Recombinant Butelase-Mediated Cyclization of the p53-Binding Domain of the Oncoprotein MdmX-Stabilized Protein Conformation as a Promising Model for Structural Investigation
journal contributionposted on 12.06.2019, 00:00 authored by Ni Pi, Meng Gao, Xiyao Cheng, Huili Liu, Zhengkun Kuang, Zixin Yang, Jing Yang, Bailing Zhang, Yao Chen, Sen Liu, Yongqi Huang, Zhengding Su
Cyclization of the polypeptide backbone has proven to be a powerful strategy for enhancing protein stability for fundamental research and pharmaceutical application. The use of such an approach is restricted by how well a targeted polypeptide can be efficiently ligated. Recently, an Asx-specific peptide ligase identified from a tropical cyclotide-producing plant and named butelase 1 exhibited excellent cyclization kinetics that cannot be matched by other known ligases, including intein, PATG, PCY1, and sortase A. In this work, we aimed to examine whether butelase 1 facilitated protein conformational stability for structural investigation. First, we successfully expressed recombinant butelase 1 (rBTase) in the yeast Pichia pastoris. Next, rBTase was shown to be highly efficient in the cyclization of the p53-binding domain (N-terminal domain) of murine double minute X (N-MdmX), an important target for designing anticancer drugs. The cyclized N-MdmX (cMdmX) exhibited increased conformational stability and improved interaction with the ligand compared with those of noncyclized N-MdmX. Importantly, the thermal melting process was completely reversible, contrary to noncyclized N-MdmX, and the melting temperature (Tm) of cMdmX was increased to 47 from 43 °C. This stable conformation of cMdmX was further confirmed by 15N–1H heteronuclear single-quantum coherence nuclear magnetic resonance (NMR) spectroscopy. The complex of cMdmX and the ligand was tested for protein crystallization, and several promising findings were revealed. Therefore, our work not only provides a recombinant version of butelase 1 but also suggests a conventional approach for preparing stable protein samples for both protein crystallization and NMR structural investigation.
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stabilityligandrBTasenoncyclized N-MdmXPCYinvestigationapproachcMdmXp 53-binding domainStructural Investigation Cyclizationp 53-Binding DomainOncoprotein MdmX-Stabilized Protein ConformationRecombinant Butelase-Mediated Cyclizationyeast Pichia pastorisAsx-specific peptide ligaseprotein crystallizationpolypeptidePATGcyclizationbutelase 1NMR