Glareh Askarieh

Self-assembly of spider silk proteins is controlled by a pH-sensitive relay.

Nature’s high-performance polymer, spider silk, consists of specific proteins, spidroins, with repetitive segments flanked by conserved non-repetitive domains. Spidroins are stored as a highly concentrated fluid dope. On silk formation, intermolecular interactions between repeat regions are established that provide strength and elasticity. How spiders manage to avoid premature spidroin aggregation before self-assembly is not yet established. A pH drop to 6.3 along the spider’s spinning apparatus, altered salt composition and shear forces are believed to trigger the conversion to solid silk, but no molecular details are known. Miniature spidroins consisting of a few repetitive spidroin segments capped by the carboxy-terminal domain form metre-long silk-like fibres irrespective of pH. We discovered that incorporation of the amino-terminal domain of major ampullate spidroin 1 from the dragline of the nursery web spider Euprosthenops australis (NT) into mini-spidroins enables immediate, charge-dependent self-assembly at pH values around 6.3, but delays aggregation above pH 7. The X-ray structure of NT, determined to 1.7 Å resolution, shows a homodimer of dipolar, antiparallel five-helix bundle subunits that lack homologues. The overall dimeric structure and observed charge distribution of NT is expected to be conserved through spider evolution and in all types of spidroins. Our results indicate a relay-like mechanism through which the N-terminal domain regulates spidroin assembly by inhibiting precocious aggregation during storage, and accelerating and directing self-assembly as the pH is lowered along the spider’s silk extrusion duct.

BRICHOS Domains Efficiently Delay Fibrillation of Amyloid beta-Peptide

Background: Alzheimer disease (AD) is associated with Aβ protein misfolding and aggregation into fibrils rich in β-sheet structure. Results: BRICHOS domains prevent fibril formation of Aβ far below the stoichiometric ratio. Conclusion: Aβ is maintained as an unstructured monomer in the presence of BRICHOS. Significance: BRICHOS domain can have a natural protective role against Aβ aggregation, which may open new routes toward AD therapy. Amyloid diseases such as Alzheimer, Parkinson, and prion diseases are associated with a specific form of protein misfolding and aggregation into oligomers and fibrils rich in β-sheet structure. The BRICHOS domain consisting of ∼100 residues is found in membrane proteins associated with degenerative and proliferative disease, including lung fibrosis (surfactant protein C precursor; pro-SP-C) and familial dementia (Bri2). We find that recombinant BRICHOS domains from Bri2 and pro-SP-C prevent fibril formation of amyloid β-peptides (Aβ40 and Aβ42) far below the stoichiometric ratio. Kinetic experiments show that a main effect of BRICHOS is to prolong the lag time in a concentration-dependent, quantitative, and reproducible manner. An ongoing aggregation process is retarded if BRICHOS is added at any time during the lag phase, but it is too late to interfere at the end of the process. Results from circular dichroism and NMR spectroscopy, as well as analytical size exclusion chromatography, imply that Aβ is maintained as an unstructured monomer during the extended lag phase in the presence of BRICHOS. Electron microscopy shows that although the process is delayed, typical amyloid fibrils are eventually formed also when BRICHOS is present. Structural BRICHOS models display a conserved array of tyrosine rings on a five-stranded β-sheet, with inter-hydroxyl distances suited for hydrogen-bonding peptides in an extended β-conformation. Our data imply that the inhibitory mechanism is reliant on BRICHOS interfering with molecular events during the lag phase.

High-resolution structure of a BRICHOS domain and its implications for anti-amyloid chaperone activity on lung surfactant protein C

BRICHOS domains are encoded in > 30 human genes, which are associated with cancer, neurodegeneration, and interstitial lung disease (ILD). The BRICHOS domain from lung surfactant protein C proprotein (proSP-C) is required for membrane insertion of SP-C and has anti-amyloid activity in vitro. Here, we report the 2.1 Å crystal structure of the human proSP-C BRICHOS domain, which, together with molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry, reveals how BRICHOS domains may mediate chaperone activity. Observation of amyloid deposits composed of mature SP-C in lung tissue samples from ILD patients with mutations in the BRICHOS domain or in its peptide-binding linker region supports the in vivo relevance of the proposed mechanism. The results indicate that ILD mutations interfering with proSP-C BRICHOS activity cause amyloid disease secondary to intramolecular chaperone malfunction.

A pH-dependent dimer lock in spider silk protein.

Spider dragline silk, one of the strongest polymers in nature, is composed of proteins termed major ampullate spidroin (MaSp) 1 and MaSp2. The N-terminal (NT) domain of MaSp1 produced by the nursery web spider Euprosthenops australis acts as a pH-sensitive relay, mediating spidroin assembly at around pH 6.3. Using amide hydrogen/deuterium exchange combined with mass spectrometry (MS), we detected pH-dependent changes in deuterium incorporation into the core of the NT domain, indicating global structural stabilization at low pH. The stabilizing effects were diminished or abolished at high ionic strength, or when the surface-exposed residues Asp40 and Glu84 had been exchanged with the corresponding amides. Nondenaturing electrospray ionization MS revealed the presence of dimers in the gas phase at pH values below--but not above--6.4, indicating a tight electrostatic association that is dependent on Asp40 and Glu84 at low pH. Results from analytical ultracentrifugation support these findings. Together, the data suggest a mechanism whereby lowering the pH to <6.4 results in structural changes and alteration of charge-mediated interactions between subunits, thereby locking the spidroin NT dimer into a tight entity important for aggregation and silk formation.

Sterilized Recombinant Spider Silk Fibers of Low Pyrogenicity

We have recently shown that it is possible to recombinantly produce a miniature spider silk protein, 4RepCT, that spontaneously self-assembles into mechanically stable macroscopic fibers (Stark, M.; Grip, S.; Rising, A.; Hedhammar, M.; Engstrom, W.; Hjalm, G.; Johansson, J. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules 2007, 8 (5), 1695-1701). When produced as a soluble fusion protein (with thioredoxin) in Escherichia coli , the spider silk protein can be subjected to several purification steps without aggregating. Here, combined purification and endotoxin removal is achieved using a simple cell wash procedure, protein affinity purification, and LPS depletion. No toxic chemicals were included in the process and the protein retained its ability to self-assemble into fibers. With this method, fibers with pyrogenicity corresponding to less than 1 EU/mg could be recovered. Moreover, the fibers could be sterilized through autoclaving with retained morphology, structure, and mechanical properties. This implies that this recombinant silk is suitable for usage as biomaterial, which is further supported by data showing that the fibers allow growth of human primary fibroblasts.

pH-Dependent Dimerization of Spider Silk N-Terminal Domain Requires Relocation of a Wedged Tryptophan Side Chain.

Formation of spider silk from its constituent proteins-spidroins-involves changes from soluble helical/coil conformations to insoluble β-sheet aggregates. This conversion needs to be regulated to avoid precocious aggregation proximally in the silk gland while still allowing rapid silk assembly in the distal parts. Lowering of pH from about 7 to 6 is apparently important for silk formation. The spidroin N-terminal domain (NT) undergoes stable dimerization and structural changes in this pH region, but the underlying mechanisms are incompletely understood. Here, we determine the NMR and crystal structures of Euprosthenops australis NT mutated in the dimer interface (A72R). Also, the NMR structure of wild-type (wt) E. australis NT at pH7.2 and 300 mM sodium chloride was determined. The wt NT and A72R structures are monomers and virtually identical, but they differ from the subunit structure of dimeric wt NT mainly by having a tryptophan (W10) buried between helix 1 and helix 3, while W10 is surface exposed in the dimer. Wedging of the W10 side chain in monomeric NT tilts helix 3 approximately 5-6Å into a position that is incompatible with that of the observed dimer structure. The structural differences between monomeric and dimeric NT domains explain the tryptophan fluorescence patterns of NT at pH7 and pH6 and indicate that the biological function of NT depends on conversion between the two conformations.

Expression, crystal structure and cellulase activity of the thermostable cellobiohydrolase Cel7A from the fungus Humicola grisea var. thermoidea.

Cellobiohydrolase Cel7A from H. grisea var. thermoidea showed a 10°C higher T m and a 75% higher yield than H. jecorina Cel7A in a performance assay at 65°C. The crystal structure at 1.8 Å resolution indicates higher flexibility in tunnel-defining loops and reveals a new loop conformation near the active centre.