scholarly journals Multiple domain interfaces mediate SARM1 autoinhibition

2021 ◽  
Vol 118 (4) ◽  
pp. e2023151118
Author(s):  
Chen Shen ◽  
Mihir Vohra ◽  
Pengfei Zhang ◽  
Xianrong Mao ◽  
Matthew D. Figley ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Peptide Mapping ◽  
Structural Basis ◽  
Axon Degeneration ◽  
Cryo Electron Microscopy ◽  
Axon Loss ◽  
Enzymatic Activation ◽  
Tir Domains ◽  
Multiple Domain ◽  
Constitutively Active

Axon degeneration is an active program of self-destruction mediated by the protein SARM1. In healthy neurons, SARM1 is autoinhibited and, upon injury autoinhibition is relieved, activating the SARM1 enzyme to deplete NAD+ and induce axon degeneration. SARM1 forms a homomultimeric octamer with each monomer composed of an N-terminal autoinhibitory ARM domain, tandem SAM domains that mediate multimerization, and a C-terminal TIR domain encoding the NADase enzyme. Here we discovered multiple intramolecular and intermolecular domain interfaces required for SARM1 autoinhibition using peptide mapping and cryo-electron microscopy (cryo-EM). We identified a candidate autoinhibitory region by screening a panel of peptides derived from the SARM1 ARM domain, identifying a peptide mediating high-affinity inhibition of the SARM1 NADase. Mutation of residues in full-length SARM1 within the region encompassed by the peptide led to loss of autoinhibition, rendering SARM1 constitutively active and inducing spontaneous NAD+ and axon loss. The cryo-EM structure of SARM1 revealed 1) a compact autoinhibited SARM1 octamer in which the TIR domains are isolated and prevented from oligomerization and enzymatic activation and 2) multiple candidate autoinhibitory interfaces among the domains. Mutational analysis demonstrated that five distinct interfaces are required for autoinhibition, including intramolecular and intermolecular ARM-SAM interfaces, an intermolecular ARM-ARM interface, and two ARM-TIR interfaces formed between a single TIR and two distinct ARM domains. These autoinhibitory regions are not redundant, as point mutants in each led to constitutively active SARM1. These studies define the structural basis for SARM1 autoinhibition and may enable the development of SARM1 inhibitors that stabilize the autoinhibited state.

scholarly journals Deamidation and glycation of a Bacillus licheniformis α-amylase during industrial fermentation can improve detergent wash performance

Amylase ◽  
2021 ◽  
Vol 5 (1) ◽  
pp. 38-49
Author(s):  
Connie Pontoppidan ◽  
Svend G. Kaasgaard ◽  
Carsten P. Sønksen ◽  
Carsten Andersen ◽  
Birte Svensson
Keyword(s):  
Bacillus Licheniformis ◽  
Mutational Analysis ◽  
Peptide Mapping ◽  
Substrate Binding ◽  
Carbohydrate Binding ◽  
Surface Binding ◽  
Lysine Residues ◽  
Tandem Mass Spectrometric ◽  
Binding Cleft ◽  
Household Detergents

Abstract The industrial thermostable Bacillus licheniformis α-amylase (BLA) has wide applications, including in household detergents, and efforts to improve its performance are continuously ongoing. BLA during the industrial production is deamidated and glycated resulting in multiple forms with different isoelectric points. Forty modified positions were identified by tandem mass spectrometric peptide mapping of BLA forms separated by isoelectric focusing. These modified 12 asparagine, 9 glutamine, 8 arginine and 11 lysine residues are mostly situated on the enzyme surface and several belong to regions involved in stability, activity and carbohydrate binding. Eight residues presumed to interact with starch at the active site and surface binding sites (SBSs) were subjected to mutational analysis. Five mutants mimicking deamidation (N→D, Q→E) at the substrate binding cleft showed moderate to no effect on thermostability and k cat and K M for maltoheptaose and amylose. Notably, the mutations improved laundry wash efficiency in detergents at pH 8.5 and 10.0. Replacing three reducing sugar reactive side chains (K→M, R→L) at a distant substrate binding region and two SBSs enhanced wash performance especially in liquid detergent at pH 8.5, slightly improved enzymatic activity and maintained thermostability. Wash performance was most improved (5-fold) for the N265D mutant near substrate binding subsite +3.

scholarly journals Structural basis for the regulation of nucleosome recognition and HDAC activity by histone deacetylase assemblies

2021 ◽  
Vol 7 (2) ◽  
pp. eabd4413
Author(s):  
Jung-Hoon Lee ◽  
Daniel Bollschweiler ◽  
Tillman Schäfer ◽  
Robert Huber
Keyword(s):  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Active Sites ◽  
Chromatin Modification ◽  
Structural Basis ◽  
Amino Terminal ◽  
Cryo Electron Microscopy ◽  
Multiple Contacts ◽  
Lysine Residues ◽  
Nucleosome Binding

The chromatin-modifying histone deacetylases (HDACs) remove acetyl groups from acetyl-lysine residues in histone amino-terminal tails, thereby mediating transcriptional repression. Structural makeup and mechanisms by which multisubunit HDAC complexes recognize nucleosomes remain elusive. Our cryo–electron microscopy structures of the yeast class II HDAC ensembles show that the HDAC protomer comprises a triangle-shaped assembly of stoichiometry Hda12-Hda2-Hda3, in which the active sites of the Hda1 dimer are freely accessible. We also observe a tetramer of protomers, where the nucleosome binding modules are inaccessible. Structural analysis of the nucleosome-bound complexes indicates how positioning of Hda1 adjacent to histone H2B affords HDAC catalysis. Moreover, it reveals how an intricate network of multiple contacts between a dimer of protomers and the nucleosome creates a platform for expansion of the HDAC activities. Our study provides comprehensive insight into the structural plasticity of the HDAC complex and its functional mechanism of chromatin modification.

scholarly journals Structural insights into the activation of human calcium-sensing receptor

2021 ◽  
Author(s):  
Xiaochen Chen ◽  
Lu Wang ◽  
Zhanyu Ding ◽  
Qianqian Cui ◽  
Li Han ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Transmembrane Domains ◽  
Structural Basis ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Cryo Electron Microscopy ◽  
Activation Mechanisms ◽  
G Protein Coupled ◽  
Structural Insights

AbstractHuman calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that maintains Ca2+ homeostasis in serum. Here, we present the cryo-electron microscopy structures of the CaSR in the inactive and active states. Complemented with previously reported crystal structures of CaSR extracellular domains, it suggests that there are three distinct conformations: inactive, intermediate and active state during the activation. We used a negative allosteric nanobody to stabilize the CaSR in the fully inactive state and found a new binding site for Ca2+ ion that acts as a composite agonist with L-amino acid to stabilize the closure of active Venus flytraps. Our data shows that the agonist binding leads to the compaction of the dimer, the proximity of the cysteine-rich domains, the large-scale transitions of 7-transmembrane domains, and the inter-and intrasubunit conformational changes of 7-transmembrane domains to accommodate the downstream transducers. Our results reveal the structural basis for activation mechanisms of the CaSR.

scholarly journals Nicotinic acid mononucleotide is an allosteric SARM1 inhibitor promoting axonal protection

2021 ◽  
Author(s):  
Yo Sasaki ◽  
Jian Zhu ◽  
Yun Shi ◽  
Weixi Gu ◽  
Bostjan Kobe ◽  
...  
Keyword(s):  
Nicotinic Acid ◽  
Neurodegenerative Disorders ◽  
Axon Degeneration ◽  
Biosynthetic Enzyme ◽  
Allosteric Site ◽  
Nicotinamide Mononucleotide ◽  
Bacterial Enzyme ◽  
Axon Loss ◽  
Metabolic Sensor

SARM1 is an inducible NAD+ hydrolase that is the central executioner of pathological axon loss. Recently, we elucidated the molecular mechanism of SARM1 activation, demonstrating that SARM1 is a metabolic sensor regulated by the levels of NAD+ and its precursor, nicotinamide mononucleotide (NMN), via their competitive binding to an allosteric site. In healthy neurons with abundant NAD+, binding of NAD+ blocks access of NMN to this allosteric site. However, with injury or disease the levels of the NAD+ biosynthetic enzyme NMNAT2 drop, increasing the NMN/NAD+ ratio and thereby promoting NMN binding to the SARM1 allosteric site, which in turn induces a conformational change activating the SARM1 NAD+ hydrolase. Hence, NAD+ metabolites both regulate the activation of SARM1 and, in turn, are regulated by the SARM1 NAD+ hydrolase. This dual upstream and downstream role for NAD+ metabolites in SARM1 function has hindered mechanistic understanding of axoprotective mechanisms that manipulate the NAD+ metabolome. Here we reevaluate two methods that potently block axon degeneration via modulation of NAD+ related metabolites, 1) the administration of the NMN biosynthesis inhibitor FK866 in conjunction with the NAD+ precursor nicotinic acid riboside (NaR) and 2) the neuronal expression of the bacterial enzyme NMN deamidase. We find that these approaches not only lead to a decrease in the levels of the SARM1 activator NMN, but also an increase in the levels of the NAD+ precursor nicotinic acid mononucleotide (NaMN). We show that NaMN competes with NMN for binding to the SARM1 allosteric site, that NaMN inhibits SARM1 activation, and that this NaMN-mediated inhibition is important for the long-term axon protection induced by these treatments. Together, these results demonstrate that the SARM1 allosteric pocket can bind a diverse set of metabolites including NMN, NAD+, and NaMN to monitor cellular NAD+ homeostasis and regulate SARM1 NAD+ hydrolase activity. The relative promiscuity of the allosteric site may enable the development of potent pharmacological inhibitors of SARM1 activation for the treatment of neurodegenerative disorders.

scholarly journals Cryo-EM structure of human mitochondrial trifunctional protein

2018 ◽  
Vol 115 (27) ◽  
pp. 7039-7044 ◽  
Author(s):  
Kai Liang ◽  
Ningning Li ◽  
Xiao Wang ◽  
Jianye Dai ◽  
Pulan Liu ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Oxidation Process ◽  
Structural Basis ◽  
Acid Oxidation ◽  
Cryo Electron Microscopy ◽  
Helical Hairpin ◽  
Tetrameric Structure ◽  
Acute Fatty Liver ◽  
Mitochondrial Trifunctional Protein

The mitochondrial trifunctional protein (TFP) catalyzes three reactions in the fatty acid β-oxidation process. Mutations in the two TFP subunits cause mitochondrial trifunctional protein deficiency and acute fatty liver of pregnancy that can lead to death. Here we report a 4.2-Å cryo-electron microscopy α2β2 tetrameric structure of the human TFP. The tetramer has a V-shaped architecture that displays a distinct assembly compared with the bacterial TFPs. A concave surface of the TFP tetramer interacts with the detergent molecules in the structure, suggesting that this region is involved in associating with the membrane. Deletion of a helical hairpin in TFPβ decreases its binding to the liposomes in vitro and reduces its membrane targeting in cells. Our results provide the structural basis for TFP function and have important implications for fatty acid oxidation related diseases.

scholarly journals Direct binding of TFEα opens DNA binding cleft of RNA polymerase

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Sung-Hoon Jun ◽  
Jaekyung Hyun ◽  
Jeong Seok Cha ◽  
Hoyoung Kim ◽  
Michael S. Bartlett ◽  
...  
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Structural Basis ◽  
Transcription Bubble ◽  
Cryo Electron Microscopy ◽  
Direct Binding ◽  
Binding Cleft ◽  
Promoter Dna ◽  
Winged Helix Domain

AbstractOpening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAP-TFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft. Positioning of promoter DNA into the cleft closes it while maintaining the TFEα interactions with the RNAP mobile modules. The structures and photo-crosslinking results also suggest that the conserved aromatic residue in the extended winged-helix domain of TFEα interacts with promoter DNA to stabilize the transcription bubble. This study provides a structural basis for the functions of TFEα and elucidates the mechanism by which the DNA binding cleft is opened during transcription initiation in the stalk-containing RNAPs, including archaeal and eukaryotic RNAPs.

scholarly journals Structures of cell wall arabinosyltransferases with the anti-tuberculosis drug ethambutol

Science ◽  
2020 ◽  
Vol 368 (6496) ◽  
pp. 1211-1219 ◽  
Author(s):  
Lu Zhang ◽  
Yao Zhao ◽  
Yan Gao ◽  
Lijie Wu ◽  
Ruogu Gao ◽  
...  
Keyword(s):  
Cell Wall ◽  
Active Site ◽  
Structural Basis ◽  
Drug Resistant ◽  
Cell Wall Synthesis ◽  
X Ray ◽  
Cryo Electron Microscopy ◽  
Donor And Acceptor ◽  
Biochemical Function ◽  
Glycosyl Donor

The arabinosyltransferases EmbA, EmbB, and EmbC are involved in Mycobacterium tuberculosis cell wall synthesis and are recognized as targets for the anti-tuberculosis drug ethambutol. In this study, we determined cryo–electron microscopy and x-ray crystal structures of mycobacterial EmbA-EmbB and EmbC-EmbC complexes in the presence of their glycosyl donor and acceptor substrates and with ethambutol. These structures show how the donor and acceptor substrates bind in the active site and how ethambutol inhibits arabinosyltransferases by binding to the same site as both substrates in EmbB and EmbC. Most drug-resistant mutations are located near the ethambutol binding site. Collectively, our work provides a structural basis for understanding the biochemical function and inhibition of arabinosyltransferases and the development of new anti-tuberculosis agents.

scholarly journals Cryo-EM structure and polymorphism of Aβ amyloid fibrils purified from Alzheimer’s brain tissue

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Marius Kollmer ◽  
William Close ◽  
Leonie Funk ◽  
Jay Rasmussen ◽  
Aref Bsoul ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Brain Tissue ◽  
Amyloid Fibrils ◽  
Structural Basis ◽  
Amyloid Angiopathy ◽  
Cryo Electron Microscopy ◽  
Aβ Amyloid ◽  
Cerebral Amyloid ◽  
Aβ Fibrils

Abstract The formation of Aβ amyloid fibrils is a neuropathological hallmark of Alzheimer’s disease and cerebral amyloid angiopathy. However, the structure of Aβ amyloid fibrils from brain tissue is poorly understood. Here we report the purification of Aβ amyloid fibrils from meningeal Alzheimer’s brain tissue and their structural analysis with cryo-electron microscopy. We show that these fibrils are polymorphic but consist of similarly structured protofilaments. Brain derived Aβ amyloid fibrils are right-hand twisted and their peptide fold differs sharply from previously analyzed Aβ fibrils that were formed in vitro. These data underscore the importance to use patient-derived amyloid fibrils when investigating the structural basis of the disease.

scholarly journals Structures of ribosome-bound initiation factor 2 reveal the mechanism of subunit association

2016 ◽  
Vol 2 (3) ◽  
pp. e1501502 ◽  
Author(s):  
Thiemo Sprink ◽  
David J. F. Ramrath ◽  
Hiroshi Yamamoto ◽  
Kaori Yamamoto ◽  
Justus Loerke ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Protein Biosynthesis ◽  
Initiation Factor ◽  
Structural Basis ◽  
Initiation Complex ◽  
Cryo Electron Microscopy ◽  
Initiation Factor 2 ◽  
High Resolution Structure ◽  
Guanosine Triphosphatase ◽  
Resolution Structure

Throughout the four phases of protein biosynthesis—initiation, elongation, termination, and recycling—the ribosome is controlled and regulated by at least one specified translational guanosine triphosphatase (trGTPase). Although the structural basis for trGTPase interaction with the ribosome has been solved for the last three steps of translation, the high-resolution structure for the key initiation trGTPase, initiation factor 2 (IF2), complexed with the ribosome, remains elusive. We determine the structure of IF2 complexed with a nonhydrolyzable guanosine triphosphate analog and initiator fMet-tRNAiMet in the context of the Escherichia coli ribosome to 3.7-Å resolution using cryo-electron microscopy. The structural analysis reveals previously unseen intrinsic conformational modes of the 70S initiation complex, establishing the mutual interplay of IF2 and initator transfer RNA (tRNA) with the ribsosome and providing the structural foundation for a mechanistic understanding of the final steps of translation initiation.

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