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Computational design of a symmetrical ?-trefoil lectin with cancer cell binding activity.

ABSTRACT: Computational protein design has advanced very rapidly over the last decade, but there remain few examples of artificial proteins with direct medical applications. This study describes a new artificial ?-trefoil lectin that recognises Burkitt's lymphoma cells, and which was designed with the intention of finding a basis for novel cancer treatments or diagnostics. The new protein, called "Mitsuba", is based on the structure of the natural shellfish lectin MytiLec-1, a member of a small lectin family that uses unique sequence motifs to bind ?-D-galactose. The three subdomains of MytiLec-1 each carry one galactose binding site, and the 149-residue protein forms a tight dimer in solution. Mitsuba (meaning "three-leaf" in Japanese) was created by symmetry constraining the structure of a MytiLec-1 subunit, resulting in a 150-residue sequence that contains three identical tandem repeats. Mitsuba-1 was expressed and crystallised to confirm the X-ray structure matches the predicted model. Mitsuba-1 recognises cancer cells that express globotriose (Gal?(1,4)Gal?(1,4)Glc) on the surface, but the cytotoxicity is abolished.

SUBMITTER: Terada D

PROVIDER: S-EPMC5517649 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Computational design of a symmetrical β-trefoil lectin with cancer cell binding activity.

Terada Daiki D Voet Arnout R D ARD Noguchi Hiroki H Kamata Kenichi K Ohki Mio M Addy Christine C Fujii Yuki Y Yamamoto Daiki D Ozeki Yasuhiro Y Tame Jeremy R H JRH Zhang Kam Y J KYJ

Scientific reports 20170719 1

Computational protein design has advanced very rapidly over the last decade, but there remain few examples of artificial proteins with direct medical applications. This study describes a new artificial β-trefoil lectin that recognises Burkitt's lymphoma cells, and which was designed with the intention of finding a basis for novel cancer treatments or diagnostics. The new protein, called "Mitsuba", is based on the structure of the natural shellfish lectin MytiLec-1, a member of a small lectin fam ...[more]

PMID: 28724971

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Project description:We describe a computational protocol, called DDMI, for redesigning scaffold proteins to bind to a specified region on a target protein. The DDMI protocol is implemented within the Rosetta molecular modeling program and uses rigid-body docking, sequence design, and gradient-based minimization of backbone and side-chain torsion angles to design low-energy interfaces between the scaffold and target protein. Iterative rounds of sequence design and conformational optimization were needed to produce models that have calculated binding energies that are similar to binding energies calculated for native complexes. We also show that additional conformation sampling with molecular dynamics can be iterated with sequence design to further lower the computed energy of the designed complexes. To experimentally test the DDMI protocol, we redesigned the human hyperplastic discs protein to bind to the kinase domain of p21-activated kinase 1 (PAK1). Six designs were experimentally characterized. Two of the designs aggregated and were not characterized further. Of the remaining four designs, three bound to the PAK1 with affinities tighter than 350 muM. The tightest binding design, named Spider Roll, bound with an affinity of 100 muM. NMR-based structure prediction of Spider Roll based on backbone and (13)C(beta) chemical shifts using the program CS-ROSETTA indicated that the architecture of human hyperplastic discs protein is preserved. Mutagenesis studies confirmed that Spider Roll binds the target patch on PAK1. Additionally, Spider Roll binds to full-length PAK1 in its activated state but does not bind PAK1 when it forms an auto-inhibited conformation that blocks the Spider Roll target site. Subsequent NMR characterization of the binding of Spider Roll to PAK1 revealed a comparably small binding 'on-rate' constant (<<10(5) M(-1) s(-1)). The ability to rationally design the site of novel protein-protein interactions is an important step towards creating new proteins that are useful as therapeutics or molecular probes.

Dataset Information

Computational design of a symmetrical ?-trefoil lectin with cancer cell binding activity.

Publications

Computational design of a symmetrical β-trefoil lectin with cancer cell binding activity.

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