Application Notes

MST untangles the intricacy of a multimeric protein complex in its native form

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©2019 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. 2 APPLICATION NOTE interact with battery of auxiliary proteins and RNA molecules. One of the essential functional elements of a eukaryotic ribosome is a P-stalk, a pentameric protein complex, composed of well conserved P1, P2 and uL10 proteins. Together with ribosomal uL10, uL11 and the sarcin-ricin loop (SRL), the P1-P2 heterodimers are part of the GTPase Associated Center (GAC) which is involved in recruitment of translational GTPases. Function of the stalk proteins is related to interaction with auxiliary factors recruited to the GAC. Structurally a C-terminal part domain (CTD) of P1-P2 dimers is very important for the function of the complex [3]. CTD is very well conserved stretch of highly acidic and hydrophobic amino acids (EEEAKEESDDDMGFGLFD) and was shown to be involved in the interaction with translational GTPases and ribosome inactivating proteins (RIPs) [4]. Ricin is a type II RIP consisting of RTA and RTB subunits. RTA removes an adenine from a universally conserved SRL in the 28S rRNA and interacts with P-proteins of the stalk to depurinate SRL both in human and yeast cell [5, 6, 7]. P-proteins of the stalk represent the primary harboring site for ricin, with binding affinities in the low nanomolar range. Moreover, deletion of P-proteins reduces SRL depurination by ricin [8, 9, 10, 16]. In this application note we present a thorough characterization study of ricin's RTA chain interaction with human P1-P2 dimer. We demonstrate how the problem of characterizing the binding affinity between the multimeric assembly P1-P2 and toxin chain was solved with the help of MST. Additionally, we show the analysis with the alternative biophysical techniques and discuss benefits of MST platform in providing solution to similar biological questions. Results To understand the binding mechanism between P1/ P2 heterodimers and RTA binding affinities between RTA and several P1/P2 variants were determined by microscale thermophoresis. The individual proteins and their mutants lacking conserved 16-amino acid residues at the C-terminus, were purified in E.coli and assembled to form heterodimers. SDS-PAGE and native mass- spectrometry were used to confirm correct assembly of heterodimers with resolution of single Dalton in respect to molecular weight of heterodimers (Figure 1). For MST experiments, RTA was labeled using NHS NTT- 647 fluorescent dye according to the manufacturer's protocol. Different P-protein complexes were titrated as ligands at concentrations starting from 50nM up to 100µM. Full-length P1-P2 heterodimer showed highest affinity toward RTA at K d = 86nM. Deletion of conserved C-terminal fragment of P2 (P1-P2ΔC) resulted in six-fold weaker affinity (K d =533 nM), when compared to native P1-P2 heterodimer. Interestingly, P2 formed homodimers showed a very weak affinity ~1-2 µM. In case of P1ΔC-P2ΔC we observed binding of this heterodimer with RTA but binding affinity could not be determined precisely, since saturation was not reached at 100 µM of ligand. RTA also did not show any interaction neither with P1ΔC-P2 heterodimer mutant nor P2ΔC-P2ΔC double homodimer mutant (Figure 2).

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