CEEPC/IPM/CMSC - Presentation Abstract

(CEEPC/IPM/CMSC 2022 - SaO-22)
The single amino acid substitutions in Mason-Pfizer Monkey Virus matrix protein modulate its proteolytic cleavage rate

Jakub Sýs 1,2 *, Markéta Častorálová 2, Jan Prchal 2,3, Tomáš Ruml 2

  1. Mass Spectrometry, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science,
  2. Department of Biochemistry and Microbiology, University of Chemistry and Technology Prague, Czech Re
  3. Laboratory of NMR Spectroscopy, University of Chemistry and Technology Prague, Czech Republic


N-terminal domain of polyprotein Gag of Mason-Pfizer Monkey Virus (M-PMV) Matrix protein (MA) is naturally myristoylated on its N-terminus. When MA is a part of polyprotein Gag, the myristate moiety is buried inside hydrophobic pocket and exposed probably upon interaction with host cell plasma membrane (PM). This mechanism called myristoyl switch does not occur in M-PMV as readily as it does in HIV-1, suggesting that it may have an important role in M-PMV maturation by regulating the MA cleavage from Gag polyprotein.
To address this hypothesis, we have treated the myristoylated (myrMAPPHis) and nonmyristoylated MAPPHis (nonmyrMAPPHis) M PMV construct, bearing the cleavage site for M-PMV viral protease (Pr13), on artificial liposomes mimicking PM by Pr13 for several time points. In contrast to rapidly degraded nonmyrMAPPHis even without liposomes, the myrMAPPHis, surprisingly, become cleaved more frequently only after addition of liposomes indicating the possible exposure of myristate upon interaction with PM.
To support our findings, we have designed and examined also four mutants of MAPPHis M PMV with single amino acid substitutions with expectation to block or, conversely, facilitate the myristoyl switch by stabilization (A79V, A79L) or destabilization (I51A, I86A) of MA M PMV hydrophobic pocket. The mutants A79V and A79L were cleaved even less effectively than myrMAPPHis as well as the degradation of mutants I51A and I86A was faster compared to nonmyrMAPPHis. The different cleavage rates of proteins were confirmed also on structural level by using method of hydrogen-deuterium exchange coupled with mass spectrometric detection. It shows that the protease cleavage site has different dynamics in proteins as a result of disruption/stabilisation of hydrophobic pocket.

* Corresponding author: Jakub.Sys@seznam.cz


The work was supported from The Czech Science Foundation (GACR;) Project: No. GA22-19250S.

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