Resolving the energy paradox of chaperone/usher-mediated fibre assembly

Abstract

Periplasmic chaperone/usher machineries are used for assembly of filamentous adhesion organelles of Gram-negative pathogens in a process that has been suggested to be driven by folding energy. Structures of mutant chaperone-subunit complexes revealed a final folding transition (condensation of the subunit hydrophobic core) on the release of organelle subunit from the chaperone-subunit pre-assembly complex and incorporation into the final fibre structure. However, in view of the large interface between chaperone and subunit in the pre-assembly complex and the reported stability of this complex, it is difficult to understand how final folding could release sufficient energy to drive assembly. In the present paper, we show the X-ray structure for a native chaperone-fibre complex that, together with thermodynamic data, shows that the final folding step is indeed an essential component of the assembly process. We show that completion of the hydrophobic core and incorporation into the fibre results in an exceptionally stable module, whereas the chaperone-subunit pre-assembly complex is greatly destabilized by the high-energy conformation of the bound subunit. This difference in stabilities creates a free energy potential that drives fibre formation.

Figure 1. Schematic illustration of DSC

(a) Hypothetical free Caf1 subunit. The non-complemented subunit contains a hydrophobic acceptor cleft with five subsites for the insertion of five side chains of a donor strand. The N-terminal donor strand segment of Caf1 is unstructured. (b) Complex with Caf1M. Two chaperone strands (A₁ and G₁) interact with edge strands of the subunit to form a superbarrel [8]. Large G₁ and A₁ (partially) donor residues insert into the acceptor cleft between the two sheets of the subunit β-sandwich such that the superbarrel acquires a fused hydrophobic core. (c) Fibre. Consecutive subunits form Ig-like fibre modules by insertion of the N-terminal G_d donor strand of one subunit into the hydrophobic acceptor cleft of a neighbouring subunit. (d) Donor strand exchange. On release of the subunit from the chaperone and incorporation into the fibre, the two sheets of the subunit β-sandwich move together to form a close packed subunit hydrophobic core. In (a), (b) and (c), side chains participating in DSC are shown as spheres with radii reflecting the size of the donating groups (methyl and larger).

Figure 2. Comparison of WT and A9R Caf1M–Caf1′–Caf1″ complexes

(a) IEF of native (left-hand lane) and mutant (right-hand lane) Caf1M–(Caf1)_n complexes accumulating in the periplasm of cells lacking the usher export channel. Bars show positions of bands of complexes with the indicated number of subunits. Most of these species have been characterized thoroughly, and structures are available for ternary and binary complexes [8,13,16]. The level of secretion of the mutant protein was lower than that of the native one in this experiment. (b) Stereo diagram of the native Caf1M–Caf1′–Caf1″ complex. Caf1M is in blue, except for G₁ and A₁ β-strands (violet). The chaperone-bound Caf1′ subunit is red [N-terminal donor strand (G_d) is orange]; the Caf1″ subunit corresponding to the tip of a growing fibre is green. N- and C-termini are labelled in the same colours as the ribbons. The asterisk in the *N″ label indicates that the N-terminal sequence of Caf1″ up to this point is removed. (c) High-resolution ‘snap-shots’ of subunit collapse upon donor strand exchange. Caf1′, Caf1″ A9R and Caf1″ WT are red, yellow and green respectively (stereo view). Complementing donor strands are shown in the same colours as the complemented subunits. To avoid clutter, only β-strands in the two sheets of each subunit are shown. Ala¹²⁵ and Asn¹³⁶ of the chaperone G₁ as well as Leu³′ and Val¹⁴′ of the subunit G_d donor strands and Arg⁹ of Caf1″ A9R are labelled.

Figure 3. Thermal denaturation of Caf1M–Caf1 and Caf1M–Caf1₂ complexes observed by differential scanning calorimetry

(a) Molar heat capacity curves for Caf1M–Caf1 at 3 mg/ml (1), Caf1M at 2.5 mg/ml (2) and (1)−(2) difference curve (3). (b) Molar heat capacity curve for Caf1M–Caf1₂ (2.2 mg/ml). (c) Transition excess heat capacity curves for Caf1M–Caf1 at 1.08 and 10.8 mg/ml. (d) Dependence of the T_m of Caf1M–Caf1 at the peak maximum on the concentration. Broken lines in (a) and (b) show linear extrapolation of pre- and post-transitional dependences to the transition area.

Figure 4. Chaperone traps a high-energy folding intermediate of the subunit

(a) The transition excess heat capacity of Caf1M (·····), Caf1M–Caf1 (—; ○ indicates fully reversible transition), Caf1M–Caf1₂ (– –), and Caf1M–Caf1₃ (–·–·–). The N-terminal donor strand of the subunit in Caf1M–Caf1 has been removed genetically and the free donor strand at the tip of Caf1M–Caf1₂ and Caf1M–Caf1₃ complexes has been removed enzymatically as described in the Materials and methods section. Complexes were studied at approximately equal concentrations (40–70 μM) to allow proper comparisons. The inset shows the excess heat capacity curve for F1 fibres isolated from the cell surface. (b) IEF of Caf1M–Caf1 and Caf1M–Caf1₂ on pH 3–9 gels (5% polyacrylamide) at the indicated temperatures (left-hand panels). SDS/PAGE (12.5% polyacrylamide) of Caf1M–Caf1₂ pre-heated at 75 and 95 °C (right-hand panel). Arrowheads at B, T and M indicate positions of focusing of the binary (Caf1M–Caf1) and ternary (Caf1M–Caf1₂) complexes, and free Caf1M respectively. Arrowheads at Caf1₂, Caf1 and M indicate bands of Caf1 dimer, Caf1 denatured monomer (with intact (upper band) and digested (low band) N-terminal donor sequence) and Caf1M respectively. (c) Scheme illustrating the sequential process of melting of Caf1M–Caf1₂. Black, Caf1M; grey, Caf1′; white, Caf1″. The donor strand of Caf1′ complementing Caf1″ is shown as a thin black line. The donor strand sequence of Caf1″ was enzymatically removed to block further polymerization (and therefore not shown).

Figure 5. Complete collapse of the subunit hydrophobic core is essential for achieving stable fibre module

The transition excess heat capacity curves of the native (dotted line) and A9R mutant (solid line) Caf1M–Caf1₂ complexes.

Figure 6. Self-complemented subunit folds into a highly stable structure

(a) Caf1-SC accumulates as a monomer in the periplasm. Elution profile of Caf1-SC loaded on a Superdex 75 HR 10/30 gel filtration column. Elution volumes for the marker proteins are indicated by arrows. B, BSA; O, ovalbumin; H, chymotrypsinogen A; M, myoglobin; R, ribonuclease A. Analysis of the selectivity curve (not shown) yields an apparent molecular mass for Caf1-SC of 23.5 kDa. The inset shows Coomassie-Blue-stained SDS/PAGE (12.5% polyacrylamide) of Caf1-SC samples pre-incubated in phosphate buffer containing different concentrations of glutaraldehyde (%). No specific cross-linking is observed. Molecular-mass sizes are given in kDa. (b) Far-UV CD spectra of Caf1-SC at different temperatures. (c) Temperature-dependence of ellipticity at 220 nm. Fraction of unfolded protein is shown in the inset. (d) and (e) Far-UV CD spectra of Caf1-SC samples containing different concentrations of GdmCl at 20 °C (d) and at 55 °C (e). Far-UV parts of spectra where the noise level was high (depending on concentration of GdmCl) are not shown. However, it was checked by multiple scans that spectra shown in (d) do not deviate more than ±250 deg·cm²·dmol⁻¹ from that recorded at 0 M GdmCl in the region 206–212 nm. (f) GdmCl-induced equilibrium transition at 55 °C monitored by measurement of ellipticity at 222 nm. ■, unfolding; ○, refolding by dilution with GdmCl-free buffer. Fraction of unfolded sample is shown in the inset. Refolding data were not used in calculations. Measurements shown in (b) and (c) were performed at a protein concentration of 0.76 mg/ml in 10 mM phosphate buffer, pH 7.1, containing 75 mM NaCl. Measurements shown in (d), (e) and (f) were performed at protein concentrations of 0.3–0.6 mg/ml in 10 mM bis-Tris/propane buffer, pH 7.1, containing 50 mM NaCl.