Structural mechanism for regulation of the AAA-ATPases RUVBL1-RUVBL2 in the R2TP co-chaperone revealed by cryo-EM

The human R2TP complex (RUVBL1-RUVBL2-RPAP3-PIH1D1) is an HSP90 co-chaperone required for the maturation of several essential multiprotein complexes, including RNA polymerase II, small nucleolar ribonucleoproteins, and PIKK complexes such as mTORC1 and ATR-ATRIP. RUVBL1-RUVBL2 AAA-ATPases are also primary components of other essential complexes such as INO80 and Tip60 remodelers. Despite recent efforts, the molecular mechanisms regulating RUVBL1-RUVBL2 in these complexes remain elusive. Here, we report cryo-EM structures of R2TP and show how access to the nucleotide-binding site of RUVBL2 is coupled to binding of the client recruitment component of R2TP (PIH1D1) to its DII domain. This interaction induces conformational rearrangements that lead to the destabilization of an N-terminal segment of RUVBL2 that acts as a gatekeeper to nucleotide exchange. This mechanism couples protein-induced motions of the DII domains with accessibility of the nucleotide-binding site in RUVBL1-RUVBL2, and it is likely a general mechanism shared with other RUVBL1-RUVBL2-containing complexes

Abstract OR-9: Cryo-EM Structure of the Reconstituted Human γ-Tubulin Ring Complex

Background: Microtubules (MTs) are essential cytoskeletal polymers that provide structural support for the cell and play important roles in cell division, motility, and intracellular transport. The γ-tubulin ring complex (γTuRC) is the major MT nucleator in animal cells. The molecular mechanism by which the γTuRC promotes MT nucleation remains poorly understood although a template-based mechanism, remains the most widely accepted (Moritz et al., 2000, Kollman et al., 2010). According to this model γTuRC, a 2 MDa multi-subunit protein complex, forms a lock washer-like structure, in which γ-tubulin molecules are arranged in a ring-shaped structure that serves as a template for the assembly of αβ-tubulin heterodimers. Methods: We have set up an in vitro system to purify the human γTuRC using infected insect cells with recombinant baculoviruses. This complex sample was subjected to cryo-EM analysis and single-particle reconstruction. Results: We have demonstrated that RUVBL1-RUVBL2 AAA-ATPase complex (RUVBL) controls the assembly and composition of γTuRC in human cells both in vivo and in vitro. Likewise, RUVBL assembles γTuRC from a minimal set of core subunits in a heterologous co-expression system. Purified, reconstituted γTuRC has nucleation activity and resembles native γTuRC (Consolati et al., 2020, Liu et al., 2020, Wieczorek et al., 2020), as revealed by its cryo-EM structure at ~4.0 Å resolution. Conclusion: We have been able to identify novel mechanistic and structural features that determine the intricate, higher-order γTuRC architecture (Zimmermann, Serna et al., 2020)

Structural basis for the inactivation of cytosolic DNA sensing by the vaccinia virus.

Detection of cytosolic DNA is a central element of the innate immunity system against viral infection. The Ku heterodimer, a component of the NHEJ pathway of DNA repair in the nucleus, functions as DNA sensor that detects dsDNA of viruses that replicate in the cytoplasm. Vaccinia virus expresses two proteins, C4 and C16, that inactivate DNA sensing and enhance virulence. The structural basis for this is unknown. Here we determine the structure of the C16 - Ku complex using cryoEM. Ku binds dsDNA by a preformed ring but C16 sterically blocks this access route, abrogating binding to a dsDNA end and its insertion into DNA-PK, thereby averting signalling into the downstream innate immunity system. C4 replicates these activities using a domain with 54% identity to C16. Our results reveal how vaccinia virus subverts the capacity of Ku to recognize viral DNA

Structural basis for the inactivation of cytosolic DNA sensing by the vaccinia virus

Detection of cytosolic DNA is a central element of the innate immunity system against viral infection. The Ku heterodimer, a component of the NHEJ pathway of DNA repair in the nucleus, functions as DNA sensor that detects dsDNA of viruses that replicate in the cytoplasm. Vaccinia virus expresses two proteins, C4 and C16, that inactivate DNA sensing and enhance virulence. The structural basis for this is unknown. Here we determine the structure of the C16 – Ku complex using cryoEM. Ku binds dsDNA by a preformed ring but C16 sterically blocks this access route, abrogating binding to a dsDNA end and its insertion into DNA-PK, thereby averting signalling into the downstream innate immunity system. C4 replicates these activities using a domain with 54% identity to C16. Our results reveal how vaccinia virus subverts the capacity of Ku to recognize viral DNA

Diagnostic and Prognostic Value of Cardiac Imaging in Amyloidosis

[Abstract] Amyloidosis is an infiltrative disease caused by extracellular protein deposition that has accumulated a lot of scientific production in recent years. Different types of amyloidosis can affect the heart. Transthyretin amyloidosis and light chain amyloidosis are the two most common types of cardiac amyloidosis. These entities have a poor prognosis, so accurate diagnostic techniques are imperative for determining an early therapeutic approach. Recent advances in cardiac imaging and diagnostic strategies show that these tools are safe and can avoid the use of invasive diagnostic techniques to histological confirmation, such as endomyocardial biopsy. We performed a review on the diagnostic and prognostic implications of different cardiac imaging techniques in cardiac amyloidosis. We mainly focus on reviewing echocardiography, cardiac magnetic resonance, computed tomography and nuclear imaging techniques and the different safety measurements that can be done with each of them

Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediated mRNA decay factor DHX34, as evidenced by Cryo-EM

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1, and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.Spanish Ministry of Science and Innovation SAF2017-82632-P Andres Lopez-Perrote Carlos F Rodriguez Marina Serna Oscar Llorca. Autonomous Government of Madrid Y2018/BIO4747 Ana Gonzalez-Corpas Oscar Llorca. Autonomous Government of Madrid P2018/NMT4443 Ana Gonzalez-Corpas Oscar Llorca MRC Core funding Javier F Caceres Spanish Ministry of Science and Innovation BES-2015-071348 Carlos F Rodriguez The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.S

Automating tasks in protein structure determination with the Clipper Python module

Scripting programming languages provide the fastest means of prototyping complex functionality. Those with a syntax and grammar resembling human language also greatly enhance the maintainability of the produced source code. Furthermore, the combination of a powerful, machine-independent scripting language with binary libraries tailored for each computer architecture allows programs to break free from the tight boundaries of efficiency traditionally associated with scripts. In the present work, we describe how an efficient C++ crystallographic library such as Clipper can be wrapped, adapted and generalised for use in both crystallographic and electron cryo-microscopy applications, scripted with the Python language. We shall also place an emphasis on best practices in automation, illustrating how this can be achieved with this new Python module. This article is protected by copyright. All rights reserved

AGT haplotype in ITGA4 gene is related to antibody-mediated rejection in heart transplant patients

[Abstract] Introduction. One of the main problems involved in heart transplantation (HT) is antibody-mediated rejection (AMR). Many aspects of AMR are still unresolved, including its etiology, diagnosis and treatment. In this project, we hypothesize that variants in genes involved in B-cell biology in HT patients can yield diagnostic and prognostic information about AMR. Methods. Genetic variants in 61 genes related to B-cell biology were analyzed by next generation sequencing in 46 HT patients, 23 with and 23 without AMR. Results. We identified 3 single nucleotide polymorphisms in ITGA4 gene (c.1845G>A, c.2633A>G, and c.2883C>T) that conformed the haplotype AGT-ITGA4. This haplotype is associated with the development of AMR. Moreover, AMR patients with the haplotype AGT-ITGA4 present lower levels of integrin α-4 in serum samples compared to the reference GAC haplotype in control patients. Conclusion. We can conclude that polymorphisms in genes related to the biology of B-cells could have an important role in the development of AMR. In fact, the AGT haplotype in ITGA4 gene could potentially increase the risk of AMR.Instituto de Salud Carlos III; PI13/0217

Cryo-electron microscopy of chromatin biology

The basic unit of chromatin, the nucleosome core particle (NCP), controls how DNA in eukaryotic cells is compacted, replicated and read. Since its discovery, biochemists have sought to understand how this protein-DNA complex can help to control so many diverse tasks. Recent electron-microscopy (EM) studies on NCP-containing assemblies have helped to describe important chromatin transactions at a molecular level. With the implementation of recent technical advances in single-particle EM, our understanding of how nucleosomes are recognized and read looks to take a leap forward. In this review, the authors highlight recent advances in the architectural understanding of chromatin biology elucidated by EM

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Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly