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 Structure of undecaprenyl pyrophosphate synthase from Acinetobacter baumannii

2018 ◽  
Vol 74 (12) ◽  
pp. 765-769 ◽  
Author(s):  
Tzu-Ping Ko ◽  
Chi-Hung Huang ◽  
Shu-Jung Lai ◽  
Yeh Chen
Keyword(s):  
Acinetobacter Baumannii ◽  
Active Site ◽  
Multidrug Resistant ◽  
Structural Basis ◽  
X Ray Diffraction ◽  
X Ray ◽  
Peptidoglycan Synthesis ◽  
C Terminus ◽  
Dimeric Protein ◽  
Β Sheet

Undecaprenyl pyrophosphate (UPP) is an important carrier of the oligosaccharide component in peptidoglycan synthesis. Inhibition of UPP synthase (UPPS) may be an effective strategy in combating the pathogen Acinetobacter baumannii, which has evolved to be multidrug-resistant. Here, A. baumannii UPPS (AbUPPS) was cloned, expressed, purified and crystallized, and its structure was determined by X-ray diffraction. Each chain of the dimeric protein folds into a central β-sheet with several surrounding α-helices, including one at the C-terminus. In the active site, two molecules of citrate interact with the side chains of the catalytic aspartate and serine. These observations may provide a structural basis for inhibitor design against AbUPPS.

scholarly journals Structure based protein engineering to confer selectivity of guanine deaminase

2014 ◽  
Vol 70 (a1) ◽  
pp. C437-C437
Author(s):  
Aruna Bitra ◽  
Ruchi Anand
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Active Site Residues ◽  
X Ray ◽  
Secondary Activity ◽  
Guanine Deaminase ◽  
Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

scholarly journals Structural basis of SARS-CoV-2 Omicron immune evasion and receptor engagement

2021 ◽  
Author(s):  
Matthew McCallum ◽  
Nadine Czudnochowski ◽  
Laura E Rosen ◽  
Samantha K Zepeda ◽  
John E Bowen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Crystal Structures ◽  
Immune Evasion ◽  
Marked Reduction ◽  
Structural Basis ◽  
X Ray ◽  
Structural Framework ◽  
Cryo Electron Microscopy ◽  
Antigenic Shift

The SARS-CoV-2 Omicron variant of concern evades antibody mediated immunity with an unprecedented magnitude due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and X-ray crystal structures of the spike and RBD bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a structural framework for understanding the marked reduction of binding of all other therapeutic mAbs leading to dampened neutralizing activity. We reveal electrostatic remodeling of the interactions within the spike and those formed between the Omicron RBD and human ACE2, likely explaining enhanced affinity for the host receptor relative to the prototypic virus.

scholarly journals Structural basis of peptidoglycan endopeptidase regulation

2019 ◽  
Author(s):  
Jung-Ho Shin ◽  
Alan G. Sulpizio ◽  
Aaron Kelley ◽  
Laura Alvarez ◽  
Shannon G. Murphy ◽  
...  
Keyword(s):  
Cell Wall ◽  
Vibrio Cholerae ◽  
Active Site ◽  
Normal Growth ◽  
Structural Basis ◽  
Bacterial Cells ◽  
Cell Wall Digestion ◽  
Novel Antibiotics

AbstractMost bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over, the latter process is mediated by PG cleavage enzymes, for example the endopeptidases (EPs). EPs themselves are essential for growth, but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae. Our data suggest that ShyA assumes two drastically different conformations; a more open form that allows for substrate binding, and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli. Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain1 from the M23 active site, likely through conformational re-arrangement in vivo.SignificanceBacteria digest their cell wall following exposure to antibiotics like penicillin. The endopeptidases (EPs) are among the proteins that catalyze cell wall digestion processes after antibiotic exposure, but we do not understand how these enzymes are regulated during normal growth. Herein, we present the structure of the major EP from the diarrheal pathogen Vibrio cholerae. Surprisingly, we find that EPs from this and other pathogens appear to be produced as a largely inactive precursor that undergoes a conformational shift exposing the active site to engage in cell wall digestion. These results enhance our understanding of how EPs are regulated and could open the door for the development of novel antibiotics that overactivate cell wall digestion processes.

scholarly journals Structures of the Human LONP1 Protease Reveal Regulatory Steps Involved in Protease Activation

2020 ◽  
Author(s):  
Mia Shin ◽  
Edmond R. Watson ◽  
Scott J. Novick ◽  
Patrick Griffin ◽  
R. Luke Wiseman ◽  
...  
Keyword(s):  
Active Site ◽  
Structural Basis ◽  
Proteolytic Activation ◽  
Mitochondrial Regulation ◽  
Cryo Electron Microscopy ◽  
Transport Chain ◽  
Protease Activation ◽  
Health And Disease ◽  
Aaa Protein ◽  
Spiral Staircase

AbstractThe human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in the pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the molecular mechanism of substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. However, unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate to increase interactions with the translocating peptide as it transits into the proteolytic chamber for proteolysis. Further, we show that substrate-bound LONP1 includes a second level of regulation at the proteolytic active site, wherein autoinhibition of the active site is only relieved by the presence of a peptide substrate. Ultimately, our results define a structural basis for human LONP1 proteolytic activation and activity, establishing a molecular framework to understand the critical importance of this protease for mitochondrial regulation in health and disease.

scholarly journals Characterizing human α-1,6-fucosyltransferase (FUT8) substrate specificity and structural similarities with related fucosyltransferases

2020 ◽  
Vol 295 (50) ◽  
pp. 17027-17045
Author(s):  
Bhargavi M. Boruah ◽  
Renuka Kadirvelraj ◽  
Lin Liu ◽  
Annapoorani Ramiah ◽  
Chao Li ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Secretory Pathway ◽  
Structural Features ◽  
Structural Basis ◽  
Substrate Affinity ◽  
Neonatal Lethality ◽  
Donor And Acceptor ◽  
Active Site Mutants ◽  
Core Fucosylation

Mammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by α-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an α-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality. Elevated core fucosylation has also been identified in several human cancers. However, the structural basis for FUT8 substrate specificity remains unknown.Here, using various crystal structures of FUT8 in complex with a donor substrate analog, and with four distinct glycan acceptors, we identify the molecular basis for FUT8 specificity and activity. The ordering of three active site loops corresponds to an increased occupancy for bound GDP, suggesting an induced-fit folding of the donor-binding subsite. Structures of the various acceptor complexes were compared with kinetic data on FUT8 active site mutants and with specificity data from a library of glycan acceptors to reveal how binding site complementarity and steric hindrance can tune substrate affinity. The FUT8 structure was also compared with other known fucosyltransferases to identify conserved and divergent structural features for donor and acceptor recognition and catalysis. These data provide insights into the evolution of modular templates for donor and acceptor recognition among GT-B fold glycosyltransferases in the synthesis of diverse glycan structures in biological systems.

scholarly journals Structural basis of a nucleosome containing histone H2A.B/H2A.Bbd that transiently associates with reorganized chromatin

2013 ◽  
Vol 3 (1) ◽  
Author(s):  
Yasuhiro Arimura ◽  
Hiroshi Kimura ◽  
Takashi Oda ◽  
Koichi Sato ◽  
Akihisa Osakabe ◽  
...  
Keyword(s):  
Dna Replication ◽  
Small Angle ◽  
Living Cells ◽  
Structural Basis ◽  
Base Pairs ◽  
X Ray ◽  
X Ray Scattering ◽  
Biochemical Function ◽  
Dna Replication And Repair ◽  
Chromatin Reorganization

Abstract Human histone H2A.B (formerly H2A.Bbd), a non-allelic H2A variant, exchanges rapidly as compared to canonical H2A and preferentially associates with actively transcribed genes. We found that H2A.B transiently accumulated at DNA replication and repair foci in living cells. To explore the biochemical function of H2A.B, we performed nucleosome reconstitution analyses using various lengths of DNA. Two types of H2A.B nucleosomes, octasome and hexasome, were formed with 116, 124, or 130 base pairs (bp) of DNA and only the octasome was formed with 136 or 146 bp DNA. In contrast, only hexasome formation was observed by canonical H2A with 116 or 124 bp DNA. A small-angle X-ray scattering analysis revealed that the H2A.B octasome is more extended, due to the flexible detachment of the DNA regions at the entry/exit sites from the histone surface. These results suggested that H2A.B rapidly and transiently forms nucleosomes with short DNA segments during chromatin reorganization.

scholarly journals Cryo-EM structure of VASH1-SVBP bound to microtubules

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Faxiang Li ◽  
Yang Li ◽  
Xuecheng Ye ◽  
Haishan Gao ◽  
Zhubing Shi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Active Site ◽  
Binding Protein ◽  
Substrate Preference ◽  
Structural Basis ◽  
Globular Domain ◽  
Specific Interactions ◽  
Cryo Electron Microscopy ◽  
Positively Charged

The dynamic tyrosination-detyrosination cycle of α-tubulin regulates microtubule functions. Perturbation of this cycle impairs mitosis, neural physiology, and cardiomyocyte contraction. The carboxypeptidases vasohibins 1 and 2 (VASH1 and VASH2), in complex with the small vasohibin-binding protein (SVBP), mediate α-tubulin detyrosination. These enzymes detyrosinate microtubules more efficiently than soluble αβ-tubulin heterodimers. The structural basis for this substrate preference is not understood. Using cryo-electron microscopy (cryo-EM), we have determined the structure of human VASH1-SVBP bound to microtubules. The acidic C-terminal tail of α-tubulin binds to a positively charged groove near the active site of VASH1. VASH1 forms multiple additional contacts with the globular domain of α-tubulin, including contacts with a second α-tubulin in an adjacent protofilament. Simultaneous engagement of two protofilaments by VASH1 can only occur within the microtubule lattice, but not with free αβ heterodimers. These lattice-specific interactions enable preferential detyrosination of microtubules by VASH1.

scholarly journals Structural basis for the hydrolytic dehalogenation of the fungicide chlorothalonil

2020 ◽  
Vol 295 (26) ◽  
pp. 8668-8677
Author(s):  
Daniel S. Catlin ◽  
Xinhang Yang ◽  
Brian Bennett ◽  
Richard C. Holz ◽  
Dali Liu
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Bound Water ◽  
Structural Basis ◽  
X Ray ◽  
Distorted Trigonal Bipyramid ◽  
Industrial Compounds ◽  
Structural Site ◽  
Trigonal Bipyramid Geometry

Cleavage of aromatic carbon–chlorine bonds is critical for the degradation of toxic industrial compounds. Here, we solved the X-ray crystal structure of chlorothalonil dehalogenase (Chd) from Pseudomonas sp. CTN-3, with 15 of its N-terminal residues truncated (ChdT), using single-wavelength anomalous dispersion refined to 1.96 Å resolution. Chd has low sequence identity (<15%) compared with all other proteins whose structures are currently available, and to the best of our knowledge, we present the first structure of a Zn(II)-dependent aromatic dehalogenase that does not require a coenzyme. ChdT forms a “head-to-tail” homodimer, formed between two α-helices from each monomer, with three Zn(II)-binding sites, two of which occupy the active sites, whereas the third anchors a structural site at the homodimer interface. The catalytic Zn(II) ions are solvent-accessible via a large hydrophobic (8.5 × 17.8 Å) opening to bulk solvent and two hydrophilic branched channels. Each active-site Zn(II) ion resides in a distorted trigonal bipyramid geometry with His117, His257, Asp116, Asn216, and a water/hydroxide as ligands. A conserved His residue, His114, is hydrogen-bonded to the Zn(II)-bound water/hydroxide and likely functions as the general acid-base. We examined substrate binding by docking chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile, TPN) into the hydrophobic channel and observed that the most energetically favorable pose includes a TPN orientation that coordinates to the active-site Zn(II) ions via a CN and that maximizes a π–π interaction with Trp227. On the basis of these results, along with previously reported kinetics data, we propose a refined catalytic mechanism for Chd-mediated TPN dehalogenation.

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