Self-association of helical peptides in a lipid environment

The self-association of two model transmembrane helical peptides, differing in their surface topography, was compared in mixed micelles containing 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate (CHAPS) and dimyristoylphosphatidylcholine (DMPC). One peptide, Ac-KKL₂₄KK-amide (L₂₄), has large, rotationally mobile leucine side chains and a relatively rough surface. The other peptide, Ac-KKLLLLLLAALLALLAALLALLLLLLKK-amide (L₁₈A₆), has a patch of small alanines on one side of the helix that forms a smooth surface. The aggregation state of the peptides was sampled by chemical cross-linking with bis-sulfosuccinimidyl suberate (B53). A monomer-aggregate association constant was obtained from the cross-linking results in the range of 2 × 10⁵ M^–1 to 3 × 10⁵ M^–1 for both peptides. Kinetics of formation of cross-linked dimers indicated that the ratio of dimerization constants for L₁₈A₆ to L₂₄ was between 10 and 20. This suggests that the alanine patch contributes about 1.5 Kcal/mol more stabilization free energy to dimer formation of L₁₈A₆ compared to L₂₄.     

Functional analysis of the chromo domain of HP1.

Heterochromatin protein 1 (HP1) is a non-histone chromosomal protein in Drosophila with dosage-dependent effects on heterochromatin-mediated gene silencing. An evolutionarily conserved amino acid sequence in the N-terminal half of HP1 (the 'chromo domain') shares > 60% sequence identity with a motif found in the Polycomb protein, a silencer of homeotic genes. We report here that point mutations in the HP1 chromo domain abolish the ability of HP1 to promote gene silencing. We show that the HP1 chromo domain, like the Polycomb chromo domain, has chromosome binding activity, but to distinct chromosomal sites. We constructed a chimeric HP1-Polycomb protein, consisting of the chromo domain of Polycomb in the context of HP1, and show that it binds to both heterochromatin and Polycomb binding sites in polytene chromosomes. In flies expressing chimeric HP1-Polycomb protein, endogenous HP1 is mislocalized to Polycomb binding sites, and endogenous polycomb is misdirected to the heterochromatic chromocenter, suggesting that both proteins are recruited to their distinct chromosomal binding sites through protein-protein contacts. Chimeric HP1-Polycomb protein expression in transgenic flies promotes heterochromatin-mediated gene silencing, supporting the view that the chromo domain homology reflects a common mechanistic basis for homeotic and heterochromatic silencing.     

Structure of the chromo barrel domain from the MOF acetyltransferase

We report here the structure of the putative chromo domain from MOF, a member of the MYST family of histone acetyltransferases that acetylates histone H4 at Lys-16 and is part of the dosage compensation complex in Drosophila. We found that the structure of this domain is a beta-barrel that is distinct from the alpha + beta fold of the canonical chromo domain. Despite the differences, there are similarities that support an evolutionary relationship between the two domains, and we propose the name "chromo barrel." The chromo barrel domains may be divided into two groups, MSL3-like and MOF-like, on the basis of whether a group of conserved aromatic residues is present or not. The structure suggests that, although the MOF-like domains may have a role in RNA binding, the MSL3-like domains could instead bind methylated residues. The MOF chromo barrel shares a common fold with other chromatin-associated modules, including the MBT-like repeat, Tudor, and PWWP domains. This structural similarity suggests a probable evolutionary pathway from these other modules to the canonical chromo domains (or vice versa) with the chromo barrel domain representing an intermediate structure.     

Self-association of collagen triple helic peptides into higher order structures

Interest in self-association of peptides and proteins is motivated by an interest in the mechanism of physiologically higher order assembly of proteins such as collagen as well as the mechanism of pathological aggregation such as beta-amyloid formation. The triple helical form of (Pro-Hyp-Gly)(10), a peptide that has proved a useful model for molecular features of collagen, was found to self-associate, and its association properties are reported here. Turbidity experiments indicate that the triple helical peptide self-assembles at neutral pH via a nucleation-growth mechanism, with a critical concentration near 1 mM. The associated form is more stable than individual molecules by about 25 degrees C, and the association is reversible. The rate of self-association increases with temperature, supporting an entropically favored process. After self-association, (Pro-Hyp-Gly)(10) forms branched filamentous structures, in contrast with the highly ordered axially periodic structure of collagen fibrils. Yet a number of characteristics of triple helix assembly for the peptide resemble those of collagen fibril formation. These include promotion of fibril formation by neutral pH and increasing temperature; inhibition by sugars; and a requirement for hydroxyproline. It is suggested that these similar features for peptide and collagen self-association are based on common lateral underlying interactions between triple helical molecules mediated by hydrogen-bonded hydration networks involving hydroxyproline.     

The HP1 chromo shadow domain binds a consensus peptide pentamer

Heterochromatin-associated protein 1 (HP1) is thought to affect chromatin structure through interactions with other proteins in heterochromatin. Chromo domains located near the amino (amino chromo) and carboxy (chromo shadow) termini of HP1 may mediate such interactions, as suggested by domain swapping, in vitro binding and 3D structural studies . Several HP1-associated proteins have been reported, providing candidates that might specifically complex with the chromo domains of HP1. However, such association studies provide little mechanistic insight and explore only a limited set of potential interactions in a largely non-competitive setting. To determine how chromo domains can selectively interact with other proteins, we probed random peptide phage display libraries using chromo domains from HP1. Our results demonstrate that a consensus pentapeptide is suffident for specific interaction with the HP1 chromo shadow domain. The pentapeptide is found in the amino acid sequence of reported HP1-associated proteins, including the shadow domain itself. Peptides that bind the shadow domain also disrupt shadow domain dimers. Our results suggest that HP1 dimerization, which is thought to mediate heterochromatin compaction and cohesion, occurs via pentapeptide binding. In general, chromo domains may function by avidly binding short peptides at the surface of chromatin-associated proteins.     

Self-association of the amino-terminal domain of the yeast TATA-binding protein

The amino-terminal domain of yeast TATA-binding protein has been proposed to play a crucial role in the self-association mechanism(s) of the full-length protein. Here we tested the ability of this domain to self-associate under a variety of solution conditions. Escherichia coli two-hybrid assays, in vitro pull-down assays, and in vitro cross-linking provided qualitative evidence for a limited and specific self-association. Sedimentation equilibrium analysis using purified protein was consistent with a monomer-dimer equilibrium with an apparent dissociation constant of approximately 8.4 microM. Higher stoichiometry associations remain possible but could not be detected by any of these methods. These results demonstrate that the minimal structure necessary for amino-terminal domain self-association must be present even in the absence of carboxyl-terminal domain structures. On the basis of these results we propose that amino-terminal domain structures contribute to the oligomerization interface of the full-length yeast TATA-binding protein.     

Molecular biology of the chromo domain: an ancient chromatin module comes of age

The chromo domain motif is found in proteins from fungi, protists, plants, fish, insects, amphibians, birds, and mammals. The chromo domain peptide fold may have its origins as a chromosomal protein in a common ancestor of archea and eukaryota, making it a particularly ancient protein structural module. Chromo domains have been found in single or multiple copies in proteins with diverse structures and activities, most or all of which are connected with chromosome structure/function. In this review, our current knowledge of chromo domain properties is summarized and a variety of contexts in which chromo domains participate in aspects of chromatin metabolism are discussed.     

Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3

Recent studies show that heterochromatin-associated protein-1 (HP1) recognizes a 'histone code' involving methylated Lys9 (methyl-K9) in histone H3. Using in situ immunofluorescence, we demonstrate that methyl-K9 H3 and HP1 co-localize to the heterochromatic regions of Drosophila polytene chromosomes. NMR spectra show that methyl-K9 binding of HP1 occurs via its chromo (chromosome organization modifier) domain. This interaction requires methyl-K9 to reside within the proper context of H3 sequence. NMR studies indicate that the methylated H3 tail binds in a groove of HP1 consisting of conserved residues. Using fluorescence anisotropy and isothermal titration calorimetry, we determined that this interaction occurs with a K(D) of approximately 100 microM, with the binding enthalpically driven. A V26M mutation in HP1, which disrupts its gene silencing function, severely destabilizes the H3-binding interface, and abolishes methyl-K9 H3 tail binding. Finally, we note that sequence diversity in chromo domains may lead to diverse functions in eukaryotic gene regulation. For example, the chromo domain of the yeast histone acetyltransferase Esa1 does not interact with methyl- K9 H3, but instead shows preference for unmodified H3 tail.     

The hinge and chromo shadow domain impart distinct targeting of HP1-like proteins

Drosophila heterochromatin-associated protein 1 (HP1) is an abundant component of heterochromatin, a highly condensed compartment of the nucleus that comprises a major fraction of complex genomes. Some organisms have been shown to harbor multiple HP1-like proteins, each exhibiting spatially distinct localization patterns within interphase nuclei. We have characterized the subnuclear localization patterns of two newly discovered Drosophila HP1-like proteins (HP1b and HP1c), comparing them with that of the originally described fly HP1 protein (here designated HP1a). While HP1a targets heterochromatin, HP1b localizes to both heterochromatin and euchromatin and HP1c is restricted exclusively to euchromatin. All HP1-like proteins contain an amino-terminal chromo domain, a connecting hinge, and a carboxyl-terminal chromo shadow domain. We expressed truncated and chimeric HP1 proteins in vivo to determine which of these segments might be responsible for heterochromatin-specific and euchromatin-specific localization. Both the HP1a hinge and chromo shadow domain independently target heterochromatin, while the HP1c chromo shadow domain is implicated solely in euchromatin localization. Comparative sequence analyses of HP1 homologs reveal a conserved sequence block within the hinge that contains an invariant sequence (KRK) and a nuclear localization motif. This block is not conserved in the HP1c hinge, possibly accounting for its failure to function as an independent targeting segment. We conclude that sequence variations within the hinge and shadow account for HP1 targeting distinctions. We propose that these targeting features allow different HP1 complexes to be distinctly sequestered in organisms that harbor multiple HP1-like proteins.     

Self-association of the low density lipoprotein receptor mediated by the cytoplasmic domain

When the low density lipoprotein (LDL) receptor was solubilized from bovine adrenal cortex membranes and subjected to electrophoresis in the absence of reducing agents, a disulfide-bonded dimeric species was demonstrated. Formation of these covalent bonds was blocked when the tissue was homogenized in the presence of sulfhydryl alkylating agents, indicating that the native receptor was self-associated noncovalently and that the disulfide bond formation occurred only after homogenization. The disulfide-linked dimers were disrupted and the receptor was restored to a monomeric form when inside-out adrenal vesicles were treated with trypsin, suggesting that the disulfide bond formation involved the 50-amino acid cytoplasmic domain of the receptor. When the receptor was solubilized from bovine adrenal cortex membranes and then purified by ion exchange and affinity chromatography, it could be covalently coupled into dimers and trimers in the presence of bivalent cross-linking agents. Receptor dimers could also be demonstrated by chemical cross-linking of intact cells that were transfected with an expressible cDNA encoding the normal human LDL receptor. Dimer formation was markedly reduced in transfected cells expressing mutated cDNAs that had premature termination codons at positions 792, 807, and 812, which produced shortened receptors that retained 2, 17, and 22 of the original 50 amino acids of the cytoplasmic domain, respectively. The first two mutant receptors, which did not form oligomers, did not enter coated pits and were not rapidly internalized by cells. However, the mutant receptor that terminates at position 812 was internalized normally even though oligomer formation was greatly reduced. Moreover, a mutant receptor with a cysteine substituted for a tyrosine at position 807, which internalized very slowly, showed a normal susceptibility to chemical cross-linking. Deletion of external domains of the LDL receptor, including the epidermal growth factor homology region and the O-linked sugar domain, did not alter susceptibility to chemical cross-linking. We conclude that the cytoplasmic domain of the LDL receptor is responsible both for self-association into oligomers and for clustering in coated pits, but the available data do not establish a causal connection between these two events.     

Structure of the chromatin binding (chromo) domain from mouse modifier protein 1.

The structure of a chromatin binding domain from mouse chromatin modifier protein 1 (MoMOD1) was determined using nuclear magnetic resonance (NMR) spectroscopy. The protein consists of an N-terminal three-stranded anti-parallel beta-sheet which folds against a C-terminal alpha-helix. The structure reveals an unexpected homology to two archaebacterial DNA binding proteins which are also involved in chromatin structure. Structural comparisons suggest that chromo domains, of which more than 40 are now known, act as protein interaction motifs and that the MoMOD1 protein acts as an adaptor mediating interactions between different proteins.     

Self-association studies on the EphB2 receptor SAM domain using analytical ultracentrifugation

The self-association behavior of the Eph-kinases SAM domain has been studied in phosphate buffer, pH 7.4, containing 0.14 M NaCl using concentration-dependent sedimentation equilibrium experiments. Only weak interactions typical for a monomer-dimer equilibrium up to at least 12 mg/mL were observed. Such concentrated solutions require a consideration of the non-ideality expressed by virial coefficients. A special centrifuge equation was used for the global analysis to estimate equilibrium constants based on the thermodynamic activities of the reactants. When neglecting this, the parameters deviate by about 20%. Association constants for dimerization of the EphB2-SAM domain vary between 163 M(-1) at 10 degrees C and 395 M(-1) at 32 degrees C, indicating hydrophobic forces are involved in the dimerization process. In solutions of about 12 mg/mL, less than 50% dimers are in solution and higher oligomers can be excluded.     

Self-association and lipid binding properties of the lipoprotein initiating domain of apolipoprotein B

The amino-terminal 20.1% of apolipoprotein B (apoB20.1; residues 1-912) is sufficient to initiate and direct the formation of nascent apoB-containing lipoprotein particles. To investigate the mechanism of initial lipid acquisition by apoB, we examined the lipid binding and interfacial properties of a carboxyl-terminal His6-tagged form of apoB20.1 (apoB20.1H). ApoB20.1H was expressed in Sf9 cells and purified by nickel affinity chromatography. ApoB20.1H was produced in a folded state as characterized by formation of intramolecular disulfide bonds and resistance to chemical reduction. Dynamic light scattering in physiological buffer indicated that purified apoB20.1H formed multimers, which were readily dissociable upon the addition of nonionic detergent (0.1% Triton X-100). ApoB20.1H was incapable of binding dimyristoylphosphatidylcholine multilamellar vesicles, unless its multimeric structure was first disrupted by guanidine hydrochloride. However, apoB20.1H multimers spontaneously dissociated and bound to the interface of naked and phospholipid-coated triolein droplets. These data reveal that the initiating domain of apoB contains solvent-accessible hydrophobic sequences, which, in the absence of a hydrophobic lipid interface or detergent, engage in self-association. The high affinity of apoB20.1H for neutral lipid is consistent with the membrane binding and desorption model of apoB-containing lipoprotein assembly.     

Biological evaluation of penetration domain and killing domain peptides

BACKGROUND: Cancer gene therapy must impact the majority of cells to be effective. Current gene delivery systems are unable to achieve sufficient transfer efficiency to the tumor cells. Cell killing can be dramatically increased through a bystander effect. Modeling the gene product with synthetic peptides can identify key elements for creating cell killing through a bystander effect. METHODS: Fluorescent labeled peptides were used for uptake kinetic studies and determination of intracellular localization in human glioblastoma cell lines, rat glioma cells lines and pressurized rat cerebral arteries. The degree of cell killing was assayed using propidium iodide coupled with fluorescence-activated cell sorting (FACS) analysis. RESULTS: Peptides derived from HIV Tat and Drosophila antennapedia homeodomain were taken up by all tumor and primary cells. Attachment of an Mdm-2-binding domain derived from P14(ARF) resulted in cell killing and was independent of domain orientation. Uptake kinetics showed rapid uptake for both tumor and primary cells equilibrating with the external media within 10 min. Intraluminal or extraluminal administration of peptides into pressurized cerebral arteries showed a lack of extravasation across the subbasement lamina. Assay of biological activity following intraluminal administration showed selective suppression of response to vasodilation with no effect on response by smooth muscle cells. CONCLUSIONS: The results from these studies identified: (1) a cell trafficking domain and a cytotoxic domain for killing brain tumor cells; (2) that cell killing was independent of the domain orientations with regard to the cell trafficking domain being at the C-terminus or N-terminus; and (3) that the dual domain peptide can also be taken up by endothelial cells as shown by the cerebral artery studies. Hence, localized expression of the cytotoxic gene has the potential to not only kill brain tumor cells, but also tumor endothelium, thus further increasing the effectiveness of the therapy.     

Self-association and mapping of the interaction domain of hepatitis E virus ORF3 protein

Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information on the basic biology of the virus exists. A small protein (approximately 13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. The N-terminal region of pORF3 is associated with the cytoskeleton using one of its hydrophobic domains. The C-terminal half of pORF3 is rich in proline residues and contains a putative src homology 3 (SH3) binding domain and a mitogen-activated protein kinase phosphorylation site. In this study, we demonstrate that pORF3 can homodimerize in vivo, using the yeast two-hybrid system. We have isolated a 43-amino-acid interaction domain of pORF3 which is capable of self-association in vivo and in vitro. The overlap of the dimerization domain with the SH3 binding and phosphorylation domains suggests that pORF3 may have a dimerization-dependent regulatory role to play in the signal transduction pathway.     

Self-association and solubility of peptides. Solvent-titration study of protected C-terminal segments of porcine secretin

Self-association of peptides (related to the C-terminal sequence of porcine secretin) in methylene chloride was disrupted by adding dimethylsulfoxide in increasing amounts. This structural transition was monitored by the disappearance of the amide-I C = O stretching band of strongly intermolecularly hydrogen-bonded molecules (1625-1630 cm-1) in the infrared absorption spectra. The effects induced by main-chain length and sequence, type of N alpha-protection, and concentration were assessed. Hexamethylphosphortriamide was compared for its structure-disrupting properties to dimethylsulfoxide. The increasing propensity to aggregate displayed by these peptides is paralleled by a decrease in their solubility. The impact of these results on the planning of peptide syntheses is briefly discussed.