bims-strubi Biomed News
on Advances in structural biology
Issue of 2022–01–02
seven papers selected by
Alessandro Grinzato, European Synchrotron Radiation Facility
  1. Flexibility of telomerase in binding the RNA template and DNA telomeric repeat.
  2. The structure of the native CNGA1/CNGB1 CNG channel from bovine retinal rods.
  3. Structure of the human cone photoreceptor cyclic nucleotide-gated channel.
  4. Imaging and visualizing SARS-CoV-2 in a new era for structural biology.
  5. Molecular structures and conformations of protocadherin-15 and its complexes on stereocilia elucidated by cryo-electron tomography.
  6. Conserved and divergent features of neuronal CaMKII holoenzyme structure, function, and high-order assembly.
  7. PDBcor: An automated correlation extraction calculator for multi-state protein structures.
  1. Proc Natl Acad Sci U S A. 2022 Jan 04. pii: e2116159118. [Epub ahead of print]119(1):
    Flexibility of telomerase in binding the RNA template and DNA telomeric repeat.
    Woo Suk Choi, Peter J Weng, Wei Yang.
      Telomerase synthesizes telomeres at the ends of linear chromosomes by repeated reverse transcription from a short RNA template. Crystal structures of Tribolium castaneum telomerase reverse transcriptase (tcTERT) and cryoelectron microscopy (cryo-EM) structures of human and Tetrahymena telomerase have revealed conserved features in the reverse-transcriptase domain, including a cavity near the DNA 3' end and snug interactions with the RNA template. For the RNA template to translocate, it needs to be unpaired and separated from the DNA product. Here we investigate the potential of the structural cavity to accommodate a looped-out DNA bulge and enable the separation of the RNA/DNA hybrid. Using tcTERT as a model system, we show that a looped-out telomeric repeat in the DNA primer can be accommodated and extended by tcTERT but not by retroviral reverse transcriptase. Mutations that reduce the cavity size reduce the ability of tcTERT to extend the looped-out DNA substrate. In agreement with cryo-EM structures of telomerases, we find that tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. We also have determined the ternary-complex structure of tcTERT including a downstream RNA/DNA hybrid at 2.0-Å resolution and shown that a downstream RNA duplex, equivalent to the 5' template-boundary element in telomerase RNA, enhances the efficiency of telomere synthesis by tcTERT. Although TERT has a preformed active site without the open-and-closed conformational changes, it contains cavities to accommodate looped-out RNA and DNA. The flexible RNA-DNA binding likely underlies the processivity of telomeric repeat addition.
    Keywords:  DNA loopout; RNA-template translocation; cavity; telomeric repeat synthesis; template-boundary element
    DOI:  https://doi.org/10.1073/pnas.2116159118
  2. Nat Struct Mol Biol. 2021 Dec 30.
    The structure of the native CNGA1/CNGB1 CNG channel from bovine retinal rods.
    Diane C A Barret, Gebhard F X Schertler, U Benjamin Kaupp, Jacopo Marino.
      In rod photoreceptors of the retina, the cyclic nucleotide-gated (CNG) channel is composed of three CNGA and one CNGB subunits, and it closes in response to light activation to generate an electrical signal that is conveyed to the brain. Here we report the cryo-EM structure of the closed state of the native rod CNG channel isolated from bovine retina. The structure reveals differences between CNGA1 and CNGB1 subunits. Three CNGA1 subunits are tethered at their C terminus by a coiled-coil region. The C-helix in the cyclic nucleotide-binding domain of CNGB1 features a different orientation from that in the three CNGA1 subunits. The arginine residue R994 of CNGB1 reaches into the ionic pathway and blocks the pore, thus introducing an additional gate, which is different from the central hydrophobic gate known from homomeric CNGA channels. These results address the long-standing question of how CNGB1 subunits contribute to the function of CNG channels in visual and olfactory neurons.
    DOI:  https://doi.org/10.1038/s41594-021-00700-8
  3. Nat Struct Mol Biol. 2021 Dec 30.
    Structure of the human cone photoreceptor cyclic nucleotide-gated channel.
    Xiangdong Zheng, Zhengshan Hu, Huan Li, Jian Yang.
      Cyclic nucleotide-gated (CNG) channels transduce light-induced chemical signals into electrical signals in retinal cone and rod photoreceptors. Structures of native CNG channels, which are heterotetramers formed by CNGA and CNGB subunits, have not been obtained. In the present study, we report a high-resolution cryo-electron microscopy structure of the human cone CNG channel in the apo closed state. The channel contains three CNGA3 and one CNGB3 subunits. Arg403 in the pore helix of CNGB3 projects into an asymmetric selectivity filter and forms hydrogen bonds with two pore-lining backbone carbonyl oxygens. Arg442 in S6 of CNGB3 protrudes into and occludes the pore below the hydrophobic cavity gate previously observed in homotetrameric CNGA channels. It is interesting that Arg403Gln is a disease mutation, and Arg442 is replaced by glutamine in some animal species with dichromatic or monochromatic vision. These and other unique structural features and the disease link conferred by CNGB3 indicate a critical role of CNGB3 in shaping cone photoresponses.
    DOI:  https://doi.org/10.1038/s41594-021-00699-y
  4. Interface Focus. 2021 Dec 06. 11(6): 20210019
    Imaging and visualizing SARS-CoV-2 in a new era for structural biology.
    Kendra E Leigh, Yorgo Modis.
      The SARS-CoV-2 pandemic has had a global impact and has put scientific endeavour in the spotlight, perhaps more than any previous viral outbreak. Fortuitously, the pandemic came at a time when decades of research in multiple scientific fields could be rapidly brought to bear, and a new generation of vaccine platforms was on the cusp of clinical maturity. SARS-CoV-2 also emerged at the inflection point of a technological revolution in macromolecular imaging by cryo-electron microscopy, fuelled by a confluence of major technological advances in sample preparation, optics, detectors and image processing software, that complemented pre-existing techniques. Together, these advances enabled us to visualize SARS-CoV-2 and its components more rapidly, in greater detail, and in a wider variety of biologically relevant contexts than would have been possible even a few years earlier. The resulting ultrastructural information on SARS-CoV-2 and how it interacts with the host cell has played a critical role in the much-needed accelerated development of COVID-19 vaccines and therapeutics. Here, we review key imaging modalities used to visualize SARS-CoV-2 and present select example data, which have provided us with an exceptionally detailed picture of this virus.
    Keywords:  SARS-CoV-2; X-ray crystallography; cryo-electron microscopy; cryo-electron tomography; multiscale imaging; structural biology
    DOI:  https://doi.org/10.1098/rsfs.2021.0019
  5. Elife. 2021 Dec 29. pii: e74512. [Epub ahead of print]10
    Molecular structures and conformations of protocadherin-15 and its complexes on stereocilia elucidated by cryo-electron tomography.
    Johannes Elferich, Sarah Clark, Jingpeng Ge, April Goehring, Aya Matsui, Eric Gouaux.
      Mechanosensory transduction (MT), the conversion of mechanical stimuli into electrical signals, underpins hearing and balance and is carried out within hair cells in the inner ear. Hair cells harbor actin-filled stereocilia, arranged in rows of descending heights, where the tips of stereocilia are connected to their taller neighbors by a filament composed of protocadherin 15 (PCDH15) and cadherin 23 (CDH23), deemed the 'tip link'. Tension exerted on the tip link opens an ion channel at the tip of the shorter stereocilia, thus converting mechanical force into an electrical signal. While biochemical and structural studies have provided insights into the molecular composition and structure of isolated portions of the tip link, the architecture, location and conformational states of intact tip links, on stereocilia, remains unknown. Here we report in situ cryo-electron microscopy imaging of the tip link in mouse stereocilia. We observe individual PCDH15 molecules at the tip and shaft of stereocilia and determine their stoichiometry, conformational heterogeneity, and their complexes with other filamentous proteins, perhaps including CDH23. The PCDH15 complexes occur in clusters, frequently with more than one copy of PCDH15 at the tip of stereocilia, suggesting that tip links might consist of more than one copy of PCDH15 complexes and, by extension, might include multiple MT complexes.
    Keywords:  molecular biophysics; mouse; neuroscience; structural biology
    DOI:  https://doi.org/10.7554/eLife.74512
  6. Cell Rep. 2021 Dec 28. pii: S2211-1247(21)01668-5. [Epub ahead of print]37(13): 110168
    Conserved and divergent features of neuronal CaMKII holoenzyme structure, function, and high-order assembly.
    Olivia R Buonarati, Adam P Miller, Steven J Coultrap, K Ulrich Bayer, Steve L Reichow.
      Neuronal CaMKII holoenzymes (α and β isoforms) enable molecular signal computation underlying learning and memory but also mediate excitotoxic neuronal death. Here, we provide a comparative analysis of these signaling devices, using single-particle electron microscopy (EM) in combination with biochemical and live-cell imaging studies. In the basal state, both isoforms assemble mainly as 12-mers (but also 14-mers and even 16-mers for the β isoform). CaMKIIα and β isoforms adopt an ensemble of extended activatable states (with average radius of 12.6 versus 16.8 nm, respectively), characterized by multiple transient intra- and inter-holoenzyme interactions associated with distinct functional properties. The extended state of CaMKIIβ allows direct resolution of intra-holoenzyme kinase domain dimers. These dimers could enable cooperative activation by calmodulin, which is observed for both isoforms. High-order CaMKII clustering mediated by inter-holoenzyme kinase domain dimerization is reduced for the β isoform for both basal and excitotoxicity-induced clusters, both in vitro and in neurons.
    Keywords:  CaMKII; activation; autophosphorylation; cell signaling; clustering; electron microscopy; holoenzyme; intrinsic disorder; structure
    DOI:  https://doi.org/10.1016/j.celrep.2021.110168
  7. Structure. 2021 Dec 21. pii: S0969-2126(21)00454-8. [Epub ahead of print]
    PDBcor: An automated correlation extraction calculator for multi-state protein structures.
    Dzmitry Ashkinadze, Piotr Klukowski, Harindranath Kadavath, Peter Güntert, Roland Riek.
      Allostery and correlated motion are key elements linking protein dynamics with the mechanisms of action of proteins. Here, we present PDBCor, an automated and unbiased method for the detection and analysis of correlated motions from experimental multi-state protein structures. It uses torsion angle and distance statistics and does not require any structure superposition. Clustering of protein conformers allows us to extract correlations in the form of mutual information based on information theory. With PDBcor, we elucidated correlated motion in the WW domain of PIN1, the protein GB3, and the enzyme cyclophilin, in line with reported findings. Correlations extracted with PDBcor can be utilized in subsequent assays including nuclear magnetic resonance (NMR) multi-state structure optimization and validation. As a guide for the interpretation of PDBcor results, we provide a series of protein structure ensembles that exhibit different levels of correlation, including non-correlated, locally correlated, and globally correlated ensembles.
    Keywords:  multi-state protein structure; protein correlations; protein structure; statistical analysis of protein structures
    DOI:  https://doi.org/10.1016/j.str.2021.12.002
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