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SUMMARYAdhesive type 1 pili from uropathogenicEscherichia colistrains are filamentous, supramolecular protein complexes consisting of a short tip fibrillum and a long, helical rod formed by up to several thousand copies of the major pilus subunit FimA. Here, we reconstituted the entire type 1 pilus rod assembly reactionin vitro, using all constituent protein subunits in the presence of the assembly platform FimD, and identified the so-far uncharacterized subunit FimI as an irreversible assembly terminator. We provide a complete, quantitative model of pilus rod assembly kinetics based on the measured rate constants of FimD-catalyzed subunit incorporation. The model reliably predicts the length distribution of assembled pilus rods as a function of the ratio between FimI and the main pilus subunit FimA and is fully consistent with the length distribution of membrane-anchored pili assembledin vivo. The results show that the natural length distribution of adhesive pili formed via the chaperone-usher pathway results from a stochastic chain termination reaction.

Sortase enzymes play an important role in Gram-positive bacteria. They are responsible for the covalent attachment of proteins to the surface of the bacteria and perform this task via a highly sequence-specific transpeptidation reaction. Since these immobilized proteins are often involved in pathogenicity of Gram-positive bacteria, characterization of this type of enzyme is also of medical relevance. Different classes of sortases (A–F) have been found, which recognize characteristic recognition sequences present in substrate proteins. Up to date, sortase A from Staphylococcus aureus, a housekeeping class A sortase, is the most thoroughly studied representative of the sortase family of enzymes. Here we report the in-depth characterization of the class F sortase from Propionibacterium acnes, a class of sortases that has not been investigated before. As Sortase F is the only transpeptidase found in the P. acnes genome, it is the housekeeping sortase of this organism. Sortase F from P. acnes shows a behavior similar to sortases from class A in terms of pH dependence, recognition sequence and catalytic activity; furthermore, its activity is independent of bivalent ions, which contrasts to sortase A from S. aureus. We demonstrate that sortase F is useful for protein engineering applications, by producing a site-specifically conjugated homogenous antibody–drug conjugate with a potency similar to that of a conjugate prepared with sortase A. Thus, the detailed characterization presented here will not only enable the development of anti-virulence agents targeting P. acnes but also provides a powerful alternative to sortase A for protein engineering applications.

We report a method to equip proteolytic nanobiocatalysts with intrinsic disulphide bond reducing properties. After immobilisation onto silica particles, selected protease enzymes are partially shielded in a nanometre-thick mercaptosilica layer acting not only as a protective system but also as a substrate reducing agent. The biocatalysts produced efficiently perform simultaneous disulphide bond reduction and protein digestion. Besides a significant simplification of the proteolysis process, this strategy allows for a drastic increase of the enzyme stability. &nbsp

Sortases are enzymes that are responsible for the attachment of secreted proteins to the cell wall of Gram-positive bacteria. Hereby, the sortases recognize short, five-residue amino acid sequences present in the target proteins and fuse them to the peptidoglycan layer via a transpeptidation reaction, creating a new peptide bond between the C-terminus of the recognition sequence and the cell wall. The transpeptidation activity of sortases is widely used in protein engineering for modification of target proteins. The majority of protocols rely on the high activity of the well-characterized Staphylococcus aureus SrtA and variants thereof, while sortases from other classes are not used for this purpose. This can be attributed to the lower activity of other sortases and to the limited sequence specificity data available for the different sortases. We set out to determine the sequence specificity of Bacillus anthracis SrtB. To this end, we developed a new method for sequence specificity determination of sortases or other bond-forming enzymes that recognize an amino acid sequence. Using mixtures of recognition peptides of limited complexity, which are reacted with biotinylated substrates, the biotinylated transpeptidation products are isolated with magnetic streptavidin beads and analyzed via liquid chromatography and mass spectrometry. With this, peptide sequences that are recognized by the sortase and function as substrates can be determined and quantified. The method, developed with the highly active evolved SrtA from S. aureus, allowed for the first time unbiased in-depth analysis of the sequence specificity for SrtB from B. anthracis, which is 10

Additional file 1: Figure S1. Poisson Statistics and enrichment of colonized microcapsules. a Shown is the probability that a colonized microcapsule is monoclonal according to the Poisson distribution. During cell encapsulation, the lower the average number of viable cells per capsule (λ), the higher the probability that a colonized microcapsule is monoclonal (red). A low average number of cells per capsule leads to a lower fraction of monoclonal microcapsules (blue) and, in turn, to a higher fraction of uncolonized microcapsules. b Scheme of the gravity-driven separation procedure for the enrichment of colonized microcapsules from a mixture of colonized and uncolonized microcapsules. Figure S2. Sortase enzymes catalyze a transpeptidation reaction. a Scheme of the transpeptidation reaction catalyzed by sortase enzymes. Sortases recognize the pentapeptide sorting motif (e.g. LPXTG here) on the target protein (acyl donor substrate); The cysteine in the active site of the enzyme performs a nucleophilic attack on the carbonyl carbon of threonine residue, breaking the peptide bond between the threonine and the glycine and forming an intermediate in which the enzyme and the target protein are linked together via a thioacyl linkage. The subsequent nucleophilic attack of the thioacyl linkage by the free amino group of an oligoglycine stretch of a cell wall component (acyl acceptor substrate) resolves the intermediate and results to the covalent attachment of the target protein to the bacterial cell wall. b Scheme of the intramolecular transpeptidation reaction catalyzed by an N-terminally-modified sortase enzyme. The glycine residue at the N-terminus acts as the acyl acceptor substrate that solves the intermediate in an intramolecular fashion. This enables the sortase enzyme to covalently capture the acyl donor substrate (e.g. a FITC-labelled pentapeptide).

Sortase enzymes play an important role in Gram-positive bacteria. They are responsible for the covalent attachment of proteins to the surface of the bacteria and perform this task via a highly sequence-specific transpeptidation reaction. Since these immobilized proteins are often involved in pathogenicity of Gram-positive bacteria, characterization of this type of enzyme is also of medical relevance. Different classes of sortases (A-F) have been found, which recognize characteristic recognition sequences present in substrate proteins. Up to date, sortase A from

Type 1 pili are filamentous organelles mediating the attachment of uropathogenic Escherichia coli to epithelial cells of host organisms. The helical pilus rod consists of up to 3000 copies of the main structural subunit FimA that interact via donor strand complementation where the incomplete Ig like fold of FimA is completed by insertion of the N terminal extension (donor strand) of the following FimA subunit. Recently it was shown that FimA also exists in a monomeric assembly incompetent form and that FimA monomers act as inhibitors of apoptosis in infected host cells. Here we present the NMR structure of monomeric wild type FimA with its natural N terminal donor strand complementing the Ig fold. Compared to FimA subunits in the assembled pilus intramolecular self complementation in the monomer stabilizes the FimA fold with significantly less interactions and the natural FimA donor strand is inserted in the opposite orientation. In addition we show that a motif of two glycine residues in the FimA donor strand separated by five residues is the prerequisite of the alternative parallel donor strand insertion mechanism in the FimA monomer and that this motif is preserved in FimA homologs of many enteroinvasive pathogens. We conclude that FimA is a unique case of a protein with alternative functionally relevant folding possibilities with the FimA polymer forming the highly stable pilus rod and the FimA monomer promoting pathogen propagation by apoptosis suppression of infected epithelial target cells. © 2013 Elsevier Ltd.

Type 1 pili from uropathogenic Escherichia coli are filamentous, noncovalent protein complexes mediating bacterial adhesion to the host tissue. All structural pilus subunits are homologous proteins sharing an invariant disulfide bridge. Here we show that disulfide bond formation in the unfolded subunits, catalyzed by the periplasmic oxidoreductase DsbA, is required for subunit recognition by the assembly chaperone FimC and for FimC-catalyzed subunit folding. FimC thus guarantees quantitative disulfide bond formation in each of the up to 3,000 subunits of the pilus. The X-ray structure of the complex between FimC and the main pilus subunit FimA and the kinetics of FimC-catalyzed FimA folding indicate that FimC accelerates folding of pilus subunits by lowering their topological complexity. The kinetic data, together with the measured in vivo concentrations of DsbA and FimC, predict an in vivo half-life of 2 s for oxidative folding of FimA in the periplasm.

An alternative to tagging: The thermodynamically most stable protein ligand complex known to date (K D=1.5×10 20M) consists of the two components FimGt and DsF portions of two different subunits of the type 1 pilus protein complex. The first technical applications of this new complex are the single step purification and identification of multiprotein complexes from cell extracts. Copyright © 2012 WILEY VCH Verlag GmbH Co. KGaA Weinheim.

Filamentous type 1 pili are responsible for attachment of uropathogenic Escherichia coli strains to host cells. They consist of a linear tip fibrillum and a helical rod formed by up to 3000 copies of the main structural pilus subunit FimA. The subunits in the pilus interact via donor strand complementation, where the incomplete, immunoglobulin-like fold of each subunit is complemented by an N-terminal donor strand of the subsequent subunit. Here, we show that folding of FimA occurs at an extremely slow rate (half-life: 1.6 h) and is catalyzed more than 400-fold by the pilus chaperone FimC. Moreover, FimA is capable of intramolecular self-complementation via its own donor strand, as evidenced by the loss of folding competence upon donor strand deletion. Folded FimA is an assembly-incompetent monomer of low thermodynamic stability (− 10.1 kJ mol − 1 ) that can be rescued for pilus assembly at 37 °C because FimC selectively pulls the fraction of unfolded FimA molecules from the FimA folding equilibrium and allows FimA refolding on its surface. Elongation of FimA at the C-terminus by its own donor strand generated a self-complemented variant (FimAa) with alternative folding possibilities that spontaneously adopts the more stable conformation (− 85.0 kJ mol − 1 ) in which the C-terminal donor strand is inserted in the opposite orientation relative to that in FimA. The solved NMR structure of FimAa revealed extensive β-sheet hydrogen bonding between the FimA pilin domain and the C-terminal donor strand and provides the basis for reconstruction of an atomic model of the pilus rod.

SummaryAdhesive type 1 pili from uropathogenic Escherichia coli strains are heat and denaturant resistant, filamentous protein complexes. Individual pilus subunits associate through “donor strand complementation,” whereby the incomplete immunoglobulin-like fold of each subunit is completed by the N-terminal extension of a neighboring subunit. We show that antiparallel donor strand insertion generally causes nonequilibrium behavior in protein folding and extreme activation energy barriers for dissociation of subunit-subunit complexes. We identify the most kinetically stable, noncovalent protein complex known to date. The complex between the pilus subunit FimG and the donor strand peptide of the subunit FimF shows an extrapolated dissociation half-life of 3 × 109 years. The 15 residue peptide forms ideal intermolecular β sheet H-bonds with FimG over 10 residues, and its hydrophobic side chains strongly interact with the hydrophobic core of FimG. The results show that kinetic stability and nonequilibrium behavior in protein folding confers infinite stability against dissociation in extracellular protein complexes.

Type 1 pili from uropathogenic Escherichia coli strains mediate bacterial attachment to target receptors on the host tissue. They are composed of up to 3000 copies of the subunit FimA, which form the stiff, helical pilus rod, and the subunits FimF, FimG, and FimH, which form the linear tip fibrillum. All subunits in the pilus interact via donor strand complementation, in which the incomplete immunoglobulin-like fold of each subunit is complemented by insertion of an N-terminal extension from the following subunit. We determined the NMR structure of a monomeric, self-complemented variant of FimF, FimF F , which has a second FimF donor strand segment fused to its C-terminus that enables intramolecular complementation of the FimF fold. NMR studies on bimolecular complexes between FimF F and donor strand-depleted variants of FimF and FimG revealed that the relative orientations of neighboring domains in the tip fibrillum cover a wide range. The data provide strong support for the intrinsic flexibility of the tip fibrillum. They lend further support to the hypothesis that this flexibility would significantly increase the probability that the adhesin at the distal end of the fibrillum successfully targets host cell receptors.

The free energy of folding of small, one-domain proteins is generally measured with denaturant-induced unfolding/refolding equilibria based on the two-state model of protein folding. There is however an increasing number of reports on proteins that do not attain their folding equilibrium and show, even after long-term incubation, unfolding transitions at high and refolding transitions at low denaturant concentrations. We present a theory for the quantitative description of this nonequilibrium behavior in protein folding, which is exclusively based on the two-state assumption and the exponential dependence of the rate constants of unfolding and refolding on denaturant concentration. Using a hyperstable variant of a pilin domain from E. coli type 1 pili that does not reach its folding equilibrium within several years, we demonstrate that the method provides reliable information on the free energy of folding, the solvent accessibility of the transition state of folding relative to the native and unfolded sta...

Adhesive type 1 pili from uropathogenic Escherichia coli strains have a crucial role during infection by mediating the attachment to and potentially the invasion of host tissue. These filamentous, highly oligomeric protein complexes are assembled by the ‘chaperone–usher’ pathway1, in which the individual pilus subunits fold in the bacterial periplasm and form stoichiometric complexes with a periplasmic chaperone molecule that is essential for pilus assembly2,3,4. The chaperone subsequently delivers the subunits to an assembly platform (usher) in the outer membrane, which mediates subunit assembly and translocation to the cell surface5,6,7,8. Here we show that the periplasmic type 1 pilus chaperone FimC binds non-native pilus subunits and accelerates folding of the subunit FimG by 100-fold. Moreover, we find that the FimC–FimG complex is formed quantitatively and very rapidly when folding of FimG is initiated in the presence of both FimC and the assembly-competent subunit FimF, even though the FimC–FimG complex is thermodynamically less stable than the FimF–FimG complex. FimC thus represents a previously unknown type of protein-folding catalyst, and simultaneously acts as a kinetic trap preventing spontaneous subunit assembly in the periplasm.

The outer membrane protein FimD represents the assembly platform of adhesive type 1 pili from Escherichia coli . FimD forms ring-shaped oligomers of 91.4 kDa subunits that recognize complexes between the pilus chaperone FimC and individual pilus subunits in the periplasm and mediate subunit translocation through the outer membrane. Here, we have identified a periplasmic domain of FimD (FimD N ) comprising the N-terminal 139 residues of FimD. Purified FimD N is a monomeric, soluble protein that specifically recognizes complexes between FimC and individual type 1 pilus subunits, but does not bind the isolated chaperone, or isolated subunits. In addition, FimD N retains the ability of FimD to recognize different chaperone-subunit complexes with different affinities, and has the highest affinity towards the FimC–FimH complex. Overexpression of FimD N in the periplasm of wild-type E. coli cells diminished incorporation of FimH at the tip of type 1 pili, while pilus assembly itself was not affected. The identification of FimD N and its ternary complexes with FimC and individual pilus subunits opens the avenue to structural characterization of critical type 1 pilus assembly intermediates.