ABSTRACT
Deposition of amyloid-ß (Aß) peptides in the brain is a hallmark of Alzheimer's disease. Aßs are generated through sequential proteolysis of the amyloid precursor protein by the γ-secretase complexes (GSECs). Aß peptide length, modulated by the Presenilin (PSEN) and APH-1 subunits of GSEC, is critical for Alzheimer's pathogenesis. Despite high relevance, mechanistic understanding of the proteolysis of Aß, and its modulation by APH-1, remain incomplete. Here, we report cryo-EM structures of human GSEC (PSEN1/APH-1B) reconstituted into lipid nanodiscs in apo form and in complex with the intermediate Aß46 substrate without cross-linking. We find that three non-conserved and structurally divergent APH-1 regions establish contacts with PSEN1, and that substrate-binding induces concerted rearrangements in one of the identified PSEN1/APH-1 interfaces, providing structural basis for APH-1 allosteric-like effects. In addition, the GSEC-Aß46 structure reveals an interaction between Aß46 and loop 1PSEN1, and identifies three other H-bonding interactions that, according to functional validation, are required for substrate recognition and efficient sequential catalysis.
Subject(s)
Amyloid Precursor Protein Secretases , Amyloid beta-Peptides , Cryoelectron Microscopy , Membrane Proteins , Presenilin-1 , Humans , Amyloid Precursor Protein Secretases/metabolism , Amyloid Precursor Protein Secretases/chemistry , Presenilin-1/metabolism , Presenilin-1/chemistry , Presenilin-1/genetics , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/chemistry , Membrane Proteins/metabolism , Membrane Proteins/chemistry , Endopeptidases/metabolism , Endopeptidases/chemistry , Amyloid beta-Protein Precursor/metabolism , Amyloid beta-Protein Precursor/chemistry , Protein Binding , Protein Isoforms/metabolism , Protein Isoforms/chemistry , Alzheimer Disease/metabolism , Peptide Fragments/metabolism , Peptide Fragments/chemistry , Peptide Hydrolases/metabolism , Peptide Hydrolases/chemistry , Models, Molecular , ProteolysisABSTRACT
Substrate channeling studies have frequently failed to provide conclusive results due to poor understanding of this subtle phenomenon. We analyzed the mechanism of NADH-channeling from D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to L-lactate Dehydrogenase (LDH) using enzymes from different cells. Enzyme kinetics studies showed that LDH activity with free NADH and GAPDH-NADH complex always take place in parallel. The channeling is observed only in assays that mimic cytosolic conditions where free NADH concentration is negligible and the GAPDH-NADH complex is dominant. Molecular dynamics and protein-protein interaction studies showed that LDH and GAPDH can form a leaky channeling complex only at the limiting NADH concentrations. Surface calculations showed that positive electric field between the NAD(H) binding sites on LDH and GAPDH tetramers can merge in the LDH-GAPDH complex. NAD(H)-channeling within the LDH-GAPDH complex can be an extension of NAD(H)-channeling within each tetramer. In the case of a transient LDH-(GAPDH-NADH) complex, the relative contribution from the channeled and the diffusive paths depends on the overlap between the off-rates for the LDH-(GAPDH-NADH) complex and the GAPDH-NADH complex. Molecular evolution or metabolic engineering protocols can exploit substrate channeling for metabolic flux control by fine-tuning substrate-binding affinity for the key enzymes in the competing reaction paths.
Subject(s)
Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , L-Lactate Dehydrogenase/metabolism , Molecular Dynamics Simulation , Animals , Binding Sites , Muscle, Skeletal/metabolism , NAD/metabolism , RabbitsABSTRACT
BACKGROUND: We use our earlier experimental studies of the catalytic mechanism of DNA methyltransferases to prepare in silico a family of novel mechanism-based inhibitors of human Dnmt1. Highly specific inhibitors of DNA methylation can be used for analysis of human epigenome and for the creation of iPS cells. RESULTS: We describe a set of adenosyl-1-methyl-pyrimidin-2-one derivatives as novel mechanism-based inhibitors of mammalian DNA methyltransferase Dnmt1. The inhibitors have been designed to bind simultaneously in the active site and the cofactor site and thus act as transition-state analogues. Molecular dynamics studies showed that the lead compound can form between 6 to 9 binding interactions with Dnmt1. QM/MM analysis showed that the upon binding to Dnmt1 the inhibitor can form a covalent adduct with active site Cys1226 and thus act as a mechanism-based suicide-inhibitor. The inhibitor can target DNA-bond and DNA-free form of Dnmt1, however the suicide-inhibition step is more likely to happen when DNA is bound to Dnmt1. The validity of presented analysis is described in detail using 69 modifications in the lead compound structure. In total 18 of the presented 69 modifications can be used to prepare a family of highly specific inhibitors that can differentiate even between closely related enzymes such as Dnmt1 and Dnmt3a DNA methyltransferases. CONCLUSIONS: Presented results can be used for preparation of some highly specific and potent inhibitors of mammalian DNA methylation with specific pharmacological properties.
