Chasper Puorger

Pilus chaperones represent a new type of protein-folding catalyst

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’ pathway, 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 assembly. The chaperone subsequently delivers the subunits to an assembly platform (usher) in the outer membrane, which mediates subunit assembly and translocation to the cell surface. 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.

Identification and Characterization of the Chaperone-Subunit Complex-binding Domain from the Type 1 Pilus Assembly Platform FimD

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.

Quality control of disulfide bond formation in pilus subunits by the chaperone FimC

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.

Intramolecular donor strand complementation in the E. coli type 1 pilus subunit FimA explains the existence of FimA monomers as off-pathway products of pilus assembly that inhibit host cell apoptosis.

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.

The Most Stable Protein–Ligand Complex: Applications for One-Step Affinity Purification and Identification of Protein Assemblies†

The purification of protein complexes and large-scale investigations of protein–protein interaction networks have been greatly facilitated through the development of a number of affinity tags such as the cmyc, FLAG, and His6 tags. [2] However, all of the currently available affinity purification systems suffer from dynamic binding equilibria and measurable dissociation rate constants which enable the competitive elution of bound target proteins when an excess of a suitable free ligand is present. This circumstance hampers the quantitative and cost-effective isolation of low-abundance protein complexes. Here, we introduce a new affinity purification system that is derived from type 1 pili of E. coli. Type 1 pili are rigid, filamentous supramolecular protein complexes which are anchored in the cell s outer membrane and extend into the extracellular space. They are composed of four structural protein subunits termed FimH, FimG, FimF, and FimA. In the assembled pilus, these subunits interact noncovalently by a mechanism called donor strand complementation, where the incomplete, immunoglobulin-like fold of one subunit is completed by an N-terminal extension, termed donor strand, of the successive subunit. The complex between FimGt, an N-terminally truncated variant of FimG lacking its own donor strand, and a peptide corresponding to the donor strand of the neighboring subunit FimF (DsF) was found to be the kinetically most stable protein–ligand complex known to date (Figure 1). Here, we establish the FimGt/DsF system for use in the affinity purification of heterooligomeric protein complexes from cell extracts. Utilizing the donor strand of FimF as the affinity tag (termed DsF tag) and FimGt as the binding partner, we demonstrate the one-step purification of DsFtagged E. coli tryptophan synthase, a heterotetrameric abba complex of low cellular abundance. We compare the performance of the DsF tag to that of other commonly used affinity tags and find that, in agreement with the high kinetic stability of the FimGt/DsF complex, enrichment of the tryptophan synthase complex is most efficient for the DsF tag. This result suggests that the DsF tag is most suitable not only for the isolation of low-abundance protein complexes but presumably also for many other technical applications such as, for example, the functional and permanent immobilization of DsF-tagged proteins on surfaces and their detection in cells and on Western blot membranes. As a prerequisite for the technical application of the FimGt/DsF system we first optimized the production of FimGt by producing it in the cytoplasm of E. coli BL21(DE3) cells in the form of inclusion bodies (Figure S1 in the Supporting Information). After solubilization of the inclusion bodies, oxidative refolding, and purification of FimGt by conventional chromatographic techniques, the final yield of purified FimGt was 35 mg per liter of bacterial culture— sufficient amounts for large-scale applications of the FimGt/ DsF system. Quantitative formation of the single, structural disulfide bond was verified by the Ellman assay. The identity of FimGt was confirmed by ESI-MS (expected/measured mass: 13656.9/13657.0 Da). To gain mechanistic insight into the binding reaction between DsF and FimGt, association kinetics were measured for DsF concentrations of 5, 10, 25 and 50 mm while the FimGt concentration was kept constant at 5 mm (Figure 2 a). The reaction rates were dependent on the DsF concentration, indicating that binding of DsF is the rate-limiting step of complex formation. The data were globally fit according to an irreversible second-order reaction and yielded a rate constant of association of (330 8.9)m 1 s . Although relatively slow, the binding of DsF to FimGt is fast enough to allow for technical applications on reasonable time scales. We determined the rate constant for spontaneous dissociation/unfolding of the FimGt/DsF complex at pH 8.0 and 25 8C to be Figure 1. Crystal structure of the FimGt/DsF complex (3BFQ.pdb). FimGt is shown as a gray surface, the DsF peptide as stick representation. Residues of DsF that point towards FimGt are in bold and their position in the structure is indicated by arrows.