Crystal structure of the PHF8 Jumonji domain, an N-methyl lysine demethylase

Crystallographic analysis of the catalytic domain of PHD finger protein 8 (PHF8), an N^ε-methyl lysine histone demethylase associated with mental retardation and cleft lip/palate, reveals a double-stranded β-helix fold with conserved Fe(II) and cosubstrate binding sites typical of the 2-oxoglutarate dependent oxygenases. The PHF8 active site is highly conserved with those of the FBXL10/11demethylases, which are also selective for the di-/mono-methylated lysine states, but differs from that of the JMJD2 demethylases which are selective for tri-/di-methylated states. The results rationalize the lack of activity for the clinically observed F279S PHF8 variant and they will help to identify inhibitors selective for specific N^ε-methyl lysine demethylase subfamilies.     

Structure/function analysis of four core ESCRT-III proteins reveals common regulatory role for extreme C-terminal domain

Endosomal sorting complex required for transport-III (ESCRT-III) is a large complex built from related ESCRT-III proteins involved in multivesicular body biogenesis. Little is known about the structure and function of this complex. Here, we compare four human ESCRT-III proteins - hVps2-1/CHMP2a, hVps24/CHMP3, hVps20/CHMP6, and hSnf7-1/CHMP4a - to each other, studying the effects of deleting predicted alpha-helical domains on their behavior in transfected cells. Surprisingly, removing approximately 40 amino acids from the C-terminus of each protein unmasks a common ability to associate with endosomal membranes and assemble into large polymeric complexes. Expressing these truncated ESCRT-III proteins in cultured cells causes ubiquitinated cargo to accumulate on enlarged endosomes and inhibits viral budding, while expressing full-length proteins does not. hVps2-1/CHMP2a lacking its C-terminal 42 amino acids further fails to bind to the AAA+ adenosine triphosphatase VPS4B/SKD1, indicating that C-terminal sequences are important for interaction of ESCRT-III proteins with VPS4. Overall, our study supports a model in which ESCRT-III proteins cycle between a default 'closed' state and an activated 'open' state under control of sequences at their C-terminus and associated factors.     

Amyloid-forming propensity of the hydrophobic non-natural amino acid on the fibril-forming core peptide of human tau

Amino acid residues with aromatic side chains, such as Tyr and Phe, are known to play essential roles in forming and stabilizing the amyloid fibrils of pathogenic polypeptides by affecting their amyloid forming propensity. We have studied the amyloid-type aggregation of peptides containing non-natural amino acid derived from a core part of human pathogenic protein, tau. The hydrophobic nature of the biphenyl group and its intermolecular aromatic interactions strongly alter their amyloid formation properties.     

Structure and specificity of several triclocarban‐binding single domain camelid antibody fragments

The variable VHH domains of camelid single chain antibodies have been useful in numerous biotechnology applications due to their simplicity, biophysical properties, and abilities to bind to their cognate antigens with high affinities and specificity. Their interactions with proteins have been well‐studied, but considerably less work has been done to characterize their ability to bind haptens. A high‐resolution structural study of three nanobodies (T4, T9, and T10) which have been shown to bind triclocarban (TCC, 3‐(4‐chlorophenyl)‐1‐(3,4‐dichlorophenyl)urea) with near‐nanomolar affinity shows that binding occurs in a tunnel largely formed by CDR1 rather than a surface or lateral binding mode seen in other nanobody‐hapten interactions. Additional significant interactions are formed with a non‐hypervariable loop, sometimes dubbed “CDR4”. A comparison of apo and holo forms of T9 and T10 shows that the binding site undergoes little conformational change upon binding of TCC. Structures of three nanobody‐TCC complexes demonstrated there was not a standard binding mode. T4 and T9 have a high degree of sequence identity and bind the hapten in a nearly identical manner, while the more divergent T10 binds TCC in a slightly displaced orientation with the urea moiety rotated approximately 180° along the long axis of the molecule. In addition to methotrexate, this is the second report of haptens binding in a tunnel formed by CDR1, suggesting that compounds with similar hydrophobicity and shape could be recognized by nanobodies in analogous fashion. Structure‐guided mutations failed to improve binding affinity for T4 and T9 underscoring the high degree of natural optimization.     

Tau paired helical filaments from Alzheimer's disease brain and assembled in vitro are based on beta-structure in the core domain

Tau protein, a neuronal microtubule-associated protein, forms insoluble fibers ("paired helical filaments") in Alzheimer's disease and other tauopathies. Conflicting views on the structure of the fibers have been proposed recently, ranging from mainly alpha-helical structure to mainly beta-sheet, or a mixture of mostly random coil and beta-sheet. We have addressed this issue by studying tau fibers immunopurified from Alzheimer brain tissue by a conformation-specific antibody and comparing them with fibers reassembled from recombinant tau or tau constructs in vitro, using a combination of electron microscopy and spectroscopic methods. Brain-derived fibers and reassembled fibers both exhibit a typical twisted appearance when examined by electron microscopy. The soluble tau protein is a natively unfolded protein dominated by random coil structure, whereas Alzheimer PHFs and reassembled fibers show a shift toward an increase in the level of beta-structure. The results support a model in which the repeat domain of tau (which lies within the core of PHFs) adopts an increasing level of beta-structure during aggregation, whereas the N- and C-terminal domains projecting away from the PHF core are mostly random coil.     

Prediction of amyloid fibril-forming proteins

In Alzheimer's disease and spongiform encephalopathies proteins transform from their native states into fibrils. We find that several amyloid-forming proteins harbor an alpha-helix in a polypeptide segment that should form a beta-strand according to secondary structure predictions. In 1324 nonredundant protein structures, 37 beta-strands with > or =7 residues were predicted in segments where the experimentally determined structures show helices. These discordances include the prion protein (helix 2, positions 179-191), the Alzheimer amyloid beta-peptide (Abeta, positions 16-23), and lung surfactant protein C (SP-C, positions 12-27). In addition, human coagulation factor XIII (positions 258-266), triacylglycerol lipase from Candida antarctica (positions 256-266), and d-alanyl-d-alanine transpeptidase from Streptomyces R61 (positions 92-106) contain a discordant helix. These proteins have not been reported to form fibrils but in this study were found to form fibrils in buffered saline at pH 7.4. By replacing valines in the discordant helical part of SP-C with leucines, an alpha-helix is found experimentally and by secondary structure predictions. This analogue does not form fibrils under conditions where SP-C forms abundant fibrils. Likewise, when Abeta residues 14-23 are removed or changed to a nondiscordant sequence, fibrils are no longer formed. We propose that alpha-helix/beta-strand-discordant stretches are associated with amyloid fibril formation.     

Molecular basis for bipartite recognition of histone H3 by the PZP domain of PHF14

Histone recognition constitutes a key epigenetic mechanism in gene regulation and cell fate decision. PHF14 is a conserved multi-PHD finger protein that has been implicated in organ development, tissue homeostasis, and tumorigenesis. Here we show that PHF14 reads unmodified histone H3_(1–34) through an integrated PHD1-ZnK-PHD2 cassette (PHF14_PZP). Our binding, structural and HDX-MS analyses revealed a feature of bipartite recognition, in which PHF14_PZP utilizes two distinct surfaces for concurrent yet separable engagement of segments H3-Nter (e.g. 1–15) and H3-middle (e.g. 14–34) of H3_(1–34). Structural studies revealed a novel histone H3 binding mode by PHD1 of PHF14_PZP, in which a PHF14-unique insertion loop but not the core β-strands of a PHD finger dominates H3K4 readout. Binding studies showed that H3-PHF14_PZP engagement is sensitive to modifications occurring to H3 R2, T3, K4, R8 and K23 but not K9 and K27, suggesting multiple layers of modification switch. Collectively, our work calls attention to PHF14 as a ‘ground’ state (unmodified) H3_(1–34) reader that can be negatively regulated by active marks, thus providing molecular insights into a repressive function of PHF14 and its derepression.     

Consensus prediction of amyloidogenic determinants in amyloid fibril-forming proteins

We combine the results of three prediction algorithms on a test set of 21 amyloidogenic proteins to predict amyloidogenic determinants. Two prediction algorithms are recently developed prediction algorithms of amyloidogenic stretches in protein sequences, whereas the third is a secondary structure prediction algorithm capable of identifying 'conformational switches' (regions that have both the propensity for alpha-helix and beta-sheet). Surprisingly, the results of prediction agree well and also agree with experimentally investigated amyloidogenic regions. Furthermore, they suggest several previously not identified amino acid stretches as potential amyloidogenic determinants. Most predicted (and experimentally observed) amyloidogenic determinants reside on the protein surface of relevant solved crystal structures. It appears that a consensus prediction algorithm is more objective than individual prediction methods alone.     

DNA-binding domain in adenovirus core protein VII

Major core protein, VII of adenovirus type 2 can be cleaved with BrCN into four fragments A, B, C and D according to the electrophoretic mobility on SDS-polyacrylamide gel. The sequential order of the fragments was ACBD from NH2-terminus to COOH-end. From the results of protein blotting experiment and nitrocellulose filter binding assay, fragments A and C, the amino end half of VII, was suggested to be DNA-binding domain. Furthermore, the binding of VII to DNA is suggested to become tighter as the fragment A moiety of pVII is processed in the virion.     

Stabilization of discordant helices in amyloid fibril-forming proteins

Several proteins and peptides that can convert from alpha-helical to beta-sheet conformation and form amyloid fibrils, including the amyloid beta-peptide (Abeta) and the prion protein, contain a discordant alpha-helix that is composed of residues that strongly favor beta-strand formation. In their native states, 37 of 38 discordant helices are now found to interact with other protein segments or with lipid membranes, but Abeta apparently lacks such interactions. The helical propensity of the Abeta discordant region (K16LVFFAED23) is increased by introducing V18A/F19A/F20A replacements, and this is associated with reduced fibril formation. Addition of the tripeptide KAD or phospho-L-serine likewise increases the alpha-helical content of Abeta(12-28) and reduces aggregation and fibril formation of Abeta(1-40), Abeta(12-28), Abeta(12-24), and Abeta(14-23). In contrast, tripeptides with all-neutral, all-acidic or all-basic side chains, as well as phosphoethanolamine, phosphocholine, and phosphoglycerol have no significant effects on Abeta secondary structure or fibril formation. These data suggest that in free Abeta, the discordant alpha-helix lacks stabilizing interactions (likely as a consequence of proteolytic removal from a membrane-associated precursor protein) and that stabilization of this helix can reduce fibril formation.     

Accessibility of ENaC extracellular domain central core residues

The epithelial Na⁺ channel (ENaC)/degenerin family has a similar extracellular architecture, where specific regulatory factors interact and alter channel gating behavior. The extracellular palm domain serves as a key link to the channel pore. In this study, we used cysteine-scanning mutagenesis to assess the functional effects of Cys-modifying reagents on palm domain β10 strand residues in mouse ENaC. Of the 13 ENaC α subunit mutants with Cys substitutions examined, only mutants at sites in the proximal region of β10 exhibited changes in channel activity in response to methanethiosulfonate reagents. Additionally, Cys substitutions at three proximal sites of β and γ subunit β10 strands also rendered mutant channels methanethiosulfonate-responsive. Moreover, multiple Cys mutants were activated by low concentrations of thiophilic Cd²⁺. Using the Na⁺ self-inhibition response to assess ENaC gating behavior, we identified four α, two β, and two γ subunit β10 strand mutations that changed the Na⁺ self-inhibition response. Our results suggest that the proximal regions of β10 strands in all three subunits are accessible to small aqueous compounds and Cd²⁺ and have a role in modulating ENaC gating. These results are consistent with a structural model of mouse ENaC that predicts the presence of aqueous tunnels adjacent to the proximal part of β10 and with previously resolved structures of a related family member where palm domain structural transitions were observed with channels in an open or closed state.     

The Structure Biology of Tau and Clue for Aggregation Inhibitor Design

The Protein Journal - Tau is a microtubule-associated protein that is mainly expressed in central and peripheral nerve systems. Tau binds to tubulin and regulates assembly and stabilization of...     

Structure of B-MLV capsid amino-terminal domain reveals key features of viral tropism, gag assembly and core formation

The Gag polyprotein is the major structural protein found in all classes of retroviruses. Interactions between Gag molecules control key events at several stages in the cycle of infection. In particular, the capsid (CA) domain of Gag mediates many of the protein-protein interactions that drive retrovirus assembly, maturation and disassembly. Moreover, in murine leukaemia virus (MLV), sequence variation in CA confers N and B tropism that determines susceptibility to the intracellular restriction factors Fv1n and Fv1b. We have determined the structure of the N-terminal domain (NtD) of CA from B-tropic MLV. A comparison of this structure with that of the NtD of CA from N-tropic MLV reveals that although the crystals belong to different space groups, CA monomers are packed with the same P6 hexagonal arrangement. Moreover, interhexamer crystal contacts between residues located at the periphery of the discs are conserved, indicating that switching of tropism does not result in large differences in the backbone conformation, nor does it alter the quaternary arrangement of the disc. We have also examined crystals of the N-tropic MLV CA containing both N- and C-terminal domains. In this case, the NtD hexamer is still present; however, the interhexamer spacing is increased and the conserved interhexamer contacts are absent. Investigation into the effects of mutation of residues that mediate interhexamer contacts reveals that amino acid substitutions at these positions cause severe defects in viral assembly, budding and Gag processing. Based on our crystal structures and mutational analysis, we propose that in MLV, interactions between the NtDs of CA are required for packing of Gag molecules in the early part of immature particle assembly. Moreover, we present a model where proteolytic cleavage at maturation results in migration of CA C-terminal domains into interstitial spaces between NtD hexamers. As a result, NtD-mediated interhexamer contacts present in the immature particle are displaced and the less densely packed lattice with increased hexamer-hexamer spacing characteristic of the viral core is produced.     

Melatonin interacts with repeat domain of Tau to mediate disaggregation of paired helical filaments

Tau is the major neuronal protein involved in the stabilization of microtubule assembly. In Alzheimer's disease, Tau self-assembles to form intracellular protein aggregates which are toxic to cells. Various methods have been tried and tested to restrain the aggregation of Tau. Most of the agents tested for this purpose have limitations in their effectiveness and availability to neuronal cells. We have tested melatonin, a neurohormone secreted by pineal gland and a well-known anti-oxidant, for its ability to interact with the repeat domain of Tau using ITC and NMR. In aggregation inhibition and disaggregation studies of repeat Tau, melatonin was found to modulate the aggregation propensity of repeat Tau at a concentration of 5000 μM and was more effective in dissolving preformed aggregates rather than acting as an aggregation inhibitor. However, there were no major conformational changes in Tau in presence of melatonin as observed by CD spectroscopy. On the basis of our findings, we are proposing a mechanism by which melatonin can interact with the repeat domain of Tau and exhibit its disaggregation effect.     

FTDP-17 mutations in Tau alter the regulation of microtubule dynamics: an "alternative core" model for normal and pathological Tau action

Mutations affecting either the structure or regulation of the microtubule-associated protein Tau cause neuronal cell death and dementia. However, the molecular mechanisms mediating these deleterious effects remain unclear. Among the most characterized activities of Tau is the ability to regulate microtubule dynamics, known to be essential for proper cell function and viability. Here we have tested the hypothesis that Tau mutations causing neurodegeneration also alter the ability of Tau to regulate the dynamic instability behaviors of microtubules. Using in vitro microtubule dynamics assays to assess average microtubule growth rates, microtubule growth rate distributions, and catastrophe frequencies, we found that all tested mutants possessing amino acid substitutions or deletions mapping to either the repeat or interrepeat regions of Tau do indeed compromise its ability to regulate microtubule dynamics. Further mutational analyses suggest a novel mechanism of Tau regulatory action based on an "alternative core" of microtubule binding and regulatory activities composed of two repeats and the interrepeat between them. In this model, the interrepeat serves as the primary regulator of microtubule dynamics, whereas the flanking repeats serve as tethers to properly position the interrepeat on the microtubule. Importantly, since there are multiple interrepeats on each Tau molecule, there are also multiple cores on each Tau molecule, each with distinct mechanistic capabilities, thereby providing significant regulatory potential. Taken together, the data are consistent with a microtubule misregulation mechanism for Tau-mediated neuronal cell death and provide a novel mechanistic model for normal and pathological Tau action.     

Truncated Tau with the Fyn-binding domain and without the microtubule-binding domain hinders the myelinating capacity of an oligodendrocyte cell line

The mechanisms underlying developmental myelination have therapeutic potential following CNS injury and degeneration. We report that transplanted central glial (CG)-4 cells had a diminished myelinating capacity in myelin-deficient (md) rats when cells express a mutated form of Tau (Tau [688]), which binds Fyn but not the microtubules. In the brain of the md rats, Tau [688]-transfected CG-4 cells displayed a decrease in cellular process outgrowth and myelination; in the spinal cord the extent of myelination rostral and caudal to the injection site was decreased. In contrast, control Tau [605]-transfected CG-4 cells formed long cellular processes and substantial areas of myelin both in the brain and spinal cord. In culture, Tau [688]-transfected CG-4 cells displayed a decrease in cellular process outgrowth, and Fyn localized largely in the cell body, not the processes. Thus, Tau in oligodendrocytes plays a key role in myelination, and a functional Tau-Fyn interaction might have therapeutic potential during demyelination and myelin repair following CNS injury and degeneration.