Recombinant Butelase-Mediated Cyclization of the p53-Binding Domain of the Oncoprotein MdmX-Stabilized Protein Conformation as a Promising Model for Structural Investigation

Abstract

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 (T m ) of cMdmX was increased to 47 from 43 °C. This stable conformation of cMdmX was further confirmed by 15 N− 1 H 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.

Asparaginyl endopeptidases: enzymology, applications and limitations

“…Butelase 1 was obtained via an arduous extraction and purification process from the native host, butterfly pea plant, for initial experiments. 23,37 Subsequently, procedures for the preparation of recombinant butelase 1 from bacteria (E. coli) 36 and yeast (P. pastoris) 73 were reported. Similarly, the preparation of recombinant OaAEP1b from E. coli culture have been reported in a number of studies.…”

“… 64 Native and engineered AEPs derived from various plants have also been successfully employed to facilitate backbone cyclisation of peptides derived from other plant and non-plant species such as kalata B1; 21–23 sunflower trypsin inhibitor 1; 23,45,72 histatin-3; 23 anti-malarial peptide R1; 39 MCoTI-II; 27 AS-48, 72 and proteins such as GFP; 10,40,49 somatropin; 49 MSP2; 39 p53 binding domain. 73 Butelase 1 was reported to generate the cyclic antimicrobial peptide θ-defensin and the cyclic conotoxin MrlA with >95% yield in 1 minute at 42 °C and pH 6.0. 45 …”

“…The cyclic protein was reported to aid structural investigations by NMR and X-ray crystallography, methods that may be hindered by conformational flexibility induced line broadening and poor crystal formation, respectively. 73 Furthermore, greater conformational stability in the cyclic protein was suggested to offer improved ligand binding due to a reduction of the associated entropic penalty. 73 Protein cyclisation by AEP was also applied to MSP2 from Plasmodium falciparum , a 25 kDa protein for malarial vaccine development.…”

Asparaginyl Ligases: New Enzymes for the Protein Engineer's Toolbox

“…Whereas butelase 1 was initially available only when purified from C. ternatea extracts, both wild‐type OaAEP1 and the C247A mutant were accessible via recombinant production in Escherichia coli [11a,18] . More recent efforts to recombinantly produce butelase 1 in E. coli and Pichia pastoris have been promising, though a direct kinetic comparison to the plant‐derived material is yet to be reported [13,21] . Notably, the recombinant production of full‐length asparaginyl ligases necessitates an activation step where auto‐cleavage to free the core domain is induced by acidification to pH 4–5.…”

Plant derived cyclic peptides: from discovery to biotechnological applications

“…However, cyclic peptides that are smaller than nine residues cannot be cyclized . To facilitate the broad utilisation of butelase1, it has been developed to be produced recombinantly in E. coli (James et al, 2019) and yeast (Pi et al, 2019).…”