Liquid-liquid crystalline phase separation of spider silk proteins.

Liquid-liquid phase separation (LLPS) of proteins can be considered an intermediate solubility regime between disperse solutions and solid fibers. While LLPS has been described for several pathogenic amyloids, recent evidence suggests that it is similarly relevant for functional amyloids. Here, we review the evidence that links spider silk proteins (spidroins) and LLPS and its role in the spinning process.

Molecular basis for different substrate-binding sites and chaperone functions of the BRICHOS domain.

Proteins can misfold into fibrillar or amorphous aggregates and molecular chaperones act as crucial guardians against these undesirable processes. The BRICHOS chaperone domain, found in several otherwise unrelated proproteins that contain amyloidogenic regions, effectively inhibits amyloid formation and toxicity but can in some cases also prevent non-fibrillar, amorphous protein aggregation. Here, we elucidate the molecular basis behind the multifaceted chaperone activities of the BRICHOS domain from the Bri2 proprotein.

Archaic chaperone-usher pili self-secrete into superelastic zigzag springs.

Adhesive pili assembled through the chaperone-usher pathway are hair-like appendages that mediate host tissue colonization and biofilm formation of Gram-negative bacteria. Archaic chaperone-usher pathway pili, the most diverse and widespread chaperone-usher pathway adhesins, are promising vaccine and drug targets owing to their prevalence in the most troublesome multidrug-resistant pathogens. However, their architecture and assembly-secretion process remain unknown.

MrpH, a new class of metal-binding adhesin, requires zinc to mediate biofilm formation.

Proteus mirabilis, a Gram-negative uropathogen, is a major causative agent in catheter-associated urinary tract infections (CAUTI). Mannose-resistant Proteus-like fimbriae (MR/P) are crucially important for P. mirabilis infectivity and are required for biofilm formation and auto-aggregation, as well as for bladder and kidney colonization.

Structure of the N-terminal domain of Euprosthenops australis dragline silk suggests that conversion of spidroin dope to spider silk involves a conserved asymmetric dimer intermediate.

Spider silk is a biomaterial with exceptional mechanical toughness, and there is great interest in developing biomimetic methods to produce engineered spider silk-based materials. However, the mechanisms that regulate the conversion of spider silk proteins (spidroins) from highly soluble dope into silk are not completely understood. The N-terminal domain (NT) of Euprosthenops australis dragline silk protein undergoes conformational and quaternary-structure changes from a monomer at a pH above 7 to a homodimer at lower pH values.

Structures of two fimbrial adhesins, AtfE and UcaD, from the uropathogen Proteus mirabilis.

The important uropathogen Proteus mirabilis encodes a record number of chaperone/usher-pathway adhesive fimbriae. Such fimbriae, which are used for adhesion to cell surfaces/tissues and for biofilm formation, are typically important virulence factors in bacterial pathogenesis. Here, the structures of the receptor-binding domains of the tip-located two-domain adhesins UcaD (1.

Diazirine-functionalized mannosides for photoaffinity labeling: trouble with FimH.

Photoaffinity labeling is frequently employed for the investigation of ligand-receptor interactions in solution. We have employed an interdisciplinary methodology to achieve facile photolabeling of the lectin FimH, which is a bacterial protein, crucial for adhesion, colonization and infection. Following our earlier work, we have here designed and synthesized diazirine-functionalized mannosides as high-affinity FimH ligands and performed an extensive study on photo-crosslinking of the best ligand (mannoside ) with a series of model peptides and FimH.

A gene encoding a potential adenosine 5'-phosphosulphate kinase is necessary for timely development of Myxococcus xanthus.

A Myxococcus xanthus gene, MXAN3487, was identified by transposon mutagenesis to be required for the expression of mcuABC, an operon coding for part of the chaperone-usher (CU) system in this bacterium. The MXAN3487 protein displays sequence and structural homology to adenosine 5'-phosphosulphate (APS) kinase family members and contains putative motifs for ATP and APS binding. Although the MXAN3487 locus is not linked to other sulphate assimilation genes, its protein product may have APS kinase activity in vivo and the importance of the ATP-binding site for activity was demonstrated.

Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains.

Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known.

Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation.

The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions.

The BRICHOS domain, amyloid fibril formation, and their relationship.

Amyloid diseases are defined by tissue deposition of insoluble, fibrillar β-sheet polymers of specific proteins, but it appears that toxic oligomeric species rather than the fibrils are the main cause of tissue degeneration. Many proteins can form amyloid-like fibrils in vitro, but only ~30 proteins have been found to cause mammalian amyloid disease, suggesting that physiological mechanisms that protect against amyloid formation exist. The transmembrane region of lung surfactant protein C precursor (proSP-C) forms amyloid-like fibrils in vitro, and SP-C amyloid has been found in lung tissue from patients with interstitial lung disease (ILD).

Crystal structure of enterotoxigenic Escherichia coli colonization factor CS6 reveals a novel type of functional assembly.

Coli surface antigen 6 (CS6) is a widely expressed enterotoxigenic Escherichia coli (ETEC) colonization factor that mediates bacterial attachment to the small intestinal epithelium. CS6 is a polymer of two protein subunits CssA and CssB, which are secreted and assembled on the cell surface via the CssC/CssD chaperone usher (CU) pathway. Here, we present an atomic resolution model for the structure of CS6 based on the results of X-ray crystallographic, spectroscopic and biochemical studies, and suggest a mechanism for CS6-mediated adhesion.

Allosteric mechanism controls traffic in the chaperone/usher pathway.

Many virulence organelles of Gram-negative bacterial pathogens are assembled via the chaperone/usher pathway. The chaperone transports organelle subunits across the periplasm to the outer membrane usher, where they are released and incorporated into growing fibers. Here, we elucidate the mechanism of the usher-targeting step in assembly of the Yersinia pestis F1 capsule at the atomic level.

BRICHOS domains efficiently delay fibrillation of amyloid β-peptide.

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 (Aβ(40) and Aβ(42)) far below the stoichiometric ratio.

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.

Large is fast, small is tight: determinants of speed and affinity in subunit capture by a periplasmic chaperone.

The chaperone/usher pathway assembles surface virulence organelles of Gram-negative bacteria, consisting of fibers of linearly polymerized protein subunits. Fiber subunits are connected through 'donor strand complementation': each subunit completes the immunoglobulin (Ig)-like fold of the neighboring subunit by donating the seventh β-strand in trans. Whereas the folding of Ig domains is a fast first-order process, folding of Ig modules into the fiber conformation is a slow second-order process.

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.

En route to photoaffinity labeling of the bacterial lectin FimH.

Mannose-specific adhesion of Escherichia coli bacteria to cell surfaces, the cause of various infections, is mediated by a fimbrial lectin, called FimH. X-ray studies have revealed a carbohydrate recognition domain (CRD) on FimH that can complex α-D-mannosides. However, as the precise nature of the ligand-receptor interactions in mannose-specific adhesion is not yet fully understood, it is of interest to identify carbohydrate recognition domains on the fimbrial lectin also in solution.

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.

Conserved hydrophobic clusters on the surface of the Caf1A usher C-terminal domain are important for F1 antigen assembly.

The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 (capsular antigen fraction 1) protein subunits. The Caf1A usher promotes polymerization of subunits and secretion of growing polymers to the cell surface.

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.

Novel insights into the biological function of mast cell carboxypeptidase A.

When mast cells are activated they can respond by releasing their secretory granule compounds, including mast cell-specific proteases of chymase, tryptase and carboxypeptidase A (MC-CPA) type. MC-CPA is a dominant protein component of the mast cell granule and the MC-CPA gene is extremely highly expressed. Despite this, relatively little has been known of its biological function.