Engineering of deltoid and reduced deltoid: Two chimeric proteins containing the oligomerization site of the hepatitis delta antigen

Hepatitis delta antigen (HDAg) must form oligomers to be biologically active. Quadrin (HDAg-(12–60)-Tyr) is a 50-residue protein segment from the oligomerization domain of HDAg. The crystal structure of quadrin shows an octamer consisting of four identical copies of a dimer containing an antiparallel -helical coiled coil. Each end of the dimer contains an oligomerization site that interacts isologously with the oligomerization site of another dimer to form a right-angled corner. The resulting quadrin octamer is a 400-residue square protein surrounding a large aqueous hole. We have designed, chemically synthesized, and characterized deltoid and reduced deltoid, two 51-residue chimeric proteins that structurally and functionally mimic one of the two oligomerization sites of the quadrin dimer. Dimerization of deltoid or reduced deltoid should emulate the dimerization of two quadrin dimers to form one right-angled corner of the square. Deltoid and reduced deltoid were designed by molecular modeling, mechanics, and dynamics and synthesized by the solid-phase method. The amino acid sequence of deltoid (GREDILEQWVSCRKKL + PKAPPEE + LRKLKKKCKKLEEDNPWLGNIKGIIGKY) is a chimera of three protein segments: HDAg-(12–28), Thermus thermophilus serine tRNA synthase-(59–65), and HDAg-(34–60)-Tyr. Cysteine (C) was introduced at two positions to explore the effects of the presence (deltoid) or absence (reduced deltoid) of an interhelical disulfide bond. Circular dichroic spectropolarimetry revealed that both synthetic proteins form an -helical structure that is stable over a wide range of pH and KCl concentrations. Size-exclusion chromatography indicated that deltoid and reduced deltoid each form a dimer. Interconversion of these monomers and dimers should be useful model systems for studying the structural features of the right-angled corners of the quadrin octamer that contribute to HDAg oligomerization. If, like quadrin, deltoid or reduced deltoid interferes with HDAg oligomerization, it might serve as a lead compound for the design of potent HDV inhibitors.     

Characterization of deltoid, a chimeric protein containing the oligomerization site of hepatitis delta antigen

Both forms of the hepatitis delta antigen (HDAg) encoded by hepatitis delta virus are active only as oligomers. Previous studies showed that quadrin, a synthetic 50-residue peptide containing residues 12-60 from the N-terminus of HDAg, interferes with HDAg oligomerization, forms an alpha-helical coiled coil in solution, and forms a novel square octamer in the crystal consisting of four antiparallel coiled-coil dimers joined at the corners by hydrophobic binding of oligomerization sites located at each end of the dimers. We designed and synthesized deltoid (CH3CO-[Cys23]HDAg-(12-27)-seryl-tRNA synthetae-(59-65)-[Cys42]HDAg-(34-60)-Tyr-NH2), a chimeric protein that structurally resembles one end of the quadrin dimer and contains a single oligomerization site. The 51-residue chain of deltoid contains a seven-residue alpha-hairpin loop in place of the remainder of the quadrin dimer plus Cys12 and Cys31 for forming an intrachain disulfide bridge. Reduced, unbridged deltoid (Tm=61 degrees C, DeltaG(H2O)=-1.7 kcal mol(-1)) was less stable to denaturation by heat or guanidine HCl than oxidized, intrachain disulfide-bridged deltoid (Tm>80 degrees C, DeltaG(H2O)=-2.6 kcal mol(-1)). Each form is an alpha-helical dimer that reversibly dissociates into two monomers (Kd=80 microM).     

Assignment of 1H, 13C and 15N resonances of the reduced human IgG1 CH3 domain

Here we report the NMR resonance assignments for the reduced form of human IgG1 C_H3 domain, a 26 kDa dimer in solution (residues 341–447). The assignments have been deposited in the BioMagResBank with a BMRB accession number of 15204.     

Oligomerization of hepatitis delta antigen is required for both the trans-activating and trans-dominant inhibitory activities of the delta antigen.

Two forms of hepatitis delta antigen (HDAg) have different roles in the replication cycle of hepatitis delta virus (HDV); the small forms trans activates HDV RNA replication, whereas the large form suppresses it but is needed for virion assembly. To understand the mechanism of these regulatory activities, we studied the possible HDAg oligomerization and its role in HDV replication. In this report, we provide direct biochemical evidence for the in vitro and in vivo formation of homodimers and heterodimers between these two HDAg species. By deletion mutagenesis, we showed that this protein interaction is mediated by the leucine zipper-like sequence residing in the N-terminal one-third of HDAg. Furthermore, site-specific mutants with various substitutions on two of the leucine residues in this stretch of sequence had reduced or no ability to form HDAg dimers. Correspondingly, the small HDAg with mutations in the leucine zipper-like sequence had reduced abilities to trans activate HDV RNA replication. Similar mutations on the leucine zipper-like sequence of the large HDAg also resulted in loss of the ability of large HDAg to inhibit HDV RNA replication. The in vivo biological activities of both forms of HDAg (trans activation and trans-dominant inhibition of HDV RNA replication, respectively) correlated with the extent of HDAg oligomerization in vitro. Thus, we conclude that the small HDAg participates in HDV RNA replication as an oligomer form and that the large HDAg inhibits HDV RNA replication as a result of its complex formation with small HDAg. A "black sheep" model for the mechanism of trans-dominant inhibition by the large HDAg is presented.     

Arginine-Rich Motifs Are Not Required for Hepatitis Delta Virus RNA Binding Activity of the Hepatitis Delta Antigen

Hepatitis delta virus (HDV) replication and packaging require interactions between the unbranched rodlike structure of HDV RNA and hepatitis delta antigen (HDAg), a basic, disordered, oligomeric protein. The tendency of the protein to bind nonspecifically to nucleic acids has impeded analysis of HDV RNA protein complexes and conclusive determination of the regions of HDAg involved in RNA binding. The most widely cited model suggests that RNA binding involves two proposed arginine-rich motifs (ARMs I and II) in the middle of HDAg. However, other studies have questioned the roles of the ARMs. Here, binding activity was analyzed in vitro using HDAg-160, a C-terminal truncation that binds with high affinity and specificity to HDV RNA segments in vitro. Mutation of the core arginines of ARM I or ARM II in HDAg-160 did not diminish binding to HDV unbranched rodlike RNA. These same mutations did not abolish the ability of full-length HDAg to inhibit HDV RNA editing in cells, an activity that involves RNA binding. Moreover, only the N-terminal region of the protein, which does not contain the ARMs, was cross-linked to a bound HDV RNA segment in vitro. These results indicate that the amino-terminal region of HDAg is in close contact with the RNA and that the proposed ARMs are not required for binding HDV RNA. Binding was not reduced by mutation of additional clusters of basic amino acids. This result is consistent with an RNA-protein complex that is formed via numerous contacts between the RNA and each HDAg monomer.     

GFT projection NMR based resonance assignment of membrane proteins: application to subunit c of <Emphasis Type="Italic">E. coli</Emphasis> F<Subscript>1</Subscript>F<Subscript>0</Subscript> ATP synthase in LPPG micelles

G-matrix FT projection NMR spectroscopy was employed for resonance assignment of the 79-residue subunit c of the Escherichia coli F₁F₀ ATP synthase embedded in micelles formed by lyso palmitoyl phosphatidyl glycerol (LPPG). Five GFT NMR experiments, that is, (3,2)D HNNCO, L-(4,3)D HNNC ^αβ C ^α, L-(4,3)D HNN(CO)C ^αβ C ^α, (4,2)D HACA(CO)NHN and (4,3)D HCCH, were acquired along with simultaneous 3D ¹⁵N, ¹³C^aliphatic, ¹³C^aromatic-resolved [¹H,¹H]-NOESY with a total measurement time of ∼43 h. Data analysis resulted in sequence specific assignments for all routinely measured backbone and ¹³C^β shifts, and for 97% of the side chain shifts. Moreover, the use of two G²FT NMR experiments, that is, (5,3)D HN{N,CO}{C ^αβ C ^α} and (5,3)D {C ^αβ C ^α}{CON}HN, was explored to break the very high chemical shift degeneracy typically encountered for membrane proteins. It is shown that the 4D and 5D spectral information obtained rapidly from GFT and G²FT NMR experiments enables one to efficiently obtain (nearly) complete resonance assignments of membrane proteins. Qi Zhang, Hanudatta S. Atreya, Douglas E. Kamen, Mark E. Girvin and Thomas Szyperski—New York Consortium on Membrane Protein Structure.     

Secondary structural modifications of Aβ(1-40) induced by multiple 2-acetoxy-4-methoxybenzyl (acetylHmb) protection

Summary Modifications to secondary structure and fibril formation caused by multiple acetylHmb backbone amide protection of Alzheimer's disease Aβ(1–40) were investigated using circular dichroism spectroscopy and electron microscopy. Penta(acetylHmb) Aβ(1–40) was observed to have a reduced ability to form α-helix and β-sheet structures under the same solution conditions as the native peptide, with α-helical propensity being reduced more significantly than β-sheet propensity. Further, acetylHmb backbone protection was found to alter Aβ(1–40) interaction with SDS-micelles by preventing α-helix formation. Aβ fibril formation, a characteristic property of this peptide, was also not observed for penta(acetylHmb) Aβ(1–40).     

Investigating the effect of a single glycine to alanine substitution on interactions of antimicrobial peptide latarcin 2a with a lipid membrane

Latarcins are linear, α-helical antimicrobial peptides purified from the venom of the Central Asian spider Lachesana tarabaevi, with lytic activity against Gram-positive and Gram-negative bacteria, erythrocytes, and yeast at micromolar concentrations. In this work, we investigated the role of the hinge in latarcin 2a (ltc2a, GLFGKLIKKFGRKAISYAVKKARGKH-COOH), which adopts a helix–hinge–helix conformation in membrane-mimicking environments, on peptide–membrane interactions and its potential effect on the selective toxicity of the peptide. A modified latarcin 2a, ltc2aG11A, obtained by replacing the glycine at position 11 with alanine (ltc2aG11A, GLFGKLIKKFARKAISYAVKKARGKH-COOH), adopts a more rigid structure due to the reduced conformational flexibility. Langmuir monolayer measurements combined with atomic force microscopy and X-ray photoemission electron microscopy (X-PEEM) indicate that both peptides bind and insert preferentially into anionic compared with zwitterionic phospholipid monolayers. Modified ltc2aG11A was found to be more disruptive of supported phospholipid bilayer modeling mammalian cell membrane. However, no considerable difference in lytic activity of the two peptides toward bacterial membrane was found. Overall the data indicate that decrease in the flexibility of ltc2a induced by the modification in the hinge region is likely to increase the peptide’s nonspecific interactions with zwitterionic cell membranes and potentially increase its toxicity against eukaryotic cells.     

Kinetic Ramifications of the Association-Dissociation Behavior of NAD Malic Enzyme : A Possible Regulatory Mechanism

NAD malic enzyme can exist in dimer, tetramer, or octamer form. Freshly prepared enzyme from Solanum tuberosum var. Chieftan exists predominantly as the octamer and during storage is progressively converted into lower molecular weight forms. High ionic strength favors dimer formation, whereas high concentrations of malate or citrate favor tetramer formation. The tetramer is the most active form, having a low K_m for malate and a high V_max. The dimer, with its high K_m and low V_max, is the least active form. Malate may regulate NAD malic enzyme by controlling its state of oligomerization.     

The tri–μ–hydrido–bis[(η5–C5Me5)aluminum(III)] theoretical study, the assets of sandwiched M2H3 (M of 13th group elements) stability

The stability of the tri–μ–hydrido–bis[(η⁵–C₅Me₅)aluminum], Cp*₂Al₂H₃, 1 is studied at B3LYP/6–311+G(d,p), CCSD(T)//B3LYP/6–311+G(d,p) and MP4//B3LYP/6–311+G(d,p) levels. The coordination between Al₂H₃ entity and both C₅(CH₃)₅ groups is ensured by strong electrostatic and orbital interactions. The orbital analysis of the interacting fragments shows that Al₂H₃ acceptor, which keeps its tribridged structure, implies the vacant ( \texta₁￠ ) \left( {{\text{a}}_1^\prime } \right) and five antibonding (a₂^￠￠ a_2^{\prime \prime } , e′ and e″) molecular orbitals to interact with two orbitals mixtures, b₁ and e" of the donors (C₅Me₅). When we take into account the solvent effect, the computation shows that 1 seems to be stable in condensed phase with a tribridged bond between the Al atoms [Cp*Al(μ-H)₃AlCp*], whereas in the gas phase, the monobridged Cp*AlH(μ-H)AlHCp* 4 is slightly favored (4 kcal mol⁻¹). We propose that 1 could be prepared thanks to Cp*Al (2) and Cp*AlH₂ (3) reaction in acidic medium. The experimental treatment of this type of metallocenes would contribute to the development of the organometallic chemistry of 13th group elements.      

High-resolution NMR structure of the antimicrobial peptide protegrin-2 in the presence of DPC micelles

PG-1 adopts a dimeric structure in dodecylphosphocholine (DPC) micelles, and a channel is formed by the association of several dimers but the molecular mechanisms of the membrane damage by non-α-helical peptides are still unknown. The formation of the PG-1 dimer is important for pore formation in the lipid bilayer, since the dimer can be regarded as the primary unit for assembly into the ordered aggregates. It was supposed that only 12 residues (RGGRL-CYCRR-RFCVC-V) are needed to endow protegrin molecules with strong antibacterial activity and that at least four additional residues are needed to add potent antifungal properties. Thus, the 16-residue protegrin (PG-2) represents the minimal structure needed for broad-spectrum antimicrobial activity encompassing bacteria and fungi. As the peptide conformation and peptide-to-membrane binding properties are very sensitive to single amino acid substitutions, the solution structure of PG-2 in solution and in a membrane mimicking environment are crucial. In order to find evidence if the oligomerization state of PG-1 in a lipid environment will be the same or not for another protegrins, we investigate in the present work the PG-2 NMR solution structure in the presence of perdeuterated DPC micelles. The NMR study reported in the present work indicates that PG-2 form a well-defined structure (PDB: 2MUH) composed of a two-stranded antiparallel β-sheet when it binds to DPC micelles.     

Band-selective 13C Homonuclear 3D Spectroscopy for Solid Proteins at High Field with Rotor-synchronized Soft Pulses

We demonstrate improved 3D ¹³C–¹³C–¹³C chemical shift correlation experiments for solid proteins, utilizing band-selective coherence transfer, scalar decoupling and homonuclear zero-quantum polarization transfer. Judicious use of selective pulses and a z-filter period suppress artifacts with a two-step phase cycle, allowing higher digital resolution in a fixed measurement time. The novel correlation of C^ali–C^ali–CX (C^ali for aliphatic carbons, CX for any carbon) reduces measurement time by an order of magnitude without sacrificing digital resolution. The experiment retains intensity from side-chain carbon resonances whose chemical shift dispersion is critical to minimize spectral degeneracy for large proteins with a predominance of secondary structure, such as β-sheet rich fibrillar proteins and α-helical membrane proteins. We demonstrate the experiment for the β1 immunoglobulin binding domain of protein G (GB1) and fibrils of the A30P mutant of α-synuclein, which is implicated in Parkinson’s disease. Selective pulses of duration comparable the rotor period give optimal performance, but must be synchronized with the spinning in non-trivial ways to minimize chemical shift anisotropy recoupling effects. Soft pulses with a small bandwidth-duration product are best for exciting the ~70 ppm bandwidth required for aliphatic-only dimensions.     

Catalysis and Selectivity in Prebiotic Synthesis: Initiation of the Formation of Oligo(U)s on Montmorillonite Clay by Adenosine-5′-methylphosphate

Adenosine-5′-methylphosphate (MepA) initiates the oligomerization of the 5′-phosphorimidazolide of uridine (ImpU) in the presence of montmorillonite clay. Longer oligomers form because the 5′-phosphate is blocked with a methyl group that prevents the formation of cyclic- and pyrophosphate-containing compounds. The MepA initiates 69–84% of the 5–9 charge oligomers, respectively. The montmorillonite catalyst also provides selectivity in the oligomerization reactions so that the main reaction pathway is MepA → MepA³′pU → MepA³′pU²′pU → MepA³′pU²′pU³′pU. MepA did not enhance the oligomerization of ImpA. The relative rates of the reactions were determined from an investigation of the products in competitive reactions. Selectivity was observed in the reaction of ImpU with equimolar amounts of MepA³′pU and MepA²′pU where the relative reaction rates are 10.3:1, respectively. In the reaction of ImpA with MepA³′pA and MepA²′pA the ImpA reacts 5.2 times faster with MepA³′pA. In the competitive reaction of ImpU and ImpA with MepA³′pA and MepA³′pU the elongation proceeds on MepA³′pA 5.6 times more rapidly than with MepA³′pU. There is no correlation between the extent of binding to the montmorillonite and reaction rates in the formation of longer oligomers. The formation of more than two sequential 2′,5′-linkages in the oligomer chain proceeds more slowly than the addition to a single 2′,5′-link or a 3′,5′-link and either chain termination or elongation by a 3′,5′-linage occurs. The central role that catalysis may have had in the prebiotic formation of biopolymers is discussed. Note added in proof: There are errors in the high resolution mass spectral data given in Section 4.2.1. The high resolution mass spectrum found for the cyclic dimer of UpUp (C-UpUp) was 657.02260. C₁₈H₂₁N₄O₁₆P₂Na₂ requires 657.02232. The high resolution mass spectrum found for the cyclic dimer of ApAp (C-ApAp) was 725.05850. C₂₀H₂₂N₁₀O₁₂P₂Na₃ requires 725.05839.     

Muscles within muscles: the neuromotor control of intra-muscular segments

The aim of this investigation was to anatomically identify, and then determine the function of, individual segments within the human deltoid muscle. The anatomical structure of the deltoid was determined through dissection and/or observation of the shoulder girdles of 11 male cadavers (aged 65–84 years). These results indicate that the deltoid consists of seven anatomical segments (D1–D7) based upon the distinctive arrangement of each segment's origin and insertion. Radiographic analysis of a cadaveric shoulder joint suggested that only the postero-medial segment D7 has a line of action directed below the shoulder joint's axis of rotation. The functional role of each individual segment was then determined utilising an electromyographic (EMG) technique. Seven miniature (1 mm active plate; 7 mm interelectrode distance) bipolar surface electrodes were positioned over the proximal portion of each segment's muscle belly in 18 male and female subjects (18–30 years). EMG waveforms were then recorded during the production of rapid isometric shoulder abduction and adduction force impulses with the shoulder joint in 40 degrees of abduction in the plane of the scapula. Each subject randomly performed 15 abduction and 15 adduction isometric force impulses following a short familiarisation period. All subjects received visual feed back on the duration and amplitude of each isometric force impulse produced via a visual force-time display which compared subject performance to a criterion force-time curve. Movement time was 400 ms (time-to-peak isometric force) at an intensity level of 50% maximal voluntary contraction. Temporal and intensity analyses of the EMG waveforms, as well as temporal analysis of the isometric force impulses, revealed the neuromotor control strategies utilised by the CNS to control the activity of each muscle segment. The results showed that segmental neuromotor control strategies differ across the breadth of the muscle and that individual segments of the deltoid can be identified as having either “prime mover”, “synergist”, “stabiliser” or “antagonist” functions; functional classifications normally associated with whole muscle function. Therefore, it was concluded that the CNS can “fine tune” the activity of at least six discrete segments within the human deltoid muscle to efficiently meet the demands of the imposed motor task. Accepted: 15 December 1997     

Isoelectric point heterogeneity of the two oligomeric forms of heart mitochondrial creatine kinase

Rabbit heart mitochondrial creatine kinase has been recently shown to exist in two oligomeric forms: a dimer and an octamer, the latter being the form associated with the inner mitochondrial membrane [(1988) Biochem.Biophys. Res. Commun. 153,1310.]. We report here on the determination of the isoelectric points (pI) of the two purified forms by thin layer isoelectric focusing. The pI of the dimer is 8.2 and that of the octamer is 8.8; the former is higher by more than one pH unit than that of the cytoplasmic form MM-CK. It is proposed that the higher pI of the octamer is responsible for its binding to the inner membrane.     

Hydrophobized Formate Dehydrogenase from Pseudomonas sp. 101 in the System of Reverse Micelles of Aerosol OT in Octane

NAD⁺-dependent formate dehydrogenase (FDH) was hydrophobized with palmitoyl chloride to give the samples with various modification degrees (2–10). The native and modified FDHs were comparatively studied in the system of reverse micelles of Aerosol OT in octane. Like the native, the modified enzyme displayed three maxima in the curve of dependence of its catalytic activity on the degree of surfactant hydration (the micelle size), which reflect the enzyme functioning in the form of a monomer, dimer, or octamer. The peak corresponding to the functioning of the FDH dimer was found to decrease along with an increase in the modification degree. Thus, the modified enzyme mainly functions in the form of monomer and octamer. The modified FDH displayed membranotropy and revealed the dependence of catalytic activity on surfactant concentration.     

Basis of the 1:1 stoichiometry of the high affinity receptor FcεRI-IgE complex

A soluble fragment of the high-affinity IgE receptor FcεRI α-chain (sFcεRIα) binds to the Fc fragment of IgE (IgE-Fc) as a 1:1 complex. IgE-Fc consists of a dimer of the Cε2, Cε3 and Cε4 domains of the ε-heavy chain of IgE. This region of IgE has been modelled on the crystal structure of the Fc region of IgG₁, which exhibits twofold rotational symmetry. This implies that IgE should be divalent with respect to its ligands. X-ray scattering studies reveal however that the twofold rotational symmetry of IgE-Fc is perturbed by a bend in the linker region between the Cε2 and Cε3 domains. The 1:1 stoichiometry could then arise from the conformational asymmetry or from steric occlusion of one of the sites by the overhanging Cε2 domains. To test this hypothesis we have expressed a recombinant ε-chain fragment containing Cε3 and Cε4. This product, Fcε3–4, is secreted from cells as a disulphide linked dimer and binds with higher affinity than either IgE or IgE-Fc to cell surface FcεRI. Titration experiments, together with molecular mass measurements of the Fcε3–4/sFcεRIα complex, reveal that Fcε3–4 binds only a single receptor molecule. This excludes the possibility that steric hindrance by Cε2 accounts for the unexpected stoichiometry. Received: 31 July 1996 / Accepted: 1 December 1996     

Automated Resonance Assignment of Proteins: 6 DAPSY-NMR

The 6-dimensional (6D) APSY-seq-HNCOCANH NMR experiment correlates two sequentially neighboring amide moieties in proteins via the C′ and C^α nuclei, with efficient suppression of the back transfer from C^α to the originating amide moiety. The automatic analysis of two-dimensional (2D) projections of this 6D experiment with the use of GAPRO (Hiller et al., 2005) provides a high-precision 6D peak list, which permits automated sequential assignments of proteins with the assignment software GARANT (Bartels et al., 1997). The procedure was applied to two proteins, the 63-residue 434-repressor(1–63) and the 115-residue TM1290. For both proteins, complete sequential assignments for all NMR-observable backbone resonances were obtained, and the polypeptide segments thus identified could be unambiguously located in the amino acid sequence. These results demonstrate that APSY-NMR spectroscopy in combination with a suitable assignment algorithm can provide fully automated sequence-specific backbone assignments of small proteins.Francesco Fiorito and Sebastian Hiller - Both authors contributed equally to this work     

α-Helical coiled-coil oligomerization domains in extracellular proteins

Subunit oligomerization of many proteins is mediated by α-helical coiled-coil domains. 3,4-Hydrophobic heptad repeat sequences, the characteristic feature of the coiled-coil protein folding motif, have been found in a wide variety of gene products including cytoskeletal, nuclear, muscle, cell surface, extracellular, plasma, bacterial, and viral proteins. Whereas the majority of coiled-coil structures is represented by intracellular α-helical bundles that contain two polypeptide chains, examples of extracellular coiled-coil proteins are fewer in number. Most proteins located in the extracellular space form three-stranded α-helical assemblies. Recently, five-stranded coiled coils have been identified in thrombospondins 3 and 4 in cartilage oligomeric matrix protein, and the formation of a heterotetramer has been observed in in vitro studies with the recombinant asialoglycoprotein receptor oligomerization domain. Coiled-coil domains in laminins and probably also in tenascins and thrombospondins are responsible for the formation of tissue-specific isoforms by selective oligomerization of different polypeptide chains.