Czech Society for Mass Spectrometry

(13^th Czech Mass Spectrometry Conference and 11^th Informal Proteomic Meeting - WeO-07)
Unraveling Oligomerization-Mediated Inhibition in GGPPS by Structural Mass Spectrometry

Jasmína Portašiková ^1,2 *, Ruba Yehia ³, Moshe Giladi ^3,4, Alan Kádek ¹, Yoni Haitin ^3,5, Petr Man ¹

1. Institute of Microbiology of the Czech Academy of Sciences, Division BioCeV, Vestec, CZ
2. Department of Biochemistry, Faculty of Science, Charles University, Prague, CZ
3. Department of Physiology and Pharmacology, Faculty of Medical and Health Sciences, Tel-Aviv, Israel
4. Tel Aviv Sourasky Medical Center, Tel Aviv, Israel,
5. Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel

Abstract

Geranylgeranyl diphosphate synthase (GGPPS) is a trans-prenyltransferase that catalyzes the production of the geranylgeranyl moiety in living organisms. In cells, the geranylgeranyl group can be attached to proteins, such as Rab or Rho proteins, enabling their interaction with membranes. Dysfunction in protein prenylation associated with GGPPS mutations has been proven responsible for the development of multiple myeloma and other malignant transformations. Therefore, GGPPS is a valuable target for anti-cancer drugs.
Oligomeric assemblies of GGPPS vary across organisms. In lower-level species, GGPPS assembles in a dimeric organization, whereas in higher-level species, such as humans, GGPPS forms a hexamer composed of three dimers. The inter-dimeric interaction is facilitated by hydrophobic interactions and hydrogen bonds.
In the past, the R235C mutation was discovered in human GGPPS of multiple myeloma patients. This mutation is localized to the active site lid region at the inter-dimeric interface. Using native MS experiments, we uncovered lower stability of the hexameric organization due to destabilized inter-dimeric interactions in the R235C mutant. Subsequently, we performed HDX-MS experiments, which showed reduced lid dynamics in the wild-type compared to the R235C mutant, thereby blocking the pathway for product release. These results indicate that faster product release in the R235C mutant leads to higher catalytic activity of the mutant protein due to decreased product inhibition. Additionally, experiments on the dimeric Y246D mutant showed even faster product release than the R235C mutant. Therefore, we conclude that these functional alterations are directly influenced by the oligomeric assembly of the protein.

* Corresponding author: portasij@natur.cuni.cz

Acknowledgement:

Financial support from the MEYS/EU OP JAK project PHOTOMACHINES - CZ.02.01.01/00/22_008/0004624 is gratefully acknowledged.