Published in Nature, 8th May 2019.
Ali D. Malay, Naoyuki Miyazaki, Artur Biela, Soumyananda Chakraborti, Karolina Majsterkiewicz, Izabela Stupka, Craig S. Kaplan, Agnieszka Kowalczyk, Bernard M.A.G. Piette, Georg K.A. Hochberg, Di Wu, Tomasz P. Wrobel, Adam Fineberg, Manish S. Kushwah, Mitja Kelemen, Primož Vavpetič, Primož Pelicon, Philipp Kukura, Justin L.P. Benesch, Kenji Iwasaki, Jonathan G. Heddle
Symmetrical protein cages have evolved to fulfil diverse roles in nature, including compartmentalization and cargo delivery, and have inspired synthetic biologists to create novel protein assemblies via the precise manipulation of protein–protein interfaces. Despite the impressive array of protein cages produced in the laboratory, the design of inducible assemblies remains challenging. Here we demonstrate an ultra-stable artificial protein cage, the assembly and disassembly of which can be controlled by metal coordination at the protein–protein interfaces. The addition of a gold (I)- triphenylphosphine compound to a cysteine-substituted, 11-mer protein ring triggers supramolecular self-assembly, which generates monodisperse cage structures with masses greater than 2 MDa. The geometry of these structures is based on the Archimedean snub cube and is, to our knowledge, unprecedented.
Cryo-electron microscopy confirms that the assemblies are held together by 120 S–Au–S staples between the protein oligomers, and exist in two chiral forms. The cage shows extreme chemical and thermal stability, yet it readily disassembles upon exposure to reducing agents. As well as gold, mercury is also found to enable formation of the protein cage. This work establishes an approach for linking protein components into robust, higher-order structures, and expands the design space available for pramolecular assemblies to include previously unexplored geometries.
Natalie J. Tatum†, Fernanda Duarte‡, Shina C. L. Kamerlin¶, and Ehmke Pohl*†∥§
† Department of Chemistry, Durham University, South Road, Durham DH1 3LE, U.K.
‡ Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.
¶ Department of Chemistry - BMC, Uppsala University, BMC Box 576, S-751 23 Uppsala, Sweden
∥ Department of Biosciences, Durham University, Durham DH1 3LE, U.K.
§ Biophysical Sciences Institute, Durham University, Durham DH1 3LE, U.K.
Transcriptional repressor EthR from Mycobacterium tuberculosis is a valuable target for antibiotic booster drugs. We previously reported a virtual screening campaign to identify EthR inhibitors for development. Two ligand binding orientations were often proposed, though only the top scoring pose was utilized for filtering of the large data set. We obtained biophysically validated hits, some of which yielded complex crystal structures. In some cases, the crystallized binding mode and top scoring mode agree, while for others an alternate ligand binding orientation was found. In this contribution, we combine rigid docking, molecular dynamics simulations, and the linear interaction energy method to calculate binding free energies and derive relative binding energies for a number of EthR inhibitors in both modes. This strategy allowed us to correctly predict the most favorable orientation. Therefore, this widely applicable approach will be suitable to triage multiple binding modes within EthR and other potential drug targets with similar characteristics.