22LX

De novo designed S-locus Protein 11 (SP11)-like protein (P6522 form)

Summary for 22LX

• Entry DOI: 10.2210/pdb22lx/pdb
• Descriptor: De novo designed SP11-like protein, SULFATE ION, ACETATE ION, ... (5 entities in total)
• Functional Keywords: s-locus protein 11, pollen determinant, de novo protein
• Biological source: synthetic construct
• Total number of polymer chains: 1
• Total formula weight: 17009.17

Authors

Miyoshi, H.,Sakuma, K.,Moriwaki, Y.,Fushinobu, S. (deposition date: 2026-01-16, release date: 2026-08-05)

Primary citation

Miyoshi, H.,Sakuma, K.,Moriwaki, Y.,Aoyama, N.,Kashima, T.,Terada, T.,Miyanaga, A.,Fushinobu, S.
Structural and Stability Analysis of de Novo Designed Proteins Incorporating a Plant Self-Incompatibility Motif.
Proteins, 2026

PubMed Abstract: Artificial protein design enables the creation of stable scaffolds beyond those evolved in nature. Incorporation of native functional motifs into de novo scaffolds provides a promising strategy to mimic natural interactions while altering structural frameworks. Here, we designed de novo S-locus Protein 11 (SP11)-like proteins by incorporating a key feature of the Brassica pollen determinant SP11, which mediates self-incompatibility through specific interaction with the pistil determinant S receptor kinase (SRK). The designed proteins included the six-amino-acid SRK-binding motif from native SP11 but lacked all disulfide bonds characteristic of the plant defensin-like fold. Three variants (SP11-A, SP11-B, SP11-C) were designed using Rosetta and ProteinMPNN sequence optimization and expressed in Escherichia coli. SP11-A and SP11-B were purified and analyzed by circular dichroism spectroscopy. SP11-A exhibited exceptional thermal and chemical stability, demonstrating the robustness of the artificial scaffold, while SP11-B had lower stability and displayed biphasic chemical unfolding behavior. The crystal structures of SP11-A in two space groups were determined at 1.50 and 2.10 Å resolution. Notably, the region derived from the native SP11 sequence exhibited two alternative main chain conformations, indicating that incorporation of the natural motif introduced local conformational frustration. Molecular dynamics analysis suggested that the two alternative main chain conformations remain kinetically stable within the 100 ns simulation timescale, without direct interconversion. Together, these results provide insight into how the incorporation of native motifs can influence local structure while preserving global scaffold stability in de novo protein design.

PubMed: 42499170
DOI: 10.1002/prot.70162

Experimental method

X-RAY DIFFRACTION (1.5 Å)