bims-strubi Biomed News
on Advances in structural biology
Issue of 2021–11–07
ten papers selected by
Alessandro Grinzato, European Synchrotron Radiation Facility
  1. Classifying Liganded States in Heterogeneous Single-Particle Cryo-EM Datasets.
  2. Structural basis of glycan276-dependent recognition by HIV-1 broadly neutralizing antibodies.
  3. Structural basis of human ghrelin receptor signaling by ghrelin and the synthetic agonist ibutamoren.
  4. Cryo-EM structures and transport mechanism of human P5B type ATPase ATP13A2.
  5. Structural mechanisms of TRPV6 inhibition by ruthenium red and econazole.
  6. Cryo-EM structure of MukBEF reveals DNA loop entrapment at chromosomal unloading sites.
  7. Surface-induced Dissociation Mass Spectrometry as a Structural Biology Tool.
  8. Microsecond melting and revitrification of cryo samples.
  9. CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage.
  10. C-Glycoside metabolism in the gut and in nature: Identification, characterization, structural analyses and distribution of C-C bond-cleaving enzymes.
  1. Microscopy (Oxf). 2021 Oct 30. pii: dfab044. [Epub ahead of print]
    Classifying Liganded States in Heterogeneous Single-Particle Cryo-EM Datasets.
    William R Arnold, Daniel Asarnow, Yifan Cheng.
      A powerful aspect of single particle cryogenic electron microscopy (cryo-EM) is its ability to determine high-resolution structures from samples containing heterogeneous mixtures of the same macromolecule in different conformational or compositional states. Beyond determining structures at higher resolutions, one outstanding question is if macromolecules with only subtle conformation differences, such as the same protein bound with different ligands in the same binding pocket, can be separated reliably, and if information concerning binding kinetics can be derived from the particle distributions of different conformations obtained in classification. In this study, we address these questions by assessing the classification of synthetic heterogeneous datasets of TRPV1 generated by combining different homogeneous experimental datasets. Our results indicate that classification can isolate highly homogeneous subsets of particle for calculating high-resolution structures containing individual ligands, but with limitations.
    Keywords:  Focused classification; TRPV1; cryo-EM; ligands; particle subtraction; structural heterogeneity
    DOI:  https://doi.org/10.1093/jmicro/dfab044
  2. Cell Rep. 2021 Nov 02. pii: S2211-1247(21)01395-4. [Epub ahead of print]37(5): 109922
    Structural basis of glycan276-dependent recognition by HIV-1 broadly neutralizing antibodies.
    Christopher A Cottrell, Kartik Manne, Rui Kong, Shuishu Wang, Tongqing Zhou, Gwo-Yu Chuang, Robert J Edwards, Rory Henderson, Katarzyna Janowska, Megan Kopp, Bob C Lin, Mark K Louder, Adam S Olia, Reda Rawi, Chen-Hsiang Shen, Justin D Taft, Jonathan L Torres, Nelson R Wu, Baoshan Zhang, Nicole A Doria-Rose, Myron S Cohen, Barton F Haynes, Lawrence Shapiro, Andrew B Ward, Priyamvada Acharya, John R Mascola, Peter D Kwong.
      Recognition of N-linked glycan at residue N276 (glycan276) at the periphery of the CD4-binding site (CD4bs) on the HIV-envelope trimer is a formidable challenge for many CD4bs-directed antibodies. To understand how this glycan can be recognized, here we isolate two lineages of glycan276-dependent CD4bs antibodies. Antibody CH540-VRC40.01 (named for donor-lineage.clone) neutralizes 81% of a panel of 208 diverse strains, while antibody CH314-VRC33.01 neutralizes 45%. Cryo-electron microscopy (cryo-EM) structures of these two antibodies and 179NC75, a previously identified glycan276-dependent CD4bs antibody, in complex with HIV-envelope trimer reveal substantially different modes of glycan276 recognition. Despite these differences, binding of glycan276-dependent antibodies maintains a glycan276 conformation similar to that observed in the absence of glycan276-binding antibodies. By contrast, glycan276-independent CD4bs antibodies, such as VRC01, displace glycan276 upon binding. These results provide a foundation for understanding antibody recognition of glycan276 and suggest its presence may be crucial for priming immunogens seeking to initiate broad CD4bs recognition.
    Keywords:  CD4 binding site; N276; antibody approach angle; antibody-antigen binding; cryo-EM; glycan conformation; glycan276; immunogen design
    DOI:  https://doi.org/10.1016/j.celrep.2021.109922
  3. Nat Commun. 2021 Nov 04. 12(1): 6410
    Structural basis of human ghrelin receptor signaling by ghrelin and the synthetic agonist ibutamoren.
    Heng Liu, Dapeng Sun, Alexander Myasnikov, Marjorie Damian, Jean-Louis Baneres, Ji Sun, Cheng Zhang.
      The hunger hormone ghrelin activates the ghrelin receptor GHSR to stimulate food intake and growth hormone secretion and regulate reward signaling. Acylation of ghrelin at Ser3 is required for its agonistic action on GHSR. Synthetic agonists of GHSR are under clinical evaluation for disorders related to appetite and growth hormone dysregulation. Here, we report high-resolution cryo-EM structures of the GHSR-Gi signaling complex with ghrelin and the non-peptide agonist ibutamoren as an investigational new drug. Our structures together with mutagenesis data reveal the molecular basis for the binding of ghrelin and ibutamoren. Structural comparison suggests a salt bridge and an aromatic cluster near the agonist-binding pocket as important structural motifs in receptor activation. Notable structural variations of the Gi and GHSR coupling are observed in our cryo-EM analysis. Our results provide a framework for understanding GHSR signaling and developing new GHSR agonist drugs.
    DOI:  https://doi.org/10.1038/s41467-021-26735-5
  4. Cell Discov. 2021 Nov 02. 7(1): 106
    Cryo-EM structures and transport mechanism of human P5B type ATPase ATP13A2.
    Xudong Chen, Mingze Zhou, Sensen Zhang, Jian Yin, Ping Zhang, Xujun Xuan, Peiyi Wang, Zhiqiang Liu, Boda Zhou, Maojun Yang.
      Polyamines are important polycations that play critical roles in mammalian cells. ATP13A2 belongs to the orphan P5B adenosine triphosphatases (ATPase) family and has been established as a lysosomal polyamine exporter to maintain the normal function of lysosomes and mitochondria. Previous studies have reported that several human neurodegenerative disorders are related to mutations in the ATP13A2 gene. However, the transport mechanism of ATP13A2 in the lysosome remains unclear. Here, we report the cryo-electron microscopy (cryo-EM) structures of three distinct intermediates of the human ATP13A2, revealing key insights into the spermine (SPM) transport cycle in the lysosome. The transmembrane domain serves as a substrate binding site and the C-terminal domain is essential for protein stability and may play a regulatory role. These findings advance our understanding of the polyamine transport mechanism, the lipid-associated regulation, and the disease-associated mutants of ATP13A2.
    DOI:  https://doi.org/10.1038/s41421-021-00334-6
  5. Nat Commun. 2021 Nov 01. 12(1): 6284
    Structural mechanisms of TRPV6 inhibition by ruthenium red and econazole.
    Arthur Neuberger, Kirill D Nadezhdin, Alexander I Sobolevsky.
      TRPV6 is a calcium-selective ion channel implicated in epithelial Ca2+ uptake. TRPV6 inhibitors are needed for the treatment of a broad range of diseases associated with disturbed calcium homeostasis, including cancers. Here we combine cryo-EM, calcium imaging, and mutagenesis to explore molecular bases of human TRPV6 inhibition by the antifungal drug econazole and the universal ion channel blocker ruthenium red (RR). Econazole binds to an allosteric site at the channel's periphery, where it replaces a lipid. In contrast, RR inhibits TRPV6 by binding in the middle of the ion channel's selectivity filter and plugging its pore like a bottle cork. Despite different binding site locations, both inhibitors induce similar conformational changes in the channel resulting in closure of the gate formed by S6 helices bundle crossing. The uncovered molecular mechanisms of TRPV6 inhibition can guide the design of a new generation of clinically useful inhibitors.
    DOI:  https://doi.org/10.1038/s41467-021-26608-x
  6. Mol Cell. 2021 Oct 30. pii: S1097-2765(21)00840-6. [Epub ahead of print]
    Cryo-EM structure of MukBEF reveals DNA loop entrapment at chromosomal unloading sites.
    Frank Bürmann, Louise F H Funke, Jason W Chin, Jan Löwe.
      The ring-like structural maintenance of chromosomes (SMC) complex MukBEF folds the genome of Escherichia coli and related bacteria into large loops, presumably by active DNA loop extrusion. MukBEF activity within the replication terminus macrodomain is suppressed by the sequence-specific unloader MatP. Here, we present the complete atomic structure of MukBEF in complex with MatP and DNA as determined by electron cryomicroscopy (cryo-EM). The complex binds two distinct DNA double helices corresponding to the arms of a plectonemic loop. MatP-bound DNA threads through the MukBEF ring, while the second DNA is clamped by the kleisin MukF, MukE, and the MukB ATPase heads. Combinatorial cysteine cross-linking confirms this topology of DNA loop entrapment in vivo. Our findings illuminate how a class of near-ubiquitous DNA organizers with important roles in genome maintenance interacts with the bacterial chromosome.
    Keywords:  MukBEF; SMC; chromosome organization; cohesin; condensin; cryo-EM; loop extrusion
    DOI:  https://doi.org/10.1016/j.molcel.2021.10.011
  7. Chem Rev. 2021 Nov 02.
    Surface-induced Dissociation Mass Spectrometry as a Structural Biology Tool.
    Dalton T Snyder, Sophie R Harvey, Vicki H Wysocki.
      Native mass spectrometry (nMS) is evolving into a workhorse for structural biology. The plethora of online and offline preparation, separation, and purification methods as well as numerous ionization techniques combined with powerful new hybrid ion mobility and mass spectrometry systems has illustrated the great potential of nMS for structural biology. Fundamental to the progression of nMS has been the development of novel activation methods for dissociating proteins and protein complexes to deduce primary, secondary, tertiary, and quaternary structure through the combined use of multiple MS/MS technologies. This review highlights the key features and advantages of surface collisions (surface-induced dissociation, SID) for probing the connectivity of subunits within protein and nucleoprotein complexes and, in particular, for solving protein structure in conjunction with complementary techniques such as cryo-EM and computational modeling. Several case studies highlight the significant role SID, and more generally nMS, will play in structural elucidation of biological assemblies in the future as the technology becomes more widely adopted. Cases are presented where SID agrees with solved crystal or cryoEM structures or provides connectivity maps that are otherwise inaccessible by "gold standard" structural biology techniques.
    DOI:  https://doi.org/10.1021/acs.chemrev.1c00309
  8. Struct Dyn. 2021 Sep;8(5): 054302
    Microsecond melting and revitrification of cryo samples.
    Jonathan M Voss, Oliver F Harder, Pavel K Olshin, Marcel Drabbels, Ulrich J Lorenz.
      The dynamics of proteins that are associated with their function typically occur on the microsecond timescale, orders of magnitude faster than the time resolution of cryo-electron microscopy. We have recently introduced a novel approach to time-resolved cryo-electron microscopy that affords microsecond time resolution. It involves melting a cryo sample with a heating laser, so as to allow dynamics of the proteins to briefly occur in the liquid phase. When the laser is turned off, the sample rapidly revitrifies, trapping the particles in their transient configurations. Precise control of the temperature evolution of the sample is crucial for such an approach to succeed. Here, we provide a detailed characterization of the heat transfer occurring under laser irradiation as well as the associated phase behavior of the cryo sample. While areas close to the laser focus undergo melting and revitrification, surrounding regions crystallize. In situ observations of these phase changes therefore provide a convenient approach for assessing the temperature reached in each melting and revitrification experiment and for adjusting the heating laser power on the fly.
    DOI:  https://doi.org/10.1063/4.0000129
  9. Nat Commun. 2021 Nov 02. 12(1): 6316
    CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage.
    Rebecca Conners, Mathew McLaren, Urszula Łapińska, Kelly Sanders, M Rhia L Stone, Mark A T Blaskovich, Stefano Pagliara, Bertram Daum, Jasna Rakonjac, Vicki A M Gold.
      The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.
    DOI:  https://doi.org/10.1038/s41467-021-26610-3
  10. Nat Commun. 2021 Nov 02. 12(1): 6294
    C-Glycoside metabolism in the gut and in nature: Identification, characterization, structural analyses and distribution of C-C bond-cleaving enzymes.
    Takahiro Mori, Takuto Kumano, Haibing He, Satomi Watanabe, Miki Senda, Toshio Moriya, Naruhiko Adachi, Sanae Hori, Yuzu Terashita, Masato Kawasaki, Yoshiteru Hashimoto, Takayoshi Awakawa, Toshiya Senda, Ikuro Abe, Michihiko Kobayashi.
      C-Glycosides, in which a sugar moiety is linked via a carbon-carbon (C-C) bond to a non-sugar moiety (aglycone), are found in our food and medicine. The C-C bond is cleaved by intestinal microbes and the resulting aglycones exert various bioactivities. Although the enzymes responsible for the reactions have been identified, their catalytic mechanisms and the generality of the reactions in nature remain to be explored. Here, we present the identification and structural basis for the activation of xenobiotic C-glycosides by heterocomplex C-deglycosylation enzymes from intestinal and soil bacteria. They are found to be metal-dependent enzymes exhibiting broad substrate specificity toward C-glycosides. X-ray crystallographic and cryo-electron microscopic analyses, as well as structure-based mutagenesis, reveal the structural details of these enzymes and the detailed catalytic mechanisms of their remarkable C-C bond cleavage reactions. Furthermore, bioinformatic and biochemical analyses suggest that the C-deglycosylation enzymes are widely distributed in the gut, soil, and marine bacteria.
    DOI:  https://doi.org/10.1038/s41467-021-26585-1
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