Computational design of self-assembling cyclic protein homo-oligomers

Abstract

Self-assembling cyclic protein homo-oligomers play important roles in biology, and the ability to generate custom homo-oligomeric structures could enable new approaches to probe biological function. Here we report a general approach to design cyclic homo-oligomers that employs a new residue-pair-transform method to assess the designability of a protein-protein interface. This method is sufficiently rapid to enable the systematic enumeration of cyclically docked arrangements of a monomer followed by sequence design of the newly formed interfaces. We use this method to design interfaces onto idealized repeat proteins that direct their assembly into complexes that possess cyclic symmetry. Of 96 designs that were characterized experimentally, 21 were found to form stable monodisperse homo-oligomers in solution, and 15 (four homodimers, six homotrimers, six homotetramers and one homopentamer) had solution small-angle X-ray scattering data consistent with the design models. X-ray crystal structures were obtained for five of the designs and each is very close to their corresponding computational model.

Figure 1

Computational design protocol. Left, starting with a monomeric protein we exhaustively sample cyclic docked configurations, score them using the RPX model and generate sequences to drive the complex formation using a full atom RosettaDesign calculation. Right, schematic representation of the RPX model scoring procedure.

Figure 2

Assessment of the solution conformation of selected cyclic oligomers. From left to right: computational model, symmetric docking energy landscape, SEC chromatogram used for molecular weight determination, and SAXS scattering profiles experimentally measured (black dots) and computed from the model (red line). “MW (design)” refers to the molecular weight of the oligomer design and “MW (MALS)” refers to the experimentally determined molecular weight. a, ank3C2_1. b, HR79C2. c, HR08C3 d, HR00C3_2. e, HR04C4_1. f, HR10C5_2. Analogous data for the nine other successful designs are provided in Sup Fig 5.

Figure 3

Comparison between the experimentally determined crystal structures and corresponding design models. Crystal structures are shown in cyan and models in gray. Left column, full model and crystal structure superposition; Right column, superposition showing hydrophobic side chains at the designed interface. a, ank3C2_1 (r.ms.d. to model 1 Å) b, ank1C2_1 (r.ms.d. to model 0.9 Å) c, 1na0C3_3 (r.ms.d. to model 1 Å) d, HR00C3_2 (r.ms.d. to model 0.9 Å) e, ank1C4_2 pair of chains (r.ms.d. to model 1.1 Å)

Figure 4

Robustness of designs to subunit extension by repeat addition. From left to right: computational model of the original design, computational model of the extended design, SEC-MALS chromatogram used for molecular weight determination (n represents number of repeat modules in each monomer; original design: solid line; extended design: dotted line), SAXS scattering profiles (original design: experimental data in black circles, computed profile in red; extended design: experimental data open circles, computed profile in cyan). a, ank1C2_1. B, HR04C4_1.