Domain-Swapped Dimeric Structure of a Stable and Functional De Novo Four-Helix Bundle Protein, WA20

Abstract

To probe the potential for activity in unevolved amino acid sequence space, we created a third generation combinatorial library of de novo four-helix bundle proteins. The "artificial superfamily" of helical bundles was designed using binary patterning of polar and nonpolar residues, and expressed in Escherichia coli from a library of synthetic genes. WA20, picked from the library, is one of the most stable proteins in the superfamily, and has rudimentary activities such as esterase and lipase. Here we report the crystal structure of WA20, determined by the multiwavelength anomalous dispersion method. Unexpectedly, the WA20 crystal structure is not a monomeric four-helix bundle, but a dimeric four-helix bundle. Each monomer comprises two long α-helices that intertwist with the helices of the other monomer. The two monomers together form a 3D domain-swapped four-helix bundle dimer. In addition, there are two hydrophobic pockets, which may potentially provide substrate binding sites. Small-angle X-ray scattering shows that the molecular weight of WA20 is ~25 kDa and the shape is rod-like (the maximum length, D(max) = ~8 nm), indicating that WA20 forms a dimeric four-helix bundle in solution. These results demonstrate that our de novo protein library contains not only simple monomeric proteins, but also stable and functional multimeric proteins.

De Novo Proteins with Life-Sustaining Functions Are Structurally Dynamic

“…The protein WA20 is also a member of the 3 rd generation library. We recently solved the crystal structure of WA20 to 2.2 Å, which revealed a 4-helix bundle comprising a domain swapped dimer 22 . Figure 1 shows the sequences of the SynRescue proteins, the control proteins S23, S824, and WA20 (1B), and the experimentally determined structures of S824 (1C) and WA20 (1D).…”

“…Three structures were determined by NMR or crystallography to reveal 4-helix bundles with hydrophobic interiors and polar surfaces, as envisioned by the binary patterned design. Two proteins from our 2 nd generation library formed monomeric 4-helix bundles 4; 21 , while an X-ray structure solved from a sequence from the 3 rd generation library revealed a domain swapped dimer 22 . We have also identified de novo proteins from these libraries that bind small molecules, including drugs and cofactors 18; 23 .…”

Self-Assembling Nano-Architectures Created from a Protein Nano-Building Block Using an Intermolecularly Folded Dimeric de Novo Protein

“…15,16 Recently, we solved the crystal structure of the de novo protein WA20, revealing an unusual 3D-domain-swapped dimeric structure with a intermolecularly folded 4-helix bundle. 5 (3D domain swapping is a mechanism of exchanging one structural domain of a protein monomer with that of the identical domain from a second monomer, resulting in an intertwined oligomer. 17,18 ) Each WA20 monomer ("nunchaku"-like structure), which comprises two long α-helices, intertwines with the helices of another monomer ( Figure 1A; and Figure S1, Supporting Information).…”

“…Schematics of construction and assemblies of the WA20-foldon fusion protein as a protein nano-building block (PNBlock). (A) Ribbon representation (see also Figure S1, Supporting Information) and schematics of the intermolecularly folded dimeric WA20 (PDB code, 3VJF), 5 shown in red, and trimeric foldon domain of T4 phage fibritin (PDB code, 1RFO), 6 shown in blue. (B) Construction of the WA20-foldon fusion protein as a PN-Block.…”

A protein constructed de novo enables cell growth by altering gene regulation

“…We have shown previously that several proteins from these binary patterned libraries fold into stable four-helix bundles, and both crystallographic and NMR structures have been determined (4)(5)(6). Moreover, in initial steps probing the potential for functional activity, proteins from these libraries were shown to bind small molecules, including cofactors, and to catalyze rudimentary reactions (7,8).…”