Runaway domain swapping in amyloid-like fibrils of T7 endonuclease I

Abstract

Amyloid fibrils are associated with >20 fatal human disorders, including Alzheimer's, Parkinson's, and prion diseases. Knowledge of how soluble proteins assemble into amyloid fibrils remains elusive despite its potential usefulness for developing diagnostics and therapeutics. In at least some fibrils, runaway domain swapping has been proposed as a possible mechanism for fibril formation. In runaway domain swapping, each protein molecule swaps a domain into the complementary domain of the adjacent molecule along the fibril. Here we show that T7 endonuclease I, a naturally domain-swapped dimeric protein, can form amyloid-like fibrils. Using protein engineering, we designed a double-cysteine mutant that forms amyloid-like fibrils in which molecules of T7 endonuclease I are linked by intermolecular disulfide bonds. Because the disulfide bonds are designed to form only at the domain-swapped dimer interface, the resulting covalently linked fibrils show that T7 endonuclease I forms fibrils by a runaway domain swap. In addition, we show that the disulfide mutant exists in two conformations, only one of which is able to form fibrils. We also find that domain-swapped dimers, if locked in a close-ended dimeric form, are unable to form fibrils. Our study provides strong evidence for runaway domain swapping in the formation of an amyloid-like fibril and, consequently, a molecular explanation for specificity and stability of fibrils. In addition, our results suggest that inhibition of fibril formation for domain-swapped proteins may be achieved by stabilizing domain-swapped dimers.

Fig. 1.

Amyloid-like properties of T7EI fibrils. (a) Native PAGE of the T7EI protein incubated at 4°C and 37°C. Elongated fibrils are formed only at 37°C and stay on the surface of the stacking gel (open arrowhead). The soluble dimer is indicated by the filled arrowhead. (b) Electron micrograph of the T7EI fibrils. (Scale bar: 100 nm.) (c) X-ray diffraction pattern of oriented T7EI fibrils. A sharp meridional reflection at ≈4.7 Å is a characteristic of cross-β structure. A weak 10.5-Å reflection is indicative of β-sheets. (d) The absorbance of CR solution alone and with T7EI protein incubated at 37°C. Binding of the fibrils results in a red shift of the absorption spectrum, characteristic of amyloid formation. (e) Fluorescence emission spectrum of thioflavin T (ThT) alone and with T7EI fibrils. Binding of amyloid-specific ThT to the fibrils gives an emission peak at 482 nm.

Fig. 2.

Design of the double-cysteine mutant L19C/S95C of T7EI. (a) Ribbon diagram of T7EI with Leu-19 and Ser-95 shown in space-filling models. (b) SDS/PAGE showing that T7EI L19C/S95C is locked as a disulfide-bridged dimer in the absence of disulfide-breaking agent DTT (lane 1) and is released into monomers in the presence of DTT (lane 2).

Fig. 3.

Demonstration of wild-type activity in the designed mutant T7EI L19C/S59C by a gel-shift assay. Fluorescently labeled DNA junction 3, a synthetic substrate for T7EI, was loaded either alone (lane 1) or with L19C/S95C protein (lane 2). The presence of L19C/S95C resulted in a more slowly migrating band that corresponds to the protein-bound junction 3 (lane 2). As a control, a duplex DNA that comprises the fluorescently labeled strand of the junction 3 and its complementary strand was loaded either alone (lane 3) or with L19C/S95C protein (lane 4). No binding of duplex DNA was detected in the presence of L19C/S95C protein.

Fig. 4.

Fibril formation of T7EI L19C/S95C requires a fibrillization-competent conformation. (a) Two dimers of the double-cysteine mutant L19C/S95C revealed by native PAGE in various concentrations of DTT. A new dimer (dimer II) appears in the presence of DTT, as shown by increasing amount of dimer II with increasing concentrations of DTT (lanes 2–5). The doubly disulfide-linked dimer in the absence of DTT is dimer I. DTT also results in the formation of a higher oligomer (lanes 4 and 5). (b) Dimer II is disulfide-linked, as shown by SDS/PAGE of L19C/S95C. Both dimer I and dimer II are seen in DTT concentrations up to 100 μM (lanes 2–4). This result indicates intermolecular disulfide bridges in both dimer I and dimer II. In the presence of 1 mM DTT (lane 5), both dimers are converted to monomers. There are also two forms of monomers in low concentrations of DTT on SDS gel (lanes 2–4) that correspond to the intramolecularly disulfide-bonded monomer (lower band) and nondisulfide-bonded monomer (upper band). (c) Dimer II but not dimer I forms fibrils, as shown by native PAGE of L19C/S95C after incubation at 37°C for 18 h. The 4°C samples (19 hours old) are loaded for comparison. In the presence of 25 μM and 50 μM DTT (lanes 4 and 6), dimer II disappears and fibrils form as shown by the high-molecular-mass band on the surface of the stacking gel and at the boundary between the stacking gel and the running gel. Dimer I does not disappear at these DTT concentrations. However, in the presence of 100 μM and 1 mM DTT (lanes 8 and 10), all dimer I disappears, indicating that the equilibrium between dimer I and dimer II is shifted toward dimer II that subsequently forms fibrils at 37°C.

Fig. 5.

Dimer II but not dimer I of T7EI L19C/S95C contains free cysteines. T7EI L19C/S95C in the presence of increasing concentrations of the reducing agent TCEP was run on a native PAGE. (Left) The gel was stained with a thiol-specific fluorescent label and scanned for fluorescence. (Right) T7EI L19C/S95C on the native gel was stained with Coomassie blue after fluorescence scanning.

Fig. 6.

Monomers of T7EI L19C/S95C are cross-linked into small oligomers and fibrils by disulfide bonds, demonstrating an organization based on a runaway domain swap. (a) SDS/PAGE of T7EI L19C/S95C incubated at 37°C for 18 h. This gel shows that the fibrils contain higher oligomers (lanes 3, 5, and 7), and these oligomers are disulfide-bridged because they are reduced to monomers by DTT (lanes 4, 6, and 8). The sample with 1,000 μM DTT does not show a lot of higher oligomers (lane 9) because excess DTT is present in the sample. (b) SDS/PAGE of L19C/S95C incubated at 37°C for 6 days. After 6 days at 37°C, the SDS-solubilized fibrils show even higher oligomers that remain at the boundary of the stacking gel and running gel (lanes 5, 7, and 9, filled arrowheads) or on the surface of the stacking gel (lane 9, open arrowhead), and most of these fibrils are converted into monomers by adding DTT in the gel-loading buffer (lanes 6, 8, and 10).

Fig. 7.

Schematic model for the fibril formation of T7EI L19C/S95C. Each subunit is colored either in red or blue. Black dots represent disulfide bonds, and SH represents free cysteine. In dimer I, both disulfide bonds are intact and the protein is locked in close-ended dimers and unable to form fibrils. In dimer II, half of the domain-swapped molecule is unlatched by the reduction of one of the two disulfide bonds. Upon incubation at 37°C, dimer II changes to an open form (denoted as dimer II*), in which half of the dimer opens up, exposing the interface that remains protected in dimer I. Open-ended dimer II* readily fibrillizes via runaway domain swapping. The hinge-loop region of the domain-swapped protein forms a zipper spine in the fibrils.