9RGV

X-ray crystal structure of a de novo designed single-chain antiparallel 3-helix coiled-coil bundle, sc-apCC3-CW1

Summary for 9RGV

• Entry DOI: 10.2210/pdb9rgv/pdb
• Descriptor: sc-apCC3-CW1 (2 entities in total)
• Functional Keywords: coiled coil, cc, de novo, de novo protein
• Biological source: synthetic construct
• Total number of polymer chains: 2
• Total formula weight: 24993.29

Authors

Leng, X.,Woolfson, D.N. (deposition date: 2025-06-08, release date: 2025-09-03, Last modification date: 2025-10-22)

Primary citation

Leng, X.,Albanese, K.I.,Golub, L.R.,Norman, A.A.,Clayden, J.,Woolfson, D.N.
De novo designed 3-helix bundle peptides and proteins with controlled topology and stability.
Chem Sci, 16:18632-18641, 2025

PubMed Abstract: Computational protein design is advancing rapidly. However, approaches and methods are needed to increase success rates and to elaborate designs. Here we describe the combination of rational and computational design to deliver three-helix bundle (3HB) peptide assemblies and single-chain proteins with control over topology and thermal stability. First, we garner sequence-to-structure relationships from antiparallel 3HBs in the Protein Data Bank. This gives core-packing rules, including layers of hydrogen-bonded polar residues, which are combined with surface-charge patterning to design complementary sequences for acidic (A), basic (B), and neutral (N) helices. By altering the design of the N helix, two sets of synthetic peptides are generated for clockwise and anticlockwise arrangements of the three-helix assemblies. Solution-phase characterisation shows that both ABN peptide mixtures form stable, heterotrimeric assemblies consistent with the targeted 'up-down-up' topologies. Next, AlphaFold2 models for both designs are used to seed computational designs of single-chain proteins where the helices are connected by loop building. Synthetic genes for these express in to yield soluble, monomeric, and thermally stable proteins. By systematically introducing additional polar layers within the core, the thermal stability of these proteins is varied without compromising the specificity of the helix-helix interactions. Chemical and thermal denaturation reveals comparable thermodynamic parameters to those of highly stable natural proteins. Four X-ray crystal structures confirm that the design models and AlphaFold2 predictions match to sub-Å accuracy.

PubMed: 40969159
DOI: 10.1039/d5sc05576h

Experimental method

X-RAY DIFFRACTION (2.1 Å)