Mutations at the hydrophobic core affect Hal3 trimer stability, reducing its Ppz1 inhibitory capacity but not its PPCDC moonlighting function

Carlos Santolaria,Diego Velázquez,Erick Strauss,Joaquín Ariño

doi:10.1038/s41598-018-32979-x

Carlos Santolaria, Diego Velázquez + Show 2 more

Open Access

https://doi.org/10.1038/s41598-018-32979-x

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Abstract

S. cerevisiae Hal3 (ScHal3) is a moonlighting protein that, is in its monomeric state, regulates the Ser/Thr protein phosphatase Ppz1, but also joins ScCab3 (and in some instances the Hal3 paralog Vhs3) to form an unusual heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. PPCDC is required for CoA biosynthesis and in most eukaryotes is a homotrimeric complex with three identical catalytic sites at the trimer interfaces. However, in S. cerevisiae the heterotrimeric arrangement results in a single functional catalytic center. Importantly, the specific structural determinants that direct Hal3’s oligomeric state and those required for Ppz1 inhibition remain largely unknown. We mutagenized residues in the predicted hydrophobic core of ScHal3 (L403–L405) and the plant Arabidopsis thaliana Hal3 (AtHal3, G115–L117) oligomers and characterized their properties as PPCDC components and, for ScHal3, also as Ppz1 inhibitor. We found that in AtHal3 these changes do not affect trimerization or PPCDC function. Similarly, mutation of ScHal3 L403 has no effect. In contrast, ScHal3 L405E fails to form homotrimers, but retains the capacity to bind Cab3—explaining its ability to rescue a hal3 vhs3 synthetically lethal mutation. Remarkably, the L405E mutation decreases Hal3’s ability to interact with and to inhibit Ppz1, confirming the importance of the oligomer/monomer equilibrium in Hal3’s Ppz1 regulating function.

Highlights

The Saccharomyces cerevisiae SIS2/HAL3 gene was simultaneously identified as a suppressor of the sit[4] deletion[1] and a regulator of salt tolerance[2]
While the mechanism of the phosphopantothenoylcysteine decarboxylase (PPCDC) catalytic reaction has been explored in detail[21,22,23,24,25], little is known about the structural determinants required for formation of Hal[3] trimers in A. thaliana or S. cerevisiae, as well as the specific elements in ScHal[3] that are necessary for its inhibitory role on Ppz[1]
Inspection of the 3D structure of Athal[3] (PDB accession numbers 1E20 and 1MVN) and of models created for the PD domain of ScHal[3], together with alignment of PPCDC sequences from bacteria to human pointed to residues G115 and L117 in AtHal[3], and the corresponding L403 and L405 in ScHal[3], as possible components of a hydrophobic core that could be important for generating and/or maintaining the trimer (Fig. 1A,B)

Summary

Introduction

The Saccharomyces cerevisiae SIS2/HAL3 gene was simultaneously identified as a suppressor of the sit[4] deletion[1] and a regulator of salt tolerance[2]. On the basis of the 3D structures of A. thaliana AtHal3a13,14 and Homo sapiens HsCoaC15 orthologs it was proposed that in the active S. cerevisiae PPCDC holoenzyme ScCab[3] provides an essential Cys[478] and a conserved Asn[442] residue These residues are necessary for catalysis and binding of the carboxylate of the substrate PPC, respectively. While mutation of ScHal[3] L403 has no effect, change of L405 to Glu alters the ability to form standard trimers and decreases the capacity of this variant to inhibit Ppz[1] Such mutated versions retain the ability to interact in vitro with Cab[3] and its expression in yeast rescues the hal[3] vhs[3] synthetically lethal phenotype, indicating a fruitful interaction with Cab[3] to form a functional PPCDC enzyme

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