bims-strubi 2021-12-05 papers

bims-strubi

Biomed News

on Advances in structural biology

Issue of 2021–12–05
nine papers selected by
Alessandro Grinzato, European Synchrotron Radiation Facility

Cryo-EM structures of τ filaments from human brain.
Conformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity.
Directed evolution of and structural insights into antibody-mediated disruption of a stable receptor-ligand complex.
The epitope arrangement on flavivirus particles contributes to Mab C10's extraordinary neutralization breadth across Zika and dengue viruses.
De novo protein design by deep network hallucination.
Structural basis for the E3 ligase activity enhancement of yeast Nse2 by SUMO-interacting motifs.
Structure and function of the poxvirus transcription machinery.
Crystal structures of phosphatidyl serine synthase PSS reveal the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases.
Molecular architecture of black widow spider neurotoxins.

Essays Biochem. 2021 Nov 30. pii: EBC20210025. [Epub ahead of print]

Cryo-EM structures of τ filaments from human brain.

Michel Goedert.

  Electron cryo-microscopy (cryo-EM) has made it possible to determine near-atomic structures of τ filaments from human brain. Previous work had shown that the cores of paired helical and straight filaments of Alzheimer's disease are made of two identical, but differently arranged C-shaped protofilaments. In recent years, cryo-EM has shown that the Alzheimer τ fold is 79 amino acids long. Five of the eight β-strands give rise to two antiparallel β-sheets, with the other three forming a β-helix. High-affinity binding sites of positron emission tomography ligand APN-1607 (PM-PBB3) are in the β-helix region. The Alzheimer fold contrasts with the 94 amino acid-long Pick fold, which is J-shaped and comprises nine β-strands that give rise to four antiparallel β-sheets, in the absence of a β-helix. Chronic traumatic encephalopathy τ fold is similar to the Alzheimer fold, but differs in the β-helix region, which is larger and contains a non-proteinaceous density that is probably hydrophobic. These folds are mostly two-layered. By contrast, the 107 amino acid τ fold of the 4R tauopathy corticobasal degeneration is four-layered and comprises 11 β-strands. It contains an internal, probably hydrophilic, density that is surrounded by τ. The τ folds described here share the presence of microtubule-binding repeats 3 and 4, as well as 10-13 amino acids after repeat 4.

Keywords:  cryo-electron microscopy; molecular conformation; tau proteins

DOI:  https://doi.org/10.1042/EBC20210025
Nat Commun. 2021 Dec 02. 12(1): 7018

Conformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity.

Tomas Kouba, Dominik Vogel, Sigurdur R Thorkelsson, Emmanuelle R J Quemin, Harry M Williams, Morlin Milewski, Carola Busch, Stephan Günther, Kay Grünewald, Maria Rosenthal, Stephen Cusack.

Lassa virus is endemic in West Africa and can cause severe hemorrhagic fever. The viral L protein transcribes and replicates the RNA genome via its RNA-dependent RNA polymerase activity. Here, we present nine cryo-EM structures of the L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved 3' and 5' promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and early elongation states, the endonuclease is inhibited by two distinct L protein peptides, whereas in the pre-initiation state it is uninhibited. In the early elongation state, a template-product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide and the full C-terminal region of the L protein, including the putative cap-binding domain, is well-ordered. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated.

DOI: https://doi.org/10.1038/s41467-021-27305-5
Nat Commun. 2021 Dec 03. 12(1): 7069

Directed evolution of and structural insights into antibody-mediated disruption of a stable receptor-ligand complex.

Luke F Pennington, Pascal Gasser, Silke Kleinboelting, Chensong Zhang, Georgios Skiniotis, Alexander Eggel, Theodore S Jardetzky.

Antibody drugs exert therapeutic effects via a range of mechanisms, including competitive inhibition, allosteric modulation, and immune effector mechanisms. Facilitated dissociation is an additional mechanism where antibody-mediated "disruption" of stable high-affinity macromolecular complexes can potentially enhance therapeutic efficacy. However, this mechanism is not well understood or utilized therapeutically. Here, we investigate and engineer the weak disruptive activity of an existing therapeutic antibody, omalizumab, which targets IgE antibodies to block the allergic response. We develop a yeast display approach to select for and engineer antibody disruptive efficiency and generate potent omalizumab variants that dissociate receptor-bound IgE. We determine a low resolution cryo-EM structure of a transient disruption intermediate containing the IgE-Fc, its partially dissociated receptor and an antibody inhibitor. Our results provide a conceptual framework for engineering disruptive inhibitors for other targets, insights into the failure in clinical trials of the previous high affinity omalizumab HAE variant and anti-IgE antibodies that safely and rapidly disarm allergic effector cells.

DOI: https://doi.org/10.1038/s41467-021-27397-z
Cell. 2021 Nov 24. pii: S0092-8674(21)01325-8. [Epub ahead of print]

The epitope arrangement on flavivirus particles contributes to Mab C10's extraordinary neutralization breadth across Zika and dengue viruses.

Arvind Sharma, Xiaokang Zhang, Wanwisa Dejnirattisai, Xinghong Dai, Danyang Gong, Wiyada Wongwiwat, Stéphane Duquerroy, Alexander Rouvinski, Marie-Christine Vaney, Pablo Guardado-Calvo, Ahmed Haouz, Patrick England, Ren Sun, Z Hong Zhou, Juthathip Mongkolsapaya, Gavin R Screaton, Felix A Rey.

  The human monoclonal antibody C10 exhibits extraordinary cross-reactivity, potently neutralizing Zika virus (ZIKV) and the four serotypes of dengue virus (DENV1-DENV4). Here we describe a comparative structure-function analysis of C10 bound to the envelope (E) protein dimers of the five viruses it neutralizes. We demonstrate that the C10 Fab has high affinity for ZIKV and DENV1 but not for DENV2, DENV3, and DENV4. We further show that the C10 interaction with the latter viruses requires an E protein conformational landscape that limits binding to only one of the three independent epitopes per virion. This limited affinity is nevertheless counterbalanced by the particle's icosahedral organization, which allows two different dimers to be reached by both Fab arms of a C10 immunoglobulin. The epitopes' geometric distribution thus confers C10 its exceptional neutralization breadth. Our results highlight the importance not only of paratope/epitope complementarity but also the topological distribution for epitope-focused vaccine design.

Keywords:  Dengue virus; Flaviviruses; X-ray crystallography; Zika virus; broadly neutralizing antibodies; cryo-EM; vaccine design

DOI:  https://doi.org/10.1016/j.cell.2021.11.010
Nature. 2021 Dec 01.

De novo protein design by deep network hallucination.

Ivan Anishchenko, Samuel J Pellock, Tamuka M Chidyausiku, Theresa A Ramelot, Sergey Ovchinnikov, Jingzhou Hao, Khushboo Bafna, Christoffer Norn, Alex Kang, Asim K Bera, Frank DiMaio, Lauren Carter, Cameron M Chow, Gaetano T Montelione, David Baker.

There has been considerable recent progress in protein structure prediction using deep neural networks to predict inter-residue distances from amino acid sequences1-3. Here we investigate whether the information captured by such networks is sufficiently rich to generate new folded proteins with sequences unrelated to those of the naturally occurring proteins used in training the models. We generate random amino acid sequences, and input them into the trRosetta structure prediction network to predict starting residue-residue distance maps, which, as expected, are quite featureless. We then carry out Monte Carlo sampling in amino acid sequence space, optimizing the contrast (Kullback-Leibler divergence) between the inter-residue distance distributions predicted by the network and background distributions averaged over all proteins. Optimization from different random starting points resulted in novel proteins spanning a wide range of sequences and predicted structures. We obtained synthetic genes encoding 129 of the network-'hallucinated' sequences, and expressed and purified the proteins in Escherichia coli; 27 of the proteins yielded monodisperse species with circular dichroism spectra consistent with the hallucinated structures. We determined the three-dimensional structures of three of the hallucinated proteins, two by X-ray crystallography and one by NMR, and these closely matched the hallucinated models. Thus, deep networks trained to predict native protein structures from their sequences can be inverted to design new proteins, and such networks and methods should contribute alongside traditional physics-based models to the de novo design of proteins with new functions.

DOI: https://doi.org/10.1038/s41586-021-04184-w
Nat Commun. 2021 Dec 01. 12(1): 7013

Structural basis for the E3 ligase activity enhancement of yeast Nse2 by SUMO-interacting motifs.

Nathalia Varejão, Jara Lascorz, Joan Codina-Fabra, Gemma Bellí, Helena Borràs-Gas, Jordi Torres-Rosell, David Reverter.

Post-translational modification of proteins by ubiquitin and ubiquitin-like modifiers, such as SUMO, are key events in protein homeostasis or DNA damage response. Smc5/6 is a nuclear multi-subunit complex that participates in the recombinational DNA repair processes and is required in the maintenance of chromosome integrity. Nse2 is a subunit of the Smc5/6 complex that possesses SUMO E3 ligase activity by the presence of a SP-RING domain that activates the E2~SUMO thioester for discharge on the substrate. Here we present the crystal structure of the SUMO E3 ligase Nse2 in complex with an E2-SUMO thioester mimetic. In addition to the interface between the SP-RING domain and the E2, the complex reveals how two SIM (SUMO-Interacting Motif) -like motifs in Nse2 are restructured upon binding the donor and E2-backside SUMO during the E3-dependent discharge reaction. Both SIM interfaces are essential in the activity of Nse2 and are required to cope with DNA damage.

DOI: https://doi.org/10.1038/s41467-021-27301-9
Enzymes. 2021 ;pii: S1874-6047(21)00005-6. [Epub ahead of print]50 1-20

Structure and function of the poxvirus transcription machinery.

Utz Fischer, Julia Bartuli, Clemens Grimm.

  Members of the Poxviridae family are large double-stranded DNA viruses that replicate exclusively in the cytoplasm of their hosts. This goes in hand with a high level of independence from the host cell, which supports transcription and replication events only in the nucleus or in DNA-containing organelles. Consequently, virus specific, rather than cellular enzymes mediate most processes involving DNA replication and mRNA synthesis. Recent technological advances allowed a detailed functional and structural investigation of the transcription machinery of the prototypic poxvirus vaccinia. The DNA-dependent RNA polymerase (RNAP) at its core displays distinct similarities to eukaryotic RNAPs. Strong idiosyncrasies, however, are apparent for viral factors that are associated with the viral RNAP during mRNA production. We expect that future studies will unravel more key aspects of poxvirus gene expression, helping also the understanding of nuclear transcription mechanisms.

Keywords:  Cryo electron microscopy; Poxviridae; Poxviruses; RNA polymerase; Transcription; Vaccinia; Virus

DOI:  https://doi.org/10.1016/bs.enz.2021.06.005
Nat Commun. 2021 Nov 30. 12(1): 6982

Crystal structures of phosphatidyl serine synthase PSS reveal the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases.

Martin Centola, Katharina van Pee, Heidi Betz, Özkan Yildiz.

Phospholipids are the major components of the membrane in all type of cells and organelles. They also are critical for cell metabolism, signal transduction, the immune system and other critical cell functions. The biosynthesis of phospholipids is a complex multi-step process with high-energy intermediates. Several enzymes in different metabolic pathways are involved in the initial phospholipid synthesis and its subsequent conversion. While the "Kennedy pathway" is the main pathway in mammalian cells, in bacteria and lower eukaryotes the precursor CDP-DAG is used in the de novo pathway by CDP-DAG alcohol O-phosphatidyl transferases to synthetize the basic lipids. Here we present the high-resolution structures of phosphatidyl serine synthase from Methanocaldococcus jannaschii crystallized in four different states. Detailed structural and functional analysis of the different structures allowed us to identify the substrate binding site and show how CDP-DAG, serine and two essential metal ions are bound and oriented relative to each other. In close proximity to the substrate binding site, two anions were identified that appear to be highly important for the reaction. The structural findings were confirmed by functional activity assays and suggest a model for the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases, which synthetize the phospholipids essential for the cells.

DOI: https://doi.org/10.1038/s41467-021-27281-w
Nat Commun. 2021 Nov 29. 12(1): 6956

Molecular architecture of black widow spider neurotoxins.

Minghao Chen, Daniel Blum, Lena Engelhard, Stefan Raunser, Richard Wagner, Christos Gatsogiannis.

Latrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (α-latrotoxin (α-LTX)), five specialized on insects (α, β, γ, δ, ε- latroinsectotoxins (LITs), and one on crustaceans (α-latrocrustatoxin (α-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca2+-conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear. Here, we report cryoEM structures of the α-LCT monomer and the δ-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel α-helices. Both domains are flexibly linked via an N-terminal α-helical domain and a small β-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between α-LCT and δ-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.

DOI: https://doi.org/10.1038/s41467-021-26562-8