bims-strubi Biomed News
on Advances in structural biology
Issue of 2021–11–21
thirteen papers selected by
Alessandro Grinzato, European Synchrotron Radiation Facility
  1. New tools for automated cryo-EM single-particle analysis in RELION-4.0.
  2. Development of a universal nanobody-binding Fab module for fiducial-assisted cryo-EM studies of membrane proteins.
  3. Structural basis of the regulation of the normal and oncogenic methylation of nucleosomal histone H3 Lys36 by NSD2.
  4. Structural and functional basis of the selectivity filter as a gate in human TRPM2 channel.
  5. The cryo-EM structure of the chloroplast ClpP complex.
  6. Probing the existence of non-thermal Terahertz radiation induced changes of the protein solution structure.
  7. Crystal structures of the elusive Rhizobium etli L-asparaginase reveal a peculiar active site.
  8. Abemaciclib is a potent inhibitor of DYRK1A and HIP kinases involved in transcriptional regulation.
  9. Structural insights into Ubr1-mediated N-degron polyubiquitination.
  10. Structure, function and pharmacology of human itch receptor complexes.
  11. Optimal structure determination from sub-optimal diffraction data.
  12. Identification of ligand binding sites in intrinsically disordered proteins with a differential binding score.
  13. DockStream: a docking wrapper to enhance de novo molecular design.
  1. Biochem J. 2021 Nov 16. pii: BCJ20210708. [Epub ahead of print]
    New tools for automated cryo-EM single-particle analysis in RELION-4.0.
    Dari Kimanius, Liyi Dong, Grigory Sharov, Takanori Nakane, Sjors H W Scheres.
      We describe new tools for the processing of electron cryo-microscopy (cryo-EM) images in the fourth major release of the RELION software. In particular, we introduce VDAM, a Variable-metric gradient Descent algorithm with Adaptive Moments estimation, for image refinement; a convolutional neural network for unsupervised selection of 2D classes; and a flexible framework for the design and execution of multiple jobs in pre-defined workflows. In addition, we present a stand-alone utility called MDCatch that links the execution of jobs within this framework with metadata gathering during microscope data acquisition. The new tools are aimed at providing fast and robust procedures for unsupervised cryo-EM structure determination, with potential applications for on-the-fly processing and the development of flexible, high-throughput structure determination pipelines. We illustrate their potential on twelve publicly available cryo-EM data sets.
    Keywords:  computational biochemistry; cryo-electron microscopy; imaging techniques; structural biology
    DOI:  https://doi.org/10.1042/BCJ20210708
  2. Proc Natl Acad Sci U S A. 2021 Nov 23. pii: e2115435118. [Epub ahead of print]118(47):
    Development of a universal nanobody-binding Fab module for fiducial-assisted cryo-EM studies of membrane proteins.
    Joël S Bloch, Somnath Mukherjee, Julia Kowal, Ekaterina V Filippova, Martina Niederer, Els Pardon, Jan Steyaert, Anthony A Kossiakoff, Kaspar P Locher.
      With conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that specifically and tightly bind these nanobodies without disturbing the nanobody-target protein interaction. Here, we describe the development of a synthetic Fab (termed NabFab) that binds the scaffold of an alpaca-derived nanobody with picomolar affinity. We demonstrate that upon complementary-determining region grafting onto this parent nanobody scaffold, nanobodies recognizing diverse target proteins and derived from llama or camel can cross-react with NabFab without loss of affinity. Using NabFab as a fiducial and size enhancer (50 kDa), we determined the high-resolution cryogenic electron microscopy (cryo-EM) structures of nanobody-bound VcNorM and ScaDMT, both small membrane proteins of ∼50 kDa. Using an additional anti-Fab nanobody further facilitated reliable initial three-dimensional structure determination from small cryo-EM test datasets. Given that NabFab is of synthetic origin, is humanized, and can be conveniently expressed in Escherichia coli in large amounts, it may be useful not only for structural biology but also for biomedical applications.
    Keywords:  antibody; cryogenic electron microscopy; membrane protein; nanobody; structure
    DOI:  https://doi.org/10.1073/pnas.2115435118
  3. Nat Commun. 2021 Nov 15. 12(1): 6605
    Structural basis of the regulation of the normal and oncogenic methylation of nucleosomal histone H3 Lys36 by NSD2.
    Ko Sato, Amarjeet Kumar, Keisuke Hamada, Chikako Okada, Asako Oguni, Ayumi Machiyama, Shun Sakuraba, Tomohiro Nishizawa, Osamu Nureki, Hidetoshi Kono, Kazuhiro Ogata, Toru Sengoku.
      Dimethylated histone H3 Lys36 (H3K36me2) regulates gene expression, and aberrant H3K36me2 upregulation, resulting from either the overexpression or point mutation of the dimethyltransferase NSD2, is found in various cancers. Here we report the cryo-electron microscopy structure of NSD2 bound to the nucleosome. Nucleosomal DNA is partially unwrapped, facilitating NSD2 access to H3K36. NSD2 interacts with DNA and H2A along with H3. The NSD2 autoinhibitory loop changes its conformation upon nucleosome binding to accommodate H3 in its substrate-binding cleft. Kinetic analysis revealed that two oncogenic mutations, E1099K and T1150A, increase NSD2 catalytic turnover. Molecular dynamics simulations suggested that in both mutants, the autoinhibitory loop adopts an open state that can accommodate H3 more often than the wild-type. We propose that E1099K and T1150A destabilize the interactions that keep the autoinhibitory loop closed, thereby enhancing catalytic turnover. Our analyses guide the development of specific inhibitors of NSD2.
    DOI:  https://doi.org/10.1038/s41467-021-26913-5
  4. Cell Rep. 2021 Nov 16. pii: S2211-1247(21)01507-2. [Epub ahead of print]37(7): 110025
    Structural and functional basis of the selectivity filter as a gate in human TRPM2 channel.
    Xiafei Yu, Yuan Xie, Xiaokang Zhang, Cheng Ma, Likun Liu, Wenxuan Zhen, Lingyi Xu, Jianmin Zhang, Yan Liang, Lixia Zhao, Xiuxia Gao, Peilin Yu, Jianhong Luo, Lin-Hua Jiang, Yan Nie, Fan Yang, Jiangtao Guo, Wei Yang.
      Transient receptor potential melastatin 2 (TRPM2), a Ca2+-permeable cation channel, is gated by intracellular adenosine diphosphate ribose (ADPR), Ca2+, warm temperature, and oxidative stress. It is critically involved in physiological and pathological processes ranging from inflammation to stroke to neurodegeneration. At present, the channel's gating and ion permeation mechanisms, such as the location and identity of the selectivity filter, remain ambiguous. Here, we report the cryo-electron microscopy (cryo-EM) structure of human TRPM2 in nanodisc in the ligand-free state. Cryo-EM map-guided computational modeling and patch-clamp recording further identify a quadruple-residue motif as the ion selectivity filter, which adopts a restrictive conformation in the closed state and acts as a gate, profoundly contrasting with its widely open conformation in the Nematostella vectensis TRPM2. Our study reveals the gating of human TRPM2 by the filter and demonstrates the feasibility of using cryo-EM in conjunction with computational modeling and functional studies to garner structural information for intrinsically dynamic but functionally important domains.
    Keywords:  Ag(+) modification; computational modeling; selectivity filter; structure of TRPM2 channel
    DOI:  https://doi.org/10.1016/j.celrep.2021.110025
  5. Nat Plants. 2021 Nov;7(11): 1505-1515
    The cryo-EM structure of the chloroplast ClpP complex.
    Ning Wang, Yifan Wang, Qian Zhao, Xiang Zhang, Chao Peng, Wenjuan Zhang, Yanan Liu, Olivier Vallon, Michael Schroda, Yao Cong, Cuimin Liu.
      Protein homoeostasis in plastids is strategically regulated by the protein quality control system involving multiple chaperones and proteases, among them the Clp protease. Here, we determined the structure of the chloroplast ClpP complex from Chlamydomonas reinhardtii by cryo-electron microscopy. ClpP contains two heptameric catalytic rings without any symmetry. The top ring contains one ClpR6, three ClpP4 and three ClpP5 subunits while the bottom ring is composed of three ClpP1C subunits and one each of the ClpR1-4 subunits. ClpR3, ClpR4 and ClpT4 subunits connect the two rings and stabilize the complex. The chloroplast Cpn11/20/23 co-chaperonin, a co-factor of Cpn60, forms a cap on the top of ClpP by protruding mobile loops into hydrophobic clefts at the surface of the top ring. The co-chaperonin repressed ClpP proteolytic activity in vitro. By regulating Cpn60 chaperone and ClpP protease activity, the co-chaperonin may play a role in coordinating protein folding and degradation in the chloroplast.
    DOI:  https://doi.org/10.1038/s41477-021-01020-x
  6. Sci Rep. 2021 Nov 16. 11(1): 22311
    Probing the existence of non-thermal Terahertz radiation induced changes of the protein solution structure.
    Martin A Schroer, Siawosch Schewa, Andrey Yu Gruzinov, Christian Rönnau, Janine Mia Lahey-Rudolph, Clement E Blanchet, Till Zickmantel, Young-Hwa Song, Dmitri I Svergun, Manfred Roessle.
      During the last decades discussions were taking place on the existence of global, non-thermal structural changes in biological macromolecules induced by Terahertz (THz) radiation. Despite numerous studies, a clear experimental proof of this effect for biological particles in solution is still missing. We developed a setup combining THz-irradiation with small angle X-ray scattering (SAXS), which is a sensitive method for detecting the expected structural changes. We investigated in detail protein systems with different shape morphologies (bovine serum albumin, microtubules), which have been proposed to be susceptible to THz-radiation, under variable parameters (THz wavelength, THz power densities up to 6.8 mW/cm2, protein concentrations). None of the studied systems and conditions revealed structural changes detectable by SAXS suggesting that the expected non-thermal THz-induced effects do not lead to alterations of the overall structures, which are revealed by scattering from dissolved macromolecules. This leaves us with the conclusion that, if such effects are present, these are either local or outside of the spectrum and power range covered by the present study.
    DOI:  https://doi.org/10.1038/s41598-021-01774-6
  7. Nat Commun. 2021 Nov 18. 12(1): 6717
    Crystal structures of the elusive Rhizobium etli L-asparaginase reveal a peculiar active site.
    Joanna I Loch, Barbara Imiolczyk, Joanna Sliwiak, Anna Wantuch, Magdalena Bejger, Miroslaw Gilski, Mariusz Jaskolski.
      Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes an essential L-asparaginase (ReAV) with no sequence homology to known enzymes with this activity. High-resolution crystal structures of ReAV show indeed a structurally distinct, dimeric enzyme, with some resemblance to glutaminases and β-lactamases. However, ReAV has no glutaminase or lactamase activity, and at pH 9 its allosteric asparaginase activity is relatively high, with Km for L-Asn at 4.2 mM and kcat of 438 s-1. The active site of ReAV, deduced from structural comparisons and confirmed by mutagenesis experiments, contains a highly specific Zn2+ binding site without a catalytic role. The extensive active site includes residues with unusual chemical properties. There are two Ser-Lys tandems, all connected through a network of H-bonds to the Zn center, and three tightly bound water molecules near Ser48, which clearly indicate the catalytic nucleophile.
    DOI:  https://doi.org/10.1038/s41467-021-27105-x
  8. Nat Commun. 2021 Nov 16. 12(1): 6607
    Abemaciclib is a potent inhibitor of DYRK1A and HIP kinases involved in transcriptional regulation.
    Ines H Kaltheuner, Kanchan Anand, Jonas Moecking, Robert Düster, Jinhua Wang, Nathanael S Gray, Matthias Geyer.
      Homeodomain-interacting protein kinases (HIPKs) belong to the CMGC kinase family and are closely related to dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). HIPKs are regulators of various signaling pathways and involved in the pathology of cancer, chronic fibrosis, diabetes, and multiple neurodegenerative diseases. Here, we report the crystal structure of HIPK3 in its apo form at 2.5 Å resolution. Recombinant HIPKs and DYRK1A are auto-activated and phosphorylate the negative elongation factor SPT5, the transcription factor c-Myc, and the C-terminal domain of RNA polymerase II, suggesting a direct function in transcriptional regulation. Based on a database search, we identified abemaciclib, an FDA-approved Cdk4/Cdk6 inhibitor used for the treatment of metastatic breast cancer, as potent inhibitor of HIPK2, HIPK3, and DYRK1A. We determined the crystal structures of HIPK3 and DYRK1A bound to abemaciclib, showing a similar binding mode to the hinge region of the kinase as observed for Cdk6. Remarkably, DYRK1A is inhibited by abemaciclib to the same extent as Cdk4/Cdk6 in vitro, raising the question of whether targeting of DYRK1A contributes to the transcriptional inhibition and therapeutic activity of abemaciclib.
    DOI:  https://doi.org/10.1038/s41467-021-26935-z
  9. Nature. 2021 Nov 17.
    Structural insights into Ubr1-mediated N-degron polyubiquitination.
    Man Pan, Qingyun Zheng, Tian Wang, Lujun Liang, Junxiong Mao, Chong Zuo, Ruichao Ding, Huasong Ai, Yuan Xie, Dong Si, Yuanyuan Yu, Lei Liu, Minglei Zhao.
      The N-degron pathway targets proteins that bear a destabilizing residue at the N terminus for proteasome-dependent degradation1. In yeast, Ubr1-a single-subunit E3 ligase-is responsible for the Arg/N-degron pathway2. How Ubr1 mediates the initiation of ubiquitination and the elongation of the ubiquitin chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here we developed chemical strategies to mimic the reaction intermediates of the first and second ubiquitin transfer steps, and determined the cryo-electron microscopy structures of Ubr1 in complex with Ubc2, ubiquitin and two N-degron peptides, representing the initiation and elongation steps of ubiquitination. Key structural elements, including a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1, were identified and characterized. These structures provide mechanistic insights into the initiation and elongation of ubiquitination catalysed by Ubr1.
    DOI:  https://doi.org/10.1038/s41586-021-04097-8
  10. Nature. 2021 Nov 17.
    Structure, function and pharmacology of human itch receptor complexes.
    Fan Yang, Lulu Guo, Yu Li, Guopeng Wang, Jia Wang, Chao Zhang, Guo-Xing Fang, Xu Chen, Lei Liu, Xu Yan, Qun Liu, Changxiu Qu, Yunfei Xu, Peng Xiao, Zhongliang Zhu, Zijian Li, Jiuyao Zhou, Xiao Yu, Ning Gao, Jin-Peng Sun.
      In the clades of animals that diverged from the bony fish, a group of Mas-related G-protein-coupled receptors (MRGPRs) evolved that have an active role in itch and allergic signals1,2. As an MRGPR, MRGPRX2 is known to sense basic secretagogues (agents that promote secretion) and is involved in itch signals and eliciting pseudoallergic reactions3-6. MRGPRX2 has been targeted by drug development efforts to prevent the side effects induced by certain drugs or to treat allergic diseases. Here we report a set of cryo-electron microscopy structures of the MRGPRX2-Gi1 trimer in complex with polycationic compound 48/80 or with inflammatory peptides. The structures of the MRGPRX2-Gi1 complex exhibited shallow, solvent-exposed ligand-binding pockets. We identified key common structural features of MRGPRX2 and describe a consensus motif for peptidic allergens. Beneath the ligand-binding pocket, the unusual kink formation at transmembrane domain 6 (TM6) and the replacement of the general toggle switch from Trp6.48 to Gly6.48 (superscript annotations as per Ballesteros-Weinstein nomenclature) suggest a distinct activation process. We characterized the interfaces of MRGPRX2 and the Gi trimer, and mapped the residues associated with key single-nucleotide polymorphisms on both the ligand and G-protein interfaces of MRGPRX2. Collectively, our results provide a structural basis for the sensing of cationic allergens by MRGPRX2, potentially facilitating the rational design of therapies to prevent unwanted pseudoallergic reactions.
    DOI:  https://doi.org/10.1038/s41586-021-04077-y
  11. Protein Sci. 2021 Nov 15.
    Optimal structure determination from sub-optimal diffraction data.
    Wladek Minor, Marcin Cymborowski, Dominika Borek, David R Cooper, Maksymilian Chruszcz, Zbyszek Otwinowski.
      Herein we present the newest version of the HKL-3000 system that integrates data collection, data reduction, phasing, model building, refinement, and validation. The system significantly accelerates the process of structure determination and has proven its high value for the determination of very high-quality structures. The heuristic for choosing the best approach for every step of structure determination for various quality samples and diffraction data has been optimized. The latest modifications increase the likelihood of a successful structure determination with challenging data. The HKL-3000 is a successor of HKL and HKL-2000 programs. The use of the HKL family of programs has been reported for over 73,000 PDB deposits, that is, almost 50% of macromolecular structures determined with X-ray diffraction. This article is protected by copyright. All rights reserved.
    Keywords:  high-quality macromolecular structures; macromolecular structure determination; optimal structure determination
    DOI:  https://doi.org/10.1002/pro.4235
  12. Sci Rep. 2021 Nov 19. 11(1): 22583
    Identification of ligand binding sites in intrinsically disordered proteins with a differential binding score.
    Qiao-Hong Chen, V V Krishnan.
      Screening ligands directly binding to an ensemble of intrinsically disordered proteins (IDP) to discover potential hits or leads for new drugs is an emerging but challenging area as IDPs lack well-defined and ordered 3D-protein structures. To explore a new IDP-based rational drug discovery strategy, a differential binding score (DIBS) is defined. The basis of DIBS is to quantitatively determine the binding preference of a ligand to an ensemble of conformations specified by IDP versus such preferences to an ensemble of random coil conformations of the same protein. Ensemble docking procedures performed on repeated sampling of conformations, and the results tested for statistical significance determine the preferential ligand binding sites of the IDP. The results of this approach closely reproduce the experimental data from recent literature on the binding of the ligand epigallocatechin gallate (EGCG) to the intrinsically disordered N-terminal domain of the tumor suppressor p53. Combining established approaches in developing a new method to screen ligands against IDPs could be valuable as a screening tool for IDP-based drug discovery.
    DOI:  https://doi.org/10.1038/s41598-021-00869-4
  13. J Cheminform. 2021 Nov 17. 13(1): 89
    DockStream: a docking wrapper to enhance de novo molecular design.
    Jeff Guo, Jon Paul Janet, Matthias R Bauer, Eva Nittinger, Kathryn A Giblin, Kostas Papadopoulos, Alexey Voronov, Atanas Patronov, Ola Engkvist, Christian Margreitter.
      Recently, we have released the de novo design platform REINVENT in version 2.0. This improved and extended iteration supports far more features and scoring function components, which allows bespoke and tailor-made protocols to maximize impact in small molecule drug discovery projects. A major obstacle of generative models is producing active compounds, in which predictive (QSAR) models have been applied to enrich target activity. However, QSAR models are inherently limited by their applicability domains. To overcome these limitations, we introduce a structure-based scoring component for REINVENT. DockStream is a flexible, stand-alone molecular docking wrapper that provides access to a collection of ligand embedders and docking backends. Using the benchmarking and analysis workflow provided in DockStream, execution and subsequent analysis of a variety of docking configurations can be automated. Docking algorithms vary greatly in performance depending on the target and the benchmarking and analysis workflow provides a streamlined solution to identifying productive docking configurations. We show that an informative docking configuration can inform the REINVENT agent to optimize towards improving docking scores using public data. With docking activated, REINVENT is able to retain key interactions in the binding site, discard molecules which do not fit the binding cavity, harness unused (sub-)pockets, and improve overall performance in the scaffold-hopping scenario. The code is freely available at https://github.com/MolecularAI/DockStream .
    Keywords:  De novo design; Generative Models; Molecular docking; Reinforcement Learning (RL); Structure-based drug discovery (SBDD)
    DOI:  https://doi.org/10.1186/s13321-021-00563-7
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