Structural and Large-scale Analysis Unveil the Intertwined Paths Promoting NMT-catalyzed Lysine and Glycine Myristoylation (original) (raw)
Related papers
Nature Communications, 2020
The promising drug target N-myristoyltransferase (NMT) catalyses an essential protein modification thought to occur exclusively at N-terminal glycines (Gly). Here, we present high-resolution human NMT1 structures co-crystallised with reactive cognate lipid and peptide substrates, revealing high-resolution snapshots of the entire catalytic mechanism from the initial to final reaction states. Structural comparisons, together with biochemical analysis, provide unforeseen details about how NMT1 reaches a catalytically competent conformation in which the reactive groups are brought into close proximity to enable catalysis. We demonstrate that this mechanism further supports efficient and unprecedented myristoylation of an N-terminal lysine side chain, providing evidence that NMT acts both as N-terminal-lysine and glycine myristoyltransferase.
Myristoylation, an Ancient Protein Modification Mirroring Eukaryogenesis and Evolution
Trends in Biochemical Sciences, 2020
N-myristoylation (MYR) is a crucial fatty acylation catalyzed by N-myristoyltransferases (NMTs) that is likely to have appeared over two billion years ago. Proteome-wide approaches have now delivered an exhaustive list of substrates undergoing MYR across approximately 2% of any proteome, with constituents, several unexpected, associated with different membrane compartments. A set of <10 proteins conserved in eukaryotes probably represents the original set of N-myristoylated targets, marking major changes occurring throughout eukaryogenesis. Recent findings have revealed unexpected mechanisms and reactivity, suggesting competition with other acylations that are likely to influence cellular homeostasis and the steady state of the modification landscape. Here, we review recent advances in NMT catalysis, substrate specificity, and MYR proteomics, and discuss concepts regarding MYR during evolution. Lipidated proteins Plasma membranes (PMs) are composed of extrinsic and intrinsic proteins (52%) and lipids (40%), the latter sustaining the overall cellular architecture. Membrane-penetrating extrinsic proteins often possess covalently linked lipids, usually fatty acids, which allow the protein to contact other intra-and extracellular proteins [1]. Protein lipidation involves amides (i.e. N-αmyristoylation, MYR, see Glossary and glycosylphosphatidylinositol (GPI) anchors), thioesters (i.e. S-palmitoylation, PAL), and thioethers (i.e. isoprenylation and farnesylation) [2]. Of these, MYR is a frequent and conserved modification specific to eukaryotes that targets major cellular components. Mapping the proteins undergoing MYR has proven challenging due to their difficult handling characteristics and amphiphilic, chimeric nature. Recently, high-end technologies have allowed the first lipidated proteome, the myristoylome, to be described in detail in various organisms recapitulating the tree of life [3]. These and other studies on myristoylome composition and genesis have also revealed (i) an unexpected novel mechanism of action of Nmyristoyltransferase (NMT), (ii) NMT substrate selectivity, and (iii) the capacity of NMT to act on N-terminal lysines (Lys) as well as the usual glycines (Gly). This review highlights the series of groundbreaking discoveries that have recently significantly advanced our knowledge about this long-studied enzyme. This includes an overview of a new NMT catalytic mechanism, substrate specificity, and proteomics, and a discussion of how a reduced set of targets is closely related to eukaryogenesis and eukaryote evolution. How NMTs catalyze MYR with high selectivity NMTs are GNAT members closely related to Nα-acetyltransferases Seventy-four crystal structures have now revealed that the C-terminal 400 amino acid-long NMT catalytic core displays a conserved 3D GCN5-related N-acetyltransferase (GNAT) core. GNATs also include the catalytic subunits of Nα-acetyltransferases (NAAs) [4], with Naa10 being the closest to NMT as it modifies N-terminal Gly [5-7]. NMTs have two adjacent GNAT domains, most likely to have arisen through duplication of a Naa10-like GNAT domain [8]. Prokaryotes do not possess NMTs, which seem to have arisen as eukaryotes evolved from the last archaeal
bioRxiv, 2021
N-myristoyltransferases (NMTs) catalyze protein myristoylation, a major and ubiquitous lipid modification. Originally thought to modify only N-terminal glycine α-amino groups (G-myristoylation), NMTs are now known to modify lysine ε-amino groups (K-myristoylation), the significance of which is uncertain. Here we exploited systematic structural proteomics analyses and a novel pipeline involving the Shigella IpaJ protease to discriminate K- and G-myristoylation with unprecedented accuracy and identify the specific features driving each modification. NMT-dependent K-myristoylation occurs post-translationally and only on lysines 1, 2, or 3 following G-myristoylation or caspase cleavage. Direct interactions between the substrate’s reactive amino group and the NMT catalytic base slow K-myristoylation catalysis. IpaJ unmasked novel K-myristoylation sites in a dozen human proteins. The unique properties of NMT-driven K-myristoylation allowed us to design potent, mechanism-based suicide NMT ...
Genome biology, 2004
We evaluated the evolutionary conservation of glycine myristoylation within eukaryotic sequences. Our large-scale cross-genome analyses, available as MYRbase, show that the functional spectrum of myristoylated proteins is currently largely underestimated. We give experimental evidence for in vitro myristoylation of selected predictions. Furthermore, we classify five membrane-attachment factors that occur most frequently in combination with, or even replacing, myristoyl anchors, as some protein family examples show.
Mapping the myristoylome through a complete understanding of protein myristoylation biochemistry
Progress in Lipid Research, 2021
Protein myristoylation is a C14 fatty acid modification found in all living organisms. Myristoylation tags either the N-terminal alpha groups of cysteine or glycine residues through amide bonds or lysine and cysteine side chains directly or indirectly via glycerol thioester and ester linkages. Before transfer to proteins, myristate must be activated into myristoyl coenzyme A in eukaryotes or, in bacteria, to derivatives like phosphatidylethanolamine. Myristate originates through de novo biosynthesis (e.g., plants), from external uptake (e.g., human tissues), or from mixed origins (e.g., unicellular organisms). Myristate usually serves as a molecular anchor, allowing tagged proteins to be targeted to membranes and travel across endomembrane networks in eukaryotes. In this review, we describe and discuss the metabolic origins of proteinbound myristate. We review strategies for in vivo protein labeling that take advantage of clickchemistry with reactive analogs, and we discuss new approaches to the proteome-wide discovery of myristate-containing proteins. The machineries of myristoylation are described, along with how protein targets can be generated directly from translating precursors or from processed proteins. Few myristoylation catalysts are currently described, with only N-myristoyltransferase described to date in eukaryotes. Finally, we describe how viruses and bacteria hijack and exploit myristoylation for their pathogenicity.
J Mol Biol, 2002
Myristoylation by the myristoyl-CoA:protein N-myristoyltransferase (NMT) is an important lipid anchor modification of eukaryotic and viral proteins. Automated prediction of N-terminal N-myristoylation from the substrate protein sequence alone is necessary for large-scale sequence annotation projects but it requires a low rate of false positive hits in addition to a sufficient sensitivity.Our previous analysis of substrate protein sequence variability, NMT sequences and 3D structures has revealed motif properties in addition to the known PROSITE motif that are utilized in a new predictor described here. The composite prediction function (with separate ad hoc parameterization (a) for queries from non-fungal eukaryotes and their viruses and (b) for sequences from fungal species) consists of terms evaluating amino acid type preferences at sequences positions close to the N terminus as well as terms penalizing deviations from the physical property pattern of amino acid side-chains encoded in multi-residue correlation within the motif sequence. The algorithm has been validated with a self-consistency and two jack-knife tests for the learning set as well as with kinetic data for model substrates. The sensitivity in recognizing documented NMT substrates is above 95 % for both taxon-specific versions. The corresponding rate of false positive prediction (for sequences with an N-terminal glycine residue) is close to 0.5 %; thus, the technique is applicable for large-scale automated sequence database annotation. The predictor is available as public WWW-server with the URL http://mendel.imp.univie.ac.at/myristate/. Additionally, we propose a version of the predictor that identifies a number of proteolytic protein processing sites at internal glycine residues and that evaluates possible N-terminal myristoylation of the protein fragments.A scan of public protein databases revealed new potential NMT targets for which the myristoyl modification may be of critical importance for biological function. Among others, the list includes kinases, phosphatases, proteasomal regulatory subunit 4, kinase interacting proteins KIP1/KIP2, protozoan flagellar proteins, homologues of mitochondrial translocase TOM40, of the neuronal calcium sensor NCS-1 and of the cytochrome c-type heme lyase CCHL. Analyses of complete eukaryote genomes indicate that about 0.5 % of all encoded proteins are apparent NMT substrates except for a higher fraction in Arabidopsis thaliana (∼0.8 %).
J Mol Biol, 2002
N-terminal N-myristoylation is a lipid anchor modification of eukaryotic and viral proteins targeting them to membrane locations, thus changing the cellular function of modified proteins. Protein myristoylation is critical in many pathways; e.g. in signal transduction, apoptosis, or alternative extracellular protein export. The myristoyl-CoA:protein N-myristoyltransferase (NMT) recognizes the sequence motif of appropriate substrate proteins at the N terminus and attaches the lipid moiety to the absolutely required N-terminal glycine residue. Reliable recognition of capacity for N-terminal myristoylation from the substrate protein sequence alone is desirable for proteome-wide function annotation projects but the existing PROSITE motif is not practical, since it produces huge numbers of false positive and even some false negative predictions.As a first step towards a new prediction method, it is necessary to refine the sequence motif coding for N-terminal N-myristoylation. Relying on the in-depth study of the amino acid sequence variability of substrate proteins, on binding site analyses in X-ray structures or 3D homology models for NMTs from various taxa, and on consideration of biochemical data extracted from the scientific literature, we found indications that, at least within a complete substrate protein, the N-terminal 17 protein residues experience different types of variability restrictions. We identified three motif regions: region 1 (positions 1-6) fitting the binding pocket; region 2 (positions 7-10) interacting with the NMT’s surface at the mouth of the catalytic cavity; and region 3 (positions 11-17) comprising a hydrophilic linker. Each region was characterized by physical requirements to single sequence positions or groups of positions regarding volume, polarity, backbone flexibility and other typical properties of amino acids (http://mendel.imp.univie.ac.at/myristate/). These specificity differences are confined partly to taxonomic ranges and are proposed for the design of NMT inhibitors in pathogenic fungal and protozoan systems including Aspergillus fumigatus, Leishmania major, Trypanosoma cruzi, Trypanosoma brucei, Giardia intestinalis, Entamoeba histolytica, Pneumocystis carinii, Strongyloides stercoralis and Schistosoma mansoni. An exhaustive search for NMT-homologues led to the discovery of two putative entomopoxviral NMTs.
N-terminal N-myristoylation of proteins: prediction of substrate proteins from amino acid sequence
Journal of Molecular Biology, 2002
Myristoylation by the myristoyl-CoA:protein N-myristoyltransferase (NMT) is an important lipid anchor modi®cation of eukaryotic and viral proteins. Automated prediction of N-terminal N-myristoylation from the substrate protein sequence alone is necessary for large-scale sequence annotation projects but it requires a low rate of false positive hits in addition to a suf®cient sensitivity.
Journal of Molecular Biology, 2002
N-terminal N-myristoylation is a lipid anchor modi®cation of eukaryotic and viral proteins targeting them to membrane locations, thus changing the cellular function of modi®ed proteins. Protein myristoylation is critical in many pathways; e.g. in signal transduction, apoptosis, or alternative extracellular protein export. The myristoyl-CoA:protein N-myristoyltransferase (NMT) recognizes the sequence motif of appropriate substrate proteins at the N terminus and attaches the lipid moiety to the absolutely required N-terminal glycine residue. Reliable recognition of capacity for N-terminal myristoylation from the substrate protein sequence alone is desirable for proteome-wide function annotation projects but the existing PROSITE motif is not practical, since it produces huge numbers of false positive and even some false negative predictions.