MPP OUTSIDE REQUEST FORM Reviewed 07/27/11 Requestor(s): Dean Appling Institution: U. Texas-Austin Request Title: Human mitochondrial proteins MTHFD1L and MTHFD2L Protein NameORF IDSource OrganismHeatTarget Request IDTarget ScorepIResiduesSize Da MTHFD1L GO Homo sapiens 16TR MTHFD2L GO Homo sapiens0TR Alt. names: MTHFD1L: Monofunctional C1-tetrahydrofolate synthase, mitochondrial | Formyltetrahydrofolate synthetase | FTHFSDC1 MTHFD2L: Probable bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase 2 | NADP-dependent methylenetetrahydrofolate dehydrogenase 2-like protein Requestor (D. Appling) notes, edited: These enzymes participate in mitochondrial tetrahydrofolate (THF)-dependent one-carbon metabolism. Under most conditions, the majority of 1C units for cytoplasmic purine, thymidylate, and methyl group biosynthesis are derived from mitochondrial formate. MTHFD1L is a monofunctional 10-formyl-THF synthetase. MTHFD2L is a bifunctional methylene-THF dehydrogenase/methenyl-THF cyclohydrolase. Together, they catalyze the oxidation of mitochondrial methylene-THF to formate, which exits the mitochondria to supply the cytoplasmic metabolic pathways. Thus, mitochondrial 1C metabolism plays a critical role in normal developmental processes, as well as disease processes. For example, common variants in the human MTHFD1L gene are associated with increased risk for cardiovascular disease and for neural tube defects. We have now confirmed with a knock-out mouse model that MTHFD1L is an essential gene, and homozygous null embryos exhibit severe neural tube defects. MTHFD2L has only recently been discovered, but given its position in the mitochondrial pathway, it is also likely to be associated with these diseases. We have generated chimeric mice on our way to knocking out MTHFD2L. Structural analysis of these two proteins will also provide insight into the evolution of this family of enzymes. MTHFD1L and MTHFD2L are related to the previously characterized MTHFD1 and MTHFD2 proteins, respectively. MTHFD2L and MTHFD2 are bifunctional enzymes composed of a single 30-kDa domain. MTHFD1L and MTHFD1 are composed of two domains, with their N-terminal domains homologous to the single domain MTHFD2/2L proteins. MTHFD1 is a trifunctional enzyme whereas MTHFD1L is monofunctional, having lost the two catalytic activities in its N- terminal domain due to several amino acid substitutions in that domain. Despite the loss of catalytic function, all vertebrate MTHFD1L proteins have retained this domain. In addition, we have evidence that these proteins exist in a multiprotein complex on the matrix side of the mitochondrial inner membrane. Structural analysis will shed important light on these protein-protein interactions. If one or both of these proteins were selected for structural analysis, I would be very interested in continued collaborative structure/function studies. Both proteins have been expressed as soluble, active proteins, MTHFD1L in E. coli and MTHFD2L in yeast (S. cerevisiae). Plasmid constructs are available for both. (continued next page)

MPP notes (dja): MTHFD1L Function and Medical Relevance - MTHFD1L is an MPP "ListB" (Most) Biological Relevance target. See reviewer notes. Physical Characteristics - The 978 aa sequence features a transit peptide (1-31) that is cleaved to create the mature protein, an ATP-binding region ( ), an inactive MTHFD /cyclohydrolase domain (31-348), and an FTHF synthetase domain ( ). This is the canonical isoform; isoform 2 is much smaller (30 kda). Two lysines are acetylated. MTHFD1L is said to form a homodimer. Similar Structures - A bacterial N(10)-formyltetrahydrofolate synthetase aligning with 53% identity over aa has been solved (1eg7a). Interactors - 11 other proteins have been demonstrated to bind to MTHFD1L. Substrates are ATP, formate, tetrahydrofolate; products are ADP, phosphate, and 10-formyltetrahydrofolate. Kms for THF monoglutamate, triglutamate, and pentaglutamate have been determined. Status - MTHFD1L was selected in List B by MPP but not yet placed in a cloning workgroup. In June 2011, NESG cloned multiple variants encompassing 4 domains or fragments, approximately residues , , , and MTHFD2L Function and Medical Relevance - See reviewer notes. Physical Characteristics - The 347 aa sequence sent by Dean Appling is 58 aa longer at the N-term than the longest isoform in Uniprot, otherwise they match. TargetP confidently predicts a transit peptide (1-8), Uniprot does not predict one. Functional domains are predicted by PFAM (1-111 and ). There are two smaller alternative splicing isoforms. Similar Structures - There are 3 prokaryotic structures with 47-49% identity over most of the length of the protein, and a human folate-dependent dehydrogenase/cyclohydrolase bifunctional enzyme with 42% identity (1a4iA) with NADPH bound. Interactors - Substrates include NAD, products NADH; Mg2+ is likely to be a cofactor. Status - MPP has not yet selected this protein. NESG selected it in May 2011 and designed constructs of residues and Suggestions 1. Get boundaries from Dean Appling for soluble, active proteins he mentions. 2.Pass info to NESG, and wait for results from their screens before doing any MPP work. Conclusions – suggestions accepted. Dja will contact Dean Appling, get boundaries, compare to NESG fragments, check on expression before MPP does any work.