The MRN complex (MRX in Saccharomyces cerevisiae, made of Mre11, Rad50 and Nbs1/Xrs2) initiates double-stranded DNA break repair and activates the Tel1/ATM kinase in the DNA damage response. Telomeres counter both outcomes at chromosome ends, partly by keeping MRN-ATM in check. We show that MRX is disabled by telomeric protein Rif2 through an N-terminal motif (MIN, MRN/X-inhibitory motif). MIN executes suppression of Tel1, DNA end-resection and non-homologous end joining by binding the Rad50 N-terminal region. Our data suggest that MIN promotes a transition within MRX that is not conductive for endonuclease activity, DNA-end tethering or Tel1 kinase activation, highlighting an Achilles’ heel in MRN, which we propose is also exploited by the RIF2 paralog ORC4 (Origin Recognition Complex 4) in Kluyveromyces lactis and the Schizosaccharomyces pombe telomeric factor Taz1, which is evolutionarily unrelated to Orc4/Rif2. This raises the possibility that analogous mechanisms might be deployed in other eukaryotes as well.

DHTKD1 is a lesser-studied E1 enzyme among the family of 2-oxoacid dehydrogenases. In complex with E2 (dihydrolipoamide succinyltransferase, DLST) and E3 (dihydrolipoamide dehydrogenase, DLD) components, DHTKD1 is involved in lysine and tryptophan catabolism by catalysing the oxidative decarboxylation of 2-oxoadipate (2OA) in mitochondria. Here, the 1.9 Å resolution crystal structure of human DHTKD1 is solved in complex with the thiamine diphosphate co-factor. The structure reveals how the DHTKD1 active site is modelled upon the well characterized homologue 2-oxoglutarate (2OG) dehydrogenase but engineered specifically to accommodate its preference for the longer substrate of 2OA over 2OG. A 4.7 Å resolution reconstruction of the human DLST catalytic core is also generated by single-particle electron microscopy, revealing a 24-mer cubic scaffold for assembling DHTKD1 and DLD protomers into a megacomplex. It is further demonstrated that missense DHTKD1 variants causing the inborn error of 2-aminoadipic and 2-oxoadipic aciduria impact on the complex formation, either directly by disrupting the interaction with DLST, or indirectly through destabilizing the DHTKD1 protein. This study provides the starting framework for developing DHTKD1 modulators to probe the intricate mitochondrial energy metabolism.

Abstract5′-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5′-aminolevulinate from glycine and succinyl-CoA. Inherited frameshift indel mutations of human erythroid-specific isozyme ALAS2, within a C-terminal (Ct) extension of its catalytic core that is only present in higher eukaryotes, lead to gain-of-function X-linked protoporphyria (XLP). Here, we report the human ALAS2 crystal structure, revealing that its Ct-extension folds onto the catalytic core, sits atop the active site, and precludes binding of substrate succinyl-CoA. The Ct-extension is therefore an autoinhibitory element that must re-orient during catalysis, as supported by molecular dynamics simulations. Our data explain how Ct deletions in XLP alleviate autoinhibition and increase enzyme activity. Crystallography-based fragment screening reveals a binding hotspot around the Ct-extension, where fragments interfere with the Ct conformational dynamics and inhibit ALAS2 activity. These fragments represent a starting point to develop ALAS2 inhibitors as substrate reduction therapy for porphyria disorders that accumulate toxic heme intermediates.