) to enhance sterol transfer.     

Modulation of the lipid binding properties of the N-terminal domain of human apolipoprotein E3.

Apolipoprotein E (apoE) plays a critical role in plasma lipid homeostasis through its function as a ligand for the low-density lipoprotein (LDL) receptor family. Receptor recognition is mediated by residues 130-150 in the independently folded, 22-kDa N-terminal (NT) domain. This elongated globular four-helix bundle undergoes a conformational change upon interaction with an appropriate lipid surface. Unlike other apolipoproteins, apoE3 NT failed to fully protect human LDL from aggregation induced by treatment with phospholipase C. Likewise, in dimyristoylglycerophosphocholine (Myr2Gro-PCho) vesicle transformation assays, 100 microg apoE3 NT induced only 15% reduction in vesicle (250 microg) light scattering intensity after 30 min. ApoE3 NT interaction with modified lipoprotein particles or Myr2Gro-PCho vesicles was concentration-dependent whereas the vesicle transformation reaction was unaffected by buffer ionic strength. In studies with the anionic phospholipid dimyristoylglycerophosphoglycerol, apoE3 NT-mediated vesicle transformation rates were enhanced > 10-fold compared with Myr2Gro-PCho and activity decreased with increasing buffer ionic strength. Solution pH had a dramatic effect on the kinetics of apoE3 NT-mediated Myr2Gro-PCho vesicle transformation with increased rates observed as a function of decreasing pH. Fluorescence studies with a single tryptophan containing apoE3 NT mutant (L155W) revealed increased solvent exposure of the protein interior at pH values below 4.0. Similarly, fluorescent dye binding experiments with 8-anilino-1-naphthalene sulfonate revealed increased exposure of apoE3 NT hydrophobic interior as a function of decreasing pH. These studies indicate that apoE3 NT lipid binding activity is modulated by lipid surface properties and protein tertiary structure.     

The N-terminal domain of annexin 2 serves as a secondary binding site during membrane bridging

Annexin A2 (AnxA2) is a Ca(2+)- and acidic phospholipid-binding protein involved in many cellular processes. It undergoes Ca(2+)-mediated membrane bridging at neutral pH and has been demonstrated to be involved in an H(+)-mediated mechanism leading to a novel AnxA2-membrane complex structure. We used fluorescence techniques to characterize this H(+)-dependent mechanism at the molecular level; in particular, the involvement of the AnxA2 N-terminal domain. This domain was labeled at Cys-8 either with acrylodan or pyrene-maleimide fluorescent probes. Steady-state and time-resolved fluorescence analysis for acrylodan and fluorescence quenching by doxyl-labeled phospholipids revealed direct interaction between the N-terminal domain and the membrane. The absence of pyrene excimer suggested that interactions between N termini are not involved in the H(+)-mediated mechanism. These findings differ from those previously observed for the Ca(2+)-mediated mechanism. Protein titration experiments showed that the protein concentration for half-maximal membrane aggregation was twice for Ca(2+)-mediated compared with H(+)-mediated aggregation, suggesting that AnxA2 was able to bridge membranes either as a dimer or as a monomer, respectively. An N-terminally deleted AnxA2 was 2-3 times less efficient than the wild-type protein for H(+)-mediated membrane aggregation. We propose a model of AnxA2-membrane assemblies, highlighting the different roles of the N-terminal domain in the H(+)- and Ca(2+)-mediated membrane bridging mechanisms.     

Glycosylation of annexin I and annexin II.

Human placental annexin I and annexin II were shown to be glycosylated by one-dimensional affinity blotting with the lectin concanavalin A, which recognizes D-mannose and D-glucose residues. Further evidence that annexin I and annexin II are glycosylated was provided by the finding that these proteins incorporated D-[2,6-3H]mannose and D-[6-3H]glucose when they were biosynthesized by the human squamous carcinoma cell line SqCC/Y1. Annexin I and annexin II could be rapidly purified from a human placental membrane extract by concanavalin A-Sepharose, which indicated that these proteins contain two biantennary mannosyl residues.