Modular repeat protein sculpting using rigid helical junctions

Abstract

The ability to precisely design large proteins with diverse shapes would enable applications ranging from the design of protein binders that wrap around their target to the positioning of multiple functional sites in specified orientations. We describe a protein backbone design method for generating a wide range of rigid fusions between helix-containing proteins and use it to design 75,000 structurally unique junctions between monomeric and homo-oligomeric de novo designed and ankyrin repeat proteins (RPs). Of the junction designs that were experimentally characterized, 82% have circular dichroism and solution small-angle X-ray scattering profiles consistent with the design models and are stable at 95 °C. Crystal structures of four designed junctions were in close agreement with the design models with rmsds ranging from 0.9 to 1.6 Å. Electron microscopic images of extended tetrameric structures and ∼10-nm-diameter “L” and “V” shapes generated using the junctions are close to the design models, demonstrating the control the rigid junctions provide for protein shape sculpting over multiple nanometer length scales.

Hierarchical design of multi-scale protein complexes by combinatorial assembly of oligomeric helical bundle and repeat protein building blocks

“…Docked configurations with linkable N-and C-termini within a distance cutoff of 9Å with interfaces predicted to yield low energy designs 25 were then subjected to Rosetta sequence design to optimize the residue identity and packing at the newly formed interface. Designs with high predicted domain-domain binding energy and shape complementarity 26 were identified, and loops connecting chain the termini were built using the ConnectChainsMover 17 . Structures with good loop geometry (passing worse9merFilter and FoldabilityFilter) were forward folded with RosettaRemodel 27 symmetrically, and those with sequences which fold into the designed structure in silico were identified.…”

“…The same set of DHRs and HBs were combinatorially fused together by overlapping the terminal helix residues in both directions ("AB": c-terminus of HB to n-terminus of DHR, "BA": n-terminus of HB to c-terminus of DHR) 17 . On the HB end, up to 4 residues were allowed to be deleted to maximize the sampling space of the fusion while maintaining the structural integrity of the oligomeric interface.…”

“…If no residues in the fused structure clash (Rosetta centroid energy < 10), sequence design was carried out at all positions within 8Å of the junction. This first step of the fusion sampling is wrapped into the Rosetta MergePDBMover 17 . After sequence design around the junction region 14,28 , fusions were then evaluated based on the number of helices interacting across the interface (at least 3), buried surface (sasa > 800) across the junction, and shape complementarity (sc > 0.6) to identify designs likely to be rigid across the junction point.…”

“…However, it can be seen that they deviate from the design model at the most distal portions of the structure. This is likely due to the inherent flexibility of the unsupported terminal helices of the DHRs 17,23,30 and lever arm effects which increase with increasing distance from the fusion site ( Figure S15).…”

“…All genes were cloned into E. coli cells (BL21 Lemo21 (DE3)) for expression, using auto-induction 38 at 18° or 37°C for [16][17][18][19][20][21][22][23][24] hours in 500mL scale. Post-induction, cultures were centrifuged at 8,000xG for 15 minutes.…”