scholarly journals Structural basis of arginine asymmetrical dimethylation by PRMT6

2016 ◽  
Vol 473 (19) ◽  
pp. 3049-3063 ◽  
Author(s):  
Hong Wu ◽  
Weihong Zheng ◽  
Mohammad S. Eram ◽  
Mynol Vhuiyan ◽  
Aiping Dong ◽  
...  
Keyword(s):  
Structural Information ◽  
Arginine Methylation ◽  
Structural Basis ◽  
Type I ◽  
Lung Cancers ◽  
Product Specificity ◽  
Repressive Mark ◽  
Methylation Product ◽  
Histone H3k4 ◽  
Cancer Types

PRMT6 is a type I protein arginine methyltransferase, generating the asymmetric dimethylarginine mark on proteins such as histone H3R2. Asymmetric dimethylation of histone H3R2 by PRMT6 acts as a repressive mark that antagonizes trimethylation of H3 lysine 4 by the MLL histone H3K4 methyltransferase. PRMT6 is overexpressed in several cancer types, including prostate, bladder and lung cancers; therefore, it is of great interest to develop potent and selective inhibitors for PRMT6. Here, we report the synthesis of a potent bisubstrate inhibitor GMS [6′-methyleneamine sinefungin, an analog of sinefungin (SNF)], and the crystal structures of human PRMT6 in complex, respectively, with S-adenosyl-L-homocysteine (SAH) and the bisubstrate inhibitor GMS that shed light on the significantly improved inhibition effect of GMS on methylation activity of PRMT6 compared with SAH and an S-adenosyl-L-methionine competitive methyltransferase inhibitor SNF. In addition, we also crystallized PRMT6 in complex with SAH and a short arginine-containing peptide. Based on the structural information here and available in the PDB database, we proposed a mechanism that can rationalize the distinctive arginine methylation product specificity of different types of arginine methyltransferases and pinpoint the structural determinant of such a specificity.

scholarly journals A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

2016 ◽  
Vol 113 (8) ◽  
pp. 2068-2073 ◽  
Author(s):  
Erik W. Debler ◽  
Kanishk Jain ◽  
Rebeccah A. Warmack ◽  
You Feng ◽  
Steven G. Clarke ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Type I ◽  
Histone H4 ◽  
Active Site Residues ◽  
Protein Arginine Methyltransferase ◽  
Arginine Methyltransferase ◽  
Product Specificity

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.

scholarly journals Structural basis underlying complex assembly and conformational transition of the type I R-M system

2017 ◽  
Vol 114 (42) ◽  
pp. 11151-11156 ◽  
Author(s):  
Yan-Ping Liu ◽  
Qun Tang ◽  
Jie-Zhong Zhang ◽  
Li-Fei Tian ◽  
Pu Gao ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Conformational Transition ◽  
Structural Information ◽  
Structural Basis ◽  
Type I ◽  
Structural Comparison ◽  
Complex Assembly ◽  
Adenosyl Methionine ◽  
Restriction Modification

Type I restriction-modification (R-M) systems are multisubunit enzymes with separate DNA-recognition (S), methylation (M), and restriction (R) subunits. Despite extensive studies spanning five decades, the detailed molecular mechanisms underlying subunit assembly and conformational transition are still unclear due to the lack of high-resolution structural information. Here, we report the atomic structure of a type I MTase complex (2M+1S) bound to DNA and cofactor S-adenosyl methionine in the “open” form. The intermolecular interactions between M and S subunits are mediated by a four-helix bundle motif, which also determines the specificity of the interaction. Structural comparison between open and previously reported low-resolution “closed” structures identifies the huge conformational changes within the MTase complex. Furthermore, biochemical results show that R subunits prefer to load onto the closed form MTase. Based on our results, we proposed an updated model for the complex assembly. The work reported here provides guidelines for future applications in molecular biology.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

scholarly journals Profiling PRMT methylome reveals roles of hnRNPA1 arginine methylation in RNA splicing and cell growth

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wen-juan Li ◽  
Yao-hui He ◽  
Jing-jing Yang ◽  
Guo-sheng Hu ◽  
Yi-an Lin ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cell Growth ◽  
Rna Splicing ◽  
Synergistic Effects ◽  
Arginine Methylation ◽  
Type I ◽  
Prostate Cancer Cells ◽  
Cancer Cell Growth ◽  
Cancer Mutations ◽  
Substrate Spectrum

AbstractNumerous substrates have been identified for Type I and II arginine methyltransferases (PRMTs). However, the full substrate spectrum of the only type III PRMT, PRMT7, and its connection to type I and II PRMT substrates remains unknown. Here, we use mass spectrometry to reveal features of PRMT7-regulated methylation. We find that PRMT7 predominantly methylates a glycine and arginine motif; multiple PRMT7-regulated arginine methylation sites are close to phosphorylations sites; methylation sites and proximal sequences are vulnerable to cancer mutations; and methylation is enriched in proteins associated with spliceosome and RNA-related pathways. We show that PRMT4/5/7-mediated arginine methylation regulates hnRNPA1 binding to RNA and several alternative splicing events. In breast, colorectal and prostate cancer cells, PRMT4/5/7 are upregulated and associated with high levels of hnRNPA1 arginine methylation and aberrant alternative splicing. Pharmacological inhibition of PRMT4/5/7 suppresses cancer cell growth and their co-inhibition shows synergistic effects, suggesting them as targets for cancer therapy.

scholarly journals Structural basis for ALK2/BMPR2 receptor complex signaling through kinase domain oligomerization

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Christopher Agnew ◽  
Pelin Ayaz ◽  
Risa Kashima ◽  
Hanna S. Loving ◽  
Prajakta Ghatpande ◽  
...  
Keyword(s):  
Md Simulations ◽  
Kinase Domain ◽  
Structural Basis ◽  
Type I ◽  
Type Ii ◽  
Exchange Mass Spectrometry ◽  
Receptor Complexes ◽  
Bmp Receptors ◽  
Morphogenetic Protein ◽  
Smad Proteins

AbstractUpon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs.

scholarly journals Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4

2004 ◽  
Vol 379 (2) ◽  
pp. 283-289 ◽  
Author(s):  
Marie-Chloé BOULANGER ◽  
Tina Branscombe MIRANDA ◽  
Steven CLARKE ◽  
Marco di FRUSCIO ◽  
Beat SUTER ◽  
...  
Keyword(s):  
Rna Binding ◽  
Developmental Stages ◽  
Rna Binding Proteins ◽  
Genetic System ◽  
Arginine Methylation ◽  
Type I ◽  
Protein Arginine Methyltransferases ◽  
Arginine Residues ◽  
Drosophila Protein

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 (Drosophilaarginine methyltransferases 1–9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.

scholarly journals Structural Basis for Linezolid Binding Site Rearrangement in the Staphylococcus aureus Ribosome

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Matthew J. Belousoff ◽  
Zohar Eyal ◽  
Mazdak Radjainia ◽  
Tofayel Ahmed ◽  
Rebecca S. Bamert ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Structural Information ◽  
Structural Basis ◽  
Common Mechanism ◽  
Resistance Mutations ◽  
Antimicrobial Drugs ◽  
Linezolid Resistance ◽  
Drug Modification ◽  
Antibiotic Resistance Mutations ◽  
Shed Light

ABSTRACT An unorthodox, surprising mechanism of resistance to the antibiotic linezolid was revealed by cryo-electron microscopy (cryo-EM) in the 70S ribosomes from a clinical isolate of Staphylococcus aureus. This high-resolution structural information demonstrated that a single amino acid deletion in ribosomal protein uL3 confers linezolid resistance despite being located 24 Å away from the linezolid binding pocket in the peptidyl-transferase center. The mutation induces a cascade of allosteric structural rearrangements of the rRNA that ultimately results in the alteration of the antibiotic binding site. IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821–832, 2015, https://doi.org/10.1038/nrd4675 ). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance. IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821–832, 2015, https://doi.org/10.1038/nrd4675 ). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance.

scholarly journals MLL1 minimal catalytic complex is a dynamic conformational ensemble susceptible to pharmacological allosteric disruption

2018 ◽  
Author(s):  
Lilia Kaustov ◽  
Alexander Lemak ◽  
Hong Wu ◽  
Marco Faini ◽  
Scott Houliston ◽  
...  
Keyword(s):  
Hybrid Methods ◽  
Structural Basis ◽  
Epigenetic Mark ◽  
Conformational Ensemble ◽  
Interaction Sites ◽  
Catalytic Function ◽  
Individual Interaction ◽  
Mixed Lineage Leukaemia ◽  
Repeat Domain ◽  
Histone H3k4

ABSTRACTHistone H3K4 methylation is an epigenetic mark associated with actively transcribed genes. This modification is catalyzed by the mixed lineage leukaemia (MLL) family of histone methyltransferases including MLL1, MLL2, MLL3, MLL4, SET1A and SET1B. Catalytic activity of MLL proteins is dependent on interactions with additional conserved proteins but the structural basis for subunit assembly and the mechanism of regulation is not well understood. We used a hybrid methods approach to study the assembly and biochemical function of the minimally active MLL1 complex (MLL1, WDR5 and RbBP5). A combination of small angle X-ray scattering (SAXS), cross-linking mass spectrometry (XL-MS), NMR spectroscopy, and computational modeling were used to generate a dynamic ensemble model in which subunits are assembled via multiple weak interaction sites. We identified a new interaction site between the MLL1 SET domain and the WD40 repeat domain of RbBP5, and demonstrate the susceptibility of the catalytic function of the complex to disruption of individual interaction sites.

scholarly journals Functional Importance of Hydrophobic Patches on the Ebola Virus VP35 IFN-Inhibitory Domain

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2316
Author(s):  
Nodoka Kasajima ◽  
Keita Matsuno ◽  
Hiroko Miyamoto ◽  
Masahiro Kajihara ◽  
Manabu Igarashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ebola Virus ◽  
Structural Information ◽  
Site Directed Mutagenesis ◽  
Genome Replication ◽  
Type I ◽  
Multifunctional Protein ◽  
Functional Sites ◽  
Inhibitory Function ◽  
Inhibitory Domain

Viral protein 35 (VP35) of Ebola virus (EBOV) is a multifunctional protein that mainly acts as a viral polymerase cofactor and an interferon antagonist. VP35 interacts with the viral nucleoprotein (NP) and double-stranded RNA for viral RNA transcription/replication and inhibition of type I interferon (IFN) production, respectively. The C-terminal portion of VP35, which is termed the IFN-inhibitory domain (IID), is important for both functions. To further identify critical regions in this domain, we analyzed the physical properties of the surface of VP35 IID, focusing on hydrophobic patches, which are expected to be functional sites that are involved in interactions with other molecules. Based on the known structural information of VP35 IID, three hydrophobic patches were identified on its surface and their biological importance was investigated using minigenome and IFN-β promoter-reporter assays. Site-directed mutagenesis revealed that some of the amino acid substitutions that were predicted to disrupt the hydrophobicity of the patches significantly decreased the efficiency of viral genome replication/transcription due to reduced interaction with NP, suggesting that the hydrophobic patches might be critical for the formation of a replication complex through the interaction with NP. It was also found that the hydrophobic patches were involved in the IFN-inhibitory function of VP35. These results highlight the importance of hydrophobic patches on the surface of EBOV VP35 IID and also indicate that patch analysis is useful for the identification of amino acid residues that directly contribute to protein functions.

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