Stepwise design of pseudosymmetric protein hetero-oligomers

Abstract

Pseudosymmetric hetero-oligomers with three or more unique subunits with overall structural (but not sequence) symmetry play key roles in biology, and systematic approaches for generating such proteins de novo would provide new routes to controlling cell signaling and designing complex protein materials. However, the de novo design of protein hetero-oligomers with three or more distinct chains with nearly identical structures is a challenging problem because it requires the accurate design of multiple protein-protein interfaces simultaneously. Here, we describe a divide-and-conquer approach that breaks the multiple-interface design challenge into a set of more tractable symmetric single-interface redesign problems, followed by structural recombination of the validated homo-oligomers into pseudosymmetric hetero-oligomers. Starting from de novo designed circular homo-oligomers composed of 9 or 24 tandemly repeated units, we redesigned the inter-subunit interfaces to generate 15 new homo-oligomers and recombined them to make 17 new hetero-oligomers, including ABC heterotrimers, A2B2 heterotetramers, and A3B3 and A2B2C2 heterohexamers which assemble with high structural specificity. The symmetric homo-oligomers and pseudosymmetric hetero-oligomers generated for each system share a common backbone, and hence are ideal building blocks for generating and functionalizing larger symmetric assemblies.

Fig. 1. Overview of pseudosymmetic recombination design approach.

(a) Two examples of natural pseudosymmetric hetero-oligomers (each pair, right) which likely arose from symmetric ancestors, resembling the paired symmetric paralogs (each pair, left), through gene duplication and diversification. Left, LSm complexes from Escherichia coli (Hfq) and Saccharomyces cerevisiae. Right, PCNA from S. cerevisiae and Sulfolbus solfataricus. (b) Diagram of our stepwise hetero-oligomer creation strategy; variant homo-oligomeric interfaces are generated from a common structure first, followed by junction splicing to assemble theminto hetero-oligomers. Cartoons represent trimers with colored interfaces that match up (separated by a small white gap in the cartoon) and have unique sequences according to their color. Interfaces on the same chain are connected via a homology region in dark gray which has the same sequence and structure across all protomer variants. In this strategy, the interface of an existing homo-oligomer (left; a homotrimer in this example) is redesigned symmetrically and the junction regions remain unchanged. Homo-oligomers that assemble correctly (left column) are cut at the same point within their common homology regions and then spliced back together, making two-chain hybrids with two different interfaces (Fig. S1b) which assemble into heterotrimers (right column). It is possible to create two different heterotrimers with the same interfaces by rearranging the order of the interfaces (Fig. S1c). (c,d) The two test scaffolds used in this work, the homotrimer BGL0 (c) and the homotetramer RTR0 (d). Each is bisected by black crosshairs which pass through the orange sphere on each chain which indicates the location of the residue (Arg82 and Leu98 for BGL0 and RTR0, respectively) within the junction region where splicing will occur (Fig. S1b).

Figure 2:. Characterization of BGL homotrimers

(a) Cartoon representation of 6 representative successful redesigns of BGL0, with two from each design set: Helix Rebuilding + C terminal attachment (HR-C), Helix Rebuilding + N terminal attachment (HR-N) and Normal Modes (NM) relaxation. Blue and red colored backbones highlight N- and C-terminal two-helix segments comprising the interface which was redesigned, and newly designed hydrogen bond networks (HBNets) are shown in sticks with the hydrogen bonds in dashed lines. Gray backbones are supporting structures or other copies of the interface. Design names in the bottom left corner of each box and box outline colors are used consistently throughout. Characterization of the six examples from (a) by (b) SEC, (c) nMS, (d) SAXS, and (e) nsEM. (b) Overlay of A280 normalized SEC traces. Samples were previously purified by SEC to remove any soluble aggregate. SEC traces of all 20 designs are provided in Supplemental Figure 2. (c) Individual mass deconvoluted nMS data of TEV-cleaved samples with all peaks that match the expected mass for each homotrimer are annotated with green circles, and homohexamers (likely to be dimers of trimers) are annotated with orange diamonds. Trace color corresponds to sample ID. See Table S7 for complete data, including m/z spectra. (d) Overlay of SAXS data. Data were scaled appropriately to align at q=0.01 for the purpose of displaying similarity of profile. Radii of gyration calculated from the SAXS profiles match the design models within 5.7Å. See Supplemental Figure 5. (e) Individual 2D class averages of nsEM data collected on each sample. Outline color corresponds to sample ID.

Figure 3.. Crystal structures of BGL homotrimers

(a,b,d) Crystal structures (white) of four different BGL homotrimers are superimposed onto the design models which are colored in teal, purple, and magenta. (left) The top-down view of the full ring with HBNets highlighted and visible through transparent cartoon models. The backbones of all crystal structures match well with the design models (<1.4 Å RMSD over all Cα atoms). (Right) Close up view(s) of each HBNet well enough resolved to build full side chains. (a) BGL06 (2.1 Å resolution; RMSD = 1.2 Å over all Cα atoms; PDB: 8E0L) and (d) BGL18 (3.0 Å resolution; RMSD = 1.4 Å over all Cα atoms; PDB: 8E0N) were both well enough resolved to model all three interfaces. BGL18 contained two copies of the full trimer in the crystal unit cell, which are structurally similar to each other (RMSD = 0.3 Å over all Cα atoms between copies). (b) One interface of BGL14 (3.0 Å resolution; RMSD = 1.3 Å over all Cα atoms; PDB: 8E12) was well resolved. (c) BGL15 (4.0Å resolution, PDB: 8E0M) had three copies of the trimer in the unit cell and all three were close to the design model (0.63 Å, 0.64 Å, and 0.66 Å RMSD over Cα atoms). See Table S2 for crystallographic details.

Figure 4:. Characterization of hetBGL heterotrimers

(a-e) Results of hetBGL03-15-18. (a) (top) Schematic showing the result of recombination between BGL03, BGL15, and BGL18. (bottom) The expression constructs for each chain as expressed tricistronically. “GFP” is superfolderGFP, included to add a large amount of mass and provide an additional spectroscopic identification, and “MP” is EHEE_rd2_0005, a stable and small protein included to add a small amount of mass. (b) SDS-PAGE of IMAC elution. M: protein ladder. E: IMAC elution. Protein size affects band staining, so it is difficult to judge stoichiometry from the band darkness/size, but the correct trend in stain density is observed for equal stoichiometry. (c) SEC trace overlaying normalized absorbances at A280 and A485. (d) Mass deconvoluted nMS data showing the intended major species (green circle, ABC) and a minor species with twice the mass, likely to be a dimer of trimers (orange diamond, A2B2C2). See Table S7 for complete data, including m/z spectra. (e) (left) Crystal structure in white (2.1 Å resolution, PDB: 8E0O) and design model in teal, purple, and magenta overlaid to show global agreement (CA-RMSD: 1.2 Å, TM-score: 0.96). Dashed boxes indicate approximate locations of zoomed-in views of the interfaces derived from BGL03, BGL15, and BGL18, showing side chains of HBNet residues and their inferred hydrogen bonds as yellow dashes. (f) Individual mass deconvoluted nMS data for all hetBGL Set 2 samples. See Table S7 for complete data, including m/z spectra.

Figure 5:. Characterization of RTR homotetramers and hetero-oligomers

(a) Cartoon representation of 3 representative successful redesigns of RTR0. Blue and purple colored backbones represent N- and C-terminal interfaces, respectively. HBNets and disulfide bonds are shown in sticks with hydrogen bonds in dashed lines. Design names (bottom left corner of each box) and box outline colors are used consistently throughout to represent the same interfaces. Characterization of the 3 examples from (a) by (b) SEC, (c) nMS, (d) SAXS, and (e) nsEM. (b) Overlay of A280 normalized SEC traces. SEC traces of all 24 designs are provided in Supplemental Figure 12. (c) Overlay of SAXS data. Radii of gyration calculated from the SAXS profiles match the design models within 7.2 Å. (d) Individual 2D class averages of nsEM data collected on each sample. Outline color corresponds to sample ID. (e) Schematics of hetRTRs with interfaces colored corresponding to interface source next to nsEM class averages of (top left) A2B2-hetRTR05–11, (top right) A52B72-hetRTR05–11noCys including overlay of the 2D class with a 2D class of RTR11 to show angular shift, (bottom left) A3B3-hetRTR05–11noCys, and (bottom right) A2B2C2-hetRTR11noCys-05-16.