Biology subjects

Marmorstein, R.

Publications and source records attributed to Marmorstein, R..

13 recordsLinked to original sources

bioRxiv2026

Mechanism of fatty acid uptake and inhibition in human FATP2

Fatty Acid Transport Protein 2 (FATP2) couples fatty acid uptake to intracellular activation and is associated with pathological lipid accumulation in cancer and nonalcoholic fatty liver disease. Here, we present cryo-electron microscopy structures of human FATP2 across its reaction cycle. Our structures suggest that FATP2 recruits fatty acids directly from the membrane interface through a hydrophobic tunnel. Catalysis involves a [~]130{degrees} rotation of the C-terminal domain, a transition trapped by the antihypertensive drugs isradipine and benidipine. Both drugs lock the enzyme in a thioester-forming state, but benidipine exhibits superior efficacy by extending a bulky moiety into the primary catalytic tunnel to sterically block substrate entry. Furthermore, we identify a product inhibition mechanism where excess acyl-CoA traps the enzyme, potentially limiting metabolic overload. These findings provide a structural framework for understanding vectorial fatty acid channeling and a scaffold for developing modulators of metabolic flux. HighlightsO_LICryo-EM structures of human FATP2 reveal a membrane-anchored lollipop topology C_LIO_LIEndogenous fatty acids within a hydrophobic tunnel delineate the fatty acid uptake pathway C_LIO_LIIsradipine and benidipine displace fatty acids to trap a non-productive conformation C_LIO_LIAcyl-CoA product inhibition may provide negative feedback via steric occlusion C_LI

biophysics↗

bioRxiv2025

Cryo-EM Structure of the Cyclase Domain and Evaluation of Substrate Channeling in a Bifunctional Class II Terpene Synthase

Copalyl diphosphate synthase from Penicillium verruculosum (PvCPS) is a bifunctional class II terpene synthase containing a prenyltransferase that produces geranylgeranyl diphosphate (GGPP) and a class II cyclase that utilizes GGPP as a substrate to generate the bicyclic diterpene copalyl diphosphate. The various stereoisomers of copalyl diphosphate establish the greater family of labdane natural products, many of which have environmental and medicinal impact. Understanding structure-function relationships in class II diterpene synthases is crucial for guiding protein engineering campaigns aimed at the generation of diverse bicyclic diterpene scaffolds. However, only a limited number of structures are available for class II cyclases from bacteria, plants, and humans, and no structures are available for a class II cyclase from a fungus. Further, bifunctional class II terpene synthases have not been investigated with regard to substrate channeling between the prenyltransferase and the cyclase. Here, we report the 2.9 [A]-resolution cryo-EM structure of the 63-kD class II cyclase domain from PvCPS. Comparisons with bacterial and plant copalyl diphosphate synthases reveal conserved residues that likely guide the formation of the bicyclic labdane core, but divergent catalytic dyads that mediate the final deprotonation step of catalysis. Substrate competition experiments reveal preferential GGPP transit from the PvCPS prenyltransferase to the cyclase, even when prepared as separate constructs. These results are consistent with a model in which transient prenyltransferase-cyclase association facilitates substrate channeling due to active site proximity.

biochemistry↗

bioRxiv2024

A role for condensin-mediator interaction in mitotic chromosomal organization

Eukaryotic genomes are organized by condensin into 3D chromosomal architectures suitable for chromosomal segregation during mitosis. However, molecular mechanisms underlying the condensin-mediated chromosomal organization remain largely unclear. Here, we investigate the role of newly identified interaction between the Cnd1 condensin and Pmc4 mediator subunits in fission yeast, Schizosaccharomyces pombe. We develop a condensin mutation, cnd1-K658E, that impairs the condensin-mediator interaction and find that this mutation diminishes condensin-mediated chromatin domains during mitosis and causes chromosomal segregation defects. The condensin-mediator interaction is involved in recruiting condensin to highly transcribed genes and mitotically activated genes, the latter of which demarcate condensin-mediated domains. Furthermore, this study predicts that mediator-driven transcription of mitotically activated genes contributes to forming domain boundaries via phase separation. This study provides a novel insight into how genome-wide gene expression during mitosis is transformed into the functional chromosomal architecture suitable for chromosomal segregation. O_FIG O_LINKSMALLFIG WIDTH=189 HEIGHT=200 SRC="FIGDIR/small/600663v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@488ab5org.highwire.dtl.DTLVardef@8a7a73org.highwire.dtl.DTLVardef@16d416eorg.highwire.dtl.DTLVardef@1e2d3db_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG In BriefMitotic chromosomes are organized by condensin into an architecture suitable for their segregation, although molecular mechanisms underlying how condensin assembles the functional chromosomal architecture remain unclear. This study with the fission yeast model organism shows that the protein interaction between condensin and mediator is involved in chromosomal segregation by forming a mitotic chromosomal architecture. A similar mechanism involving the condensin-mediator interaction is likely conserved in human cells, implying the evolutionary conservation of this mechanism. HighlightsO_LIThe Cnd1 condensin subunit interacts with the Pmc4 mediator component in fission yeast. C_LIO_LIThe condensin-mediator interaction is required to recruit condensin to highly transcribed and mitotically activated genes. C_LIO_LIThe condensin-mediator interaction is required to form chromatin domains during mitosis. C_LIO_LIMediator participates in the boundary formation of mitotic chromatin domains. C_LI

genomics↗

bioRxiv2024

Engineering Substrate Channeling in Assembly-Line Terpene Biosynthesis

Fusicoccadiene synthase from P. amygdala (PaFS) is a bifunctional assembly-line terpene synthase containing a prenyltransferase domain that generates geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate (DMAPP) and three equivalents of isopentenyl diphosphate (IPP), and a cyclase domain that converts GGPP into fusicoccadiene, a precursor of the diterpene glycoside Fusicoccin A. The two catalytic domains are linked by a flexible 69-residue polypeptide segment. The prenyltransferase domain mediates oligomerization to form predominantly octamers, and cyclase domains are randomly splayed out around the prenyltransferase core. Previous studies suggest that substrate channeling is operative in catalysis, since most of the GGPP formed by the prenyltransferase remains on the protein for the cyclization reaction. Here, we demonstrate that the flexible linker is not required for substrate channeling, nor must the prenyltransferase and cyclase domains be covalently linked to sustain substrate channeling. Moreover, substrate competition experiments with other diterpene cyclases indicate that the PaFS prenyltransferase and cyclase domains are preferential partners regardless of whether they are covalently linked or not. The cryo-EM structure of engineered "linkerless" construct PaFSLL, in which the 69-residue linker is spliced out and replaced with the tripeptide PTQ, reveals that cyclase pairs associate with all four sides of the prenyltransferase octamer. Taken together, these results suggest that optimal substrate channeling is achieved when a cyclase domain associates with the side of the prenyltransferase octamer, regardless of whether the two domains are covalently linked and regardless of whether this interaction is transient or locked in place.

biochemistry↗

bioRxiv2024

Molecular Basis for RNA Cytidine Acetylation by NAT10

Human NAT10 acetylates the N4 position of cytidine in RNA, predominantly on rRNA and tRNA, to facilitate ribosome biogenesis and protein translation. NAT10 has been proposed as a therapeutic target in cancers as well as aging-associated pathologies such as Hutchinson-Gilford Progeria Syndrome (HGPS). The [~]120 kDa NAT10 protein uses its acetyl-CoA-dependent acetyltransferase, ATP-dependent helicase, and RNA binding domains in concert to mediate RNA-specific N4-cytidine acetylation. While the biochemical activity of NAT10 is well known, the molecular basis for catalysis of eukaryotic RNA acetylation remains relatively undefined. To provide molecular insights into the RNA-specific acetylation by NAT10, we determined the single particle cryo-EM structures of Chaetomium thermophilum NAT10 (CtNAT10) bound to a bisubstrate cytidine-CoA probe with and without ADP. The structures reveal that NAT10 forms a symmetrical heart-shaped dimer with conserved functional domains surrounding the acetyltransferase active sites harboring the cytidine-CoA probe. Structure-based mutagenesis with analysis of mutants in vitro supports the catalytic role of two conserved active site residues (His548 and Tyr549 in CtNAT10), and two basic patches, both proximal and distal to the active site for RNA-specific acetylation. Yeast complementation analyses and senescence assays in human cells also implicates NAT10 catalytic activity in yeast thermoadaptation and cellular senescence. Comparison of the NAT10 structure to protein lysine and N-terminal acetyltransferase enzymes reveals an unusually open active site suggesting that these enzymes have been evolutionarily tailored for RNA recognition and cytidine-specific acetylation.

biophysics↗

bioRxiv2023

Semi-synthetic CoA-alpha-Synuclein Constructs Trap N-terminal Acetyltransferase NatB for Binding Mechanism Studies

N-terminal acetylation is a chemical modification carried out by N-terminal acetyltransferases (NATs). A major member of this enzyme family, NatB, acts on much of the human proteome, including -synuclein (S), a synaptic protein that mediates vesicle trafficking. NatB acetylation of S modulates its lipid vesicle binding properties and amyloid fibril formation, which underlies its role in the pathogenesis of Parkinsons disease. Although the molecular details of the interaction between human NatB (hNatB) and the N-terminus of S have been resolved, whether the remainder of the protein plays a role in interacting with the enzyme is unknown. Here we execute the first synthesis, by native chemical ligation, of a bisubstrate inhibitor of NatB consisting of coenzyme A and full-length human S, additionally incorporating two fluorescent probes for studies of conformational dynamics. We use cryo-electron microscopy (cryo-EM) to characterize the structural features of the hNatB/inhibitor complex and show that, beyond the first few residues, S remains disordered when in complex with hNatB. We further probe changes in the S conformation by single molecule Forster resonance energy transfer (smFRET) to reveal that the C-terminus expands when bound to hNatB. Computational models based on the cryo-EM and smFRET data help to explain the conformational changes and their implications for hNatB substrate recognition and specific inhibition of the interaction with S. Beyond the study of S and NatB, these experiments illustrate valuable strategies for the study of challenging structural biology targets through a combination of protein semi-synthesis, cryo-EM, smFRET, and computational modeling.

biochemistry↗

bioRxiv2021

Molecular mechanism of N-terminal acetylation by the ternary NatC complex

Protein N-terminal acetylation is predominantly a ribosome-associated modification, with NatA-E serving as the major enzymes. NatC is the most unusual of these enzymes, containing one Naa30 catalytic subunit and two auxiliary subunits, Naa35 and Naa38; and substrate specificity profile that overlaps with NatE. Here, we report the Cryo-EM structure of S. pombe NatC with a NatE/C-type bisubstrate analogue and inositol hexaphosphate (IP6), and associated biochemistry studies. We find that the presence of three subunits is a prerequisite for normal NatC acetylation activity in yeast and that IP6 binds tightly to NatC to stabilize the complex. We also describe the molecular basis for IP6-mediated NatC complex stabilization and the overlapping yet distinct substrate profiles of NatC and NatE.

biophysics↗

bioRxiv2021

Novel bisubstrate inhibitors for protein N-terminal acetyltransferase D

Protein N-terminal acetyltransferase D (NatD, NAA40) that specifically acetylates the alpha-N-terminus of histone H4 and H2A has been implicated in various diseases, but no inhibitor has been reported for this important enzyme. Based on the acetyl transfer mechanism of NatD, we designed and prepared a series of highly potent NatD bisubstrate inhibitors by covalently linking coenzyme A to different peptide substrates via an acetyl or propionyl spacer. The most potent bisubstrate inhibitor displayed an apparent Ki value of 1.0 nM. Biochemical studies indicated that bisubstrate inhibitor is competitive to the peptide substrate and noncompetitive to the cofactor, suggesting NatD undergoes an ordered Bi-Bi mechanism. We also demonstrated that these inhibitors are highly specific towards NatD, displaying about 1,000-fold selectivity over other closely related acetyltransferases. High-resolution crystal structures of NatD bound to two of these inhibitors revealed the molecular basis for their selectivity and inhibition mechanism, providing a rational path for future inhibitor development.

biochemistry↗

bioRxiv2020

Naa12 rescues embryonic lethality in Naa10-Deficient Mice in the amino-terminal acetylation pathway

Amino-terminal acetylation is catalyzed by a set of N-terminal acetyltransferases (NATs). The NatA complex (including X-linked Naa10 and Naa15) is the major acetyltransferase, with 40-50% of all mammalian proteins being potential substrates. However, the overall role of amino-terminal acetylation on a whole-organism level is poorly understood, particularly in mammals. Male mice lacking Naa10 show no globally apparent in vivo amino-terminal acetylation impairment and do not exhibit complete embryonic lethality. Rather Naa10 nulls display increased neonatal lethality, and the majority of surviving undersized mutants exhibit a combination of hydrocephaly, cardiac defects, homeotic anterior transformation, piebaldism and urogenital anomalies. Naa12 is a previously unannotated Naa10-like paralogue with NAT activity that genetically compensates for Naa10. Mice deficient for Naa12 have no apparent phenotype, whereas mice deficient for Naa10 and Naa12 display embryonic lethality. The discovery of Naa12 adds to the currently known machinery involved in amino-terminal acetylation in mice.

developmental biology↗

bioRxiv2020

Identification and Characterization of a B-Raf Kinase Alpha Helix Critical for the Activity of MEK Kinase in MAPK Signaling

In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and [~]3% of all cancers and many drugs target the ATP-binding site of the enzyme for its inhibition. Since B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an alpha helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We performed binding experiments between B-Raf mutants and MEK using pull downs and biolayer interferometry, and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this alpha helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf alpha helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas.

biochemistry↗

bioRxiv2020

Molecular Basis for N-terminal Alpha-Synuclein Acetylation by Human NatB

NatB is one of three major N-terminal acetyltransferase (NAT) complexes (NatA-NatC), which co-translationally acetylate the N-termini of eukaryotic proteins. Its substrates account for about 21% of the human proteome, including well known proteins such as actin, tropomyosin, CDK2, and -synuclein (Syn). Human NatB (hNatB) mediated N-terminal acetylation of Syn has been demonstrated to play key roles in Parkinsons disease pathogenesis and as a potential therapeutic target for hepatocellular carcinoma. Here we report the cryo-EM structure of hNatB bound to a CoA-Syn conjugate, together with structure-guided analysis of mutational effects on catalysis. This analysis reveals functionally important differences with human NatA and Candida albicans NatB, resolves key hNatB protein determinants for Syn N-terminal acetylation, and identifies important residues for substrate-specific recognition and acetylation by NatB enzymes. These studies have implications for developing small molecule NatB probes and for understanding the mode of substrate selection by NAT enzymes.

biophysics↗

bioRxiv2020

Structural and molecular determinants of CCS-mediated copper activation of MEK1/2

SummaryNormal physiology relies on the precise coordination of intracellular signal transduction pathways that respond to nutrient availability to balance cell growth and cell death. We recently established a critical mechanistic function for the redox-active micronutrient copper (Cu) in the canonical mitogen activated protein kinase (MAPK) pathway at the level of MEK1 and MEK2. Here we report the X-ray crystal structure of Cu-MEK1 and reveal active site chemical ligands and oxidation state specificity for MEK1 Cu coordination. Mechanistically, the Cu chaperone CCS selectively bound to and facilitated Cu transfer to MEK1. Mutations in MEK1 that disrupt Cu(I) affinity or a CCS small molecule inhibitor reduced Cu-stimulated MEK1 kinase activity. These atomic and molecular level data provide the first mechanistic insights of Cu kinase signaling and could be exploited for the development of novel MEK1/2 inhibitors that either target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.Competing Interest StatementD.C.B holds ownership in Merlon Inc. D.C.B. is an inventor on the patent application 20150017261 entitled Methods of treating and preventing cancer by disrupting the binding of copper in the MAP kinase pathway. No potential conflicts of interest were disclosed by the other authors.View Full Text

biochemistry↗

bioRxiv2020

Tyrosine phosphorylation of ACLY regulates lipid metabolism and oncogenesis in ALK positive Anaplastic Large Cell Lymphoma

A fundamental requirement for growth of rapidly proliferating cells is metabolic adaptation to promote synthesis of biomass1. ATP citrate lyase (ACLY) is a critical enzyme responsible for synthesis of cytosolic acetyl-CoA, the key building component for de novo fatty acid synthesis and links vital pathways such as carbohydrate and lipid metabolism2. The mechanisms of ACLY regulation are not completely understood and the regulation of ACLY function by tyrosine phosphorylation is unknown. Here we show using mass-spectrometry-driven phosphoproteomics and metabolomics that ACLY is phosphorylated and functionally regulated at an evolutionary conserved residue, Y682. Physiologic signals promoting rapid cell growth such as epidermal growth factor stimulation in epithelial cells and T-cell receptor activation in primary human T-cells result in rapid phosphorylation of ACLY at Y682. In vitro kinase assays demonstrate that Y682 is directly phosphorylated by multiple tyrosine kinases, including ALK, ROS1, SRC, JAK2 and LTK. Oncogenically activating structural alterations such as gene-fusions, amplification or point mutations of ALK tyrosine kinase result in constitutive phosphorylation of ACLY in diverse forms of primary human cancer such as lung cancer, anaplastic large cell lymphoma (ALCL) and neuroblastoma. Expression of a phosphorylation-defective ACLY-Y682F mutant in NPM-ALK+ ALCL decreases ACLY activity and attenuates lipid synthesis. Metabolomic analyses reveal that ACLY-Y682F expression results in increased {beta}-oxidation of 13C-oleic acid-labeled fatty acid with increased labeling of +2-citrate (p<0.01) and +18-oleyol carnitine (p<0.001). Similarly, oxygen consumption rate (OCR) is significantly increased in cells expressing ACLY-Y682F (p<0.001). Moreover, expression of ACLY-Y682F dramatically decreases cell proliferation, impairs clonogenicity and abrogates tumor growth in vivo. Our results reveal a novel mechanism for direct ACLY regulation that is subverted by multiple oncogenically-activated tyrosine kinases in diverse human cancers. These findings have significant implications for novel therapies targeting ACLY in cancer and metabolism.

cancer biology↗