ABSTRACT H3K9 methylation (H3K9me) specifies the establishment and maintenance of transcriptionally silent epigenetic states or heterochromatin. The enzymatic erasure of histone modifications is widely assumed to be the primary mechanism that reverses epigenetic silencing. Here, we reveal an inversion of this paradigm where a putative histone demethylase Epe1 in fission yeast, has a non-enzymatic function that opposes heterochromatin assembly. An auto-inhibitory conformation regulates the non-enzymatic properties of Epe1 and licenses its interaction with Swi6HP1. Mutations that map to the putative catalytic JmjC domain preserve Epe1 in an auto-inhibited state which disrupts its localization to sites of heterochromatin formation and interaction with Swi6HP1. H3K9me relieves Epe1 from auto-inhibition and stimulates its ability to form an inhibitory complex with Swi6HP1in vitro and in cells. Our work reveals that histone modifications, rather than being passive scaffolds, actively participate in regulating the assembly of specific epigenetic complexes in cells.
The α-aminoadipate pathway of lysine biosynthesis is modulated at the transcriptional and biochemical levels by feedback inhibition. The first enzyme in the α-aminoadipate pathway, homocitrate synthase (HCS), is the target of the feedback regulation and is strongly inhibited by l-lysine. Here we report the structure of Schizosaccharomyces pombe HCS (SpHCS) in complex with l-lysine. The structure illustrates that the amino acid directly competes with the substrate 2-oxoglutarate for binding within the active site of HCS. Differential recognition of the substrate and inhibitor is achieved via a switch position within the (α/β)8 TIM barrel of the enzyme that can distinguish between the C5-carboxylate group of 2-oxoglutarate and the ϵ-ammonium group of l-lysine. In vitro and in vivo assays demonstrate that mutations of the switch residues, which interact with the l-lysine ϵ-ammonium group, abrogate feedback inhibition, as do substitutions of residues within the C-terminal domain that were identified in a previous study of l-lysine-insensitive HCS mutants in Saccharomyces cerevisiae. Together, these results yield new insights into the mechanism of feedback regulation of an enzyme central to lysine biosynthesis. The α-aminoadipate pathway of lysine biosynthesis is modulated at the transcriptional and biochemical levels by feedback inhibition. The first enzyme in the α-aminoadipate pathway, homocitrate synthase (HCS), is the target of the feedback regulation and is strongly inhibited by l-lysine. Here we report the structure of Schizosaccharomyces pombe HCS (SpHCS) in complex with l-lysine. The structure illustrates that the amino acid directly competes with the substrate 2-oxoglutarate for binding within the active site of HCS. Differential recognition of the substrate and inhibitor is achieved via a switch position within the (α/β)8 TIM barrel of the enzyme that can distinguish between the C5-carboxylate group of 2-oxoglutarate and the ϵ-ammonium group of l-lysine. In vitro and in vivo assays demonstrate that mutations of the switch residues, which interact with the l-lysine ϵ-ammonium group, abrogate feedback inhibition, as do substitutions of residues within the C-terminal domain that were identified in a previous study of l-lysine-insensitive HCS mutants in Saccharomyces cerevisiae. Together, these results yield new insights into the mechanism of feedback regulation of an enzyme central to lysine biosynthesis.
Highly effective thrombin inhibitors have been obtained by preparing boronic acid analogues of m-cyano-substituted phenylalanine and its incorporation into peptides. The cyano group enhances binding by several orders of magnitude. For example, Ac-(D)Phe-Pro-boroPheOH binds to thrombin with a Ki of 320 nM and the Ki of Ac-(D)Phe-Pro-boroPhe(m-CN)-OH is 0.79 nM. Protein crystal structure determination of trypsin complexed to H-(D)Phe-Pro-boroPhe(m-CN)-OH indicates that the aromatic side chain is bound in the P1 binding site and that the cyano group can act as a H-bond acceptor for the amide proton of Gly219. Enhanced binding for inhibitors containing the m-cyano group was observed for coagulation factor Xa and for the factor VIIa.tissue factor complex [Ki values of Ac-(D)Phe-Pro-boroPhe(mCN)-OH are 760 and 3.3 nM, respectively]. This result is consistent with the sequence homology of these two enzymes in the P1 binding site. Two enzymes lacking the strict homology in the P1 binding site, pancreatic kallikrein and chymotrypsin, did not exhibit significantly enhanced binding.
