X‐ray structure of Saccharomyces cerevisiae homologous mitochondrial matrix factor 1 (Hmf1)

Introduction. Homologous mitochondrial matrix factor 1 Hmf1 (also known as YEO7 and YER057c) is a cytoplasmic homolog of Saccharomyces cerevisiae mitochondrial matrix factor 1 Mmf1, which has been proposed to serve as a sensor for isoleucine deficiency and a regulator of branched-chain amino acid transaminases. Both proteins belong to the YjgF/YER057c/UK114 protein superfamily, which has been highly conserved among eubacteria, archaea, and eukaryotes. This family is characterized by a C-terminal signature sequence of [PA]-[ASTPV]-R[SACVF]-x-[LIVMFY]-x(2)-[GSAKR]-x-[LMVA]-x(5,8)[LIVM]-E-[MI] (Fig. 1), and its members are found in all three living kingdoms as independent domains having an average molecular weight of 15 kDa (Fig. 1). Superfamily members found in all three living kingdoms exist as independent domains having an average molecular weight of 15 kDa (Fig. 1). Some genomes even contain multiple paralogs (e.g., four in Escherichia coli and two in S. cerevisiae). Despite the high degree of sequence conservation, functional studies have documented that members of this protein superfamily perform a variety of biochemical functions. Mammalian YER057c family members play roles in protein translation (human UK14, rat UK14), modulation of calpain affinity for calcium (bovine UK14), and heat-shock response (mouse HR12). Chronic administration of rat protein UK14 curtails the development of diabetes and adjuvant-induced arthritis. Human UK14 (also known as UK-114 and p14.5) was characterized as a tumor antigen expressed by various malignant neoplasms and was observed to be upregulated during cell differentiation. Bacterial members of the family affect biosynthetic pathways. Bacillus subtilis YABJ regulates purine biosynthesis by binding to the purine repressor purR and possibly stabilizing its association with DNA, whereas Lactococcus lactis ALDR and Salmonella thyphimurium YJGF block an intermediate step in the isoleucine biosynthetic pathway. The two paralogs present in S. cerevisiae, mitochondrial matrix factor 1 (Mmf1 or YIL051c) and homologous mitochondrial matrix factor 1 (Hmf1), share 71% sequence identity and have also been implicated in the transamination step of isoleucine biosynthesis plus maintenance of mitochondrial DNA (mtDNA). Mmf1 localizes to the mitochondria because of the presence of a 16-residue targeting leader peptide (Fig. 1), where it associates with mtDNA structures. Hmf1 is found mainly in the cytoplasm. Yil051c mutants are isoleucine auxotrophs in which mitochondrial respiratory activity is dramatically decreased and mtDNA is eventually lost. Deletion of the yer057c gene does not produce an obvious phenotype. Nevertheless, when fused to a mitochondrial targeting leader sequence, Hmf1 complements the yil051c deletion, indicating that Hmf1 and Mmf1 can perform similar functions, but in distinct cellular compartments. In addition, the human homolog can rescue the yil051c deletion phenotype. These results strongly suggest that the function of these homologs has been conserved, at least in part, among eukaryotes. This article reports the X-ray structure of Hmf1, which represents the first structure of a eukaryotic member of the YjgF/YER057c/UK114 superfamily. Hmf1 is a homotrimer that folds into a triangular, pseudo / barrel with narrow, deep grooves located at the intermonomer surfaces. On the basis of the high structural similarity of Hmf1 to B. subtilis chorismate mutase as well as sequence conservation, it is suggested that these intermonomer grooves represent ligand-binding regions, and possibly, enzyme active sites. Materials and Methods. Hmf1 represents target P003 of the New York Structural Genomics Research Consortium.