Complex stability and dynamic subunit interchange modulates the disparate activities of the yeast moonlighting proteins Hal3 and Vhs3.

J Albert Abrie,Erick Strauss,Cristina Molero,Joaquín Ariño

doi:10.1038/srep15774

J Albert Abrie, Erick Strauss + Show 2 more

Open Access

https://doi.org/10.1038/srep15774

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Abstract

Saccharomyces cerevisiae Hal3 and Vhs3 are moonlighting proteins, acting both as inhibitors of the serine/threonine protein phosphatase Ppz1 and as subunits (together with Cab3) of the unique heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme of Hemiascomycetous yeast. Both these roles are essential: PPCDC catalyses the third step of coenzyme A biosynthesis, while Ppz1 inhibition is required for regulation of monovalent cation homeostasis. However, the mechanisms by which these proteins’ disparate activities are regulated are not well understood. The PPCDC domains (PDs) of Hal3, Vhs3 and Cab3 constitute the minimum requirement for these proteins to show both PPCDC activity and, in the case of Hal3 and Vhs3, to bind to Ppz1. Using these PD proteins as a model system to study the possibility of dynamic interchange between these roles, we provide evidence that Hal3 binds Ppz1 as a monomer (1:1 stoichiometry), requiring it to de-oligomerize from its usual homo- and heterotrimeric states (the latter having PPCDC activity). This de-oligomerization is made possible by structural features that set Hal3 apart from Vhs3, increasing its ability to undergo monomer exchange. These findings suggest that oligomer interchange may be a significant factor in the functional regulation of these proteins and their various unrelated (moonlighting) functions.

Highlights

Decarboxylase (PPCDC) enzyme that catalyses the third step of the universal coenzyme A (CoA) biosynthetic pathway, namely the decarboxylation of 4′ -phosphopantothenoylcysteine (PPC) to form 4′ -phosphopantetheine (PPantSH) (Fig. 1b)
The disparate and non-overlapping activities of the S. cerevisiae Hal[3] and Vhs[3] proteins— either as constituents of a unique heterotrimeric PPCDC involved in CoA biosynthesis, or as regulatory subunits of the Ppz[1] phosphatase—clearly identifies them as moonlighting proteins, the lack of structural information on these proteins made it difficult to rationalize the molecular basis whereby these activities are differentiated
In this study we investigated the activity, structure, thermal stability and exchange reactions of hetero-trimers of the Hal[3], Vhs[3] and Cab[3] PPCDC domain (PD) in an attempt to gain a better understanding of the factors that affect the formation of the active PPCDC enzyme, and whether oligomerization dynamics could act as a putative regulatory mechanism

Summary

Introduction

Decarboxylase (PPCDC) enzyme that catalyses the third step of the universal coenzyme A (CoA) biosynthetic pathway, namely the decarboxylation of 4′ -phosphopantothenoylcysteine (PPC) to form 4′ -phosphopantetheine (PPantSH) (Fig. 1b). Mutagenesis studies of Hal3’s PD indicated that some of the residues that are functionally important for Ppz[1] inhibition are associated with, not essential for, PPCDC function[5,6] It still remained unknown whether Hal[3] interacts with Ppz[1] as a monomer or as a trimer. Our results show that there are significant structural differences between Hal[3] and Vhs[3], and that oligomeric mixtures made up of these proteins exhibit different thermal stabilities based on their composition, as well as varying abilities to undergo monomer exchange These findings point to the dynamic interchange of oligomer subunits as a significant factor in the functional regulation of these proteins’ moonlighting functions

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