Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins

Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles - including their nucleotide-free states - at ~7Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin-microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface

Conformational changes during pore formation by the perforin-related protein pleurotolysin

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ~70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function

TEMPy: a Python library for assessment of 3D electron microscopy density fits

Three-dimensional electron microscopy (3D EM) is currently one of the most promising techniques used to study macromolecular assemblies. Rigid and flexible fitting of atomic models into density maps is often essential to gain further insights into the assemblies they represent. Currently, tools that facilitate the assessment of fitted atomic models and maps are needed. TEMPy – Template and EM comparison using Python – is a toolkit designed for this purpose. The library includes a set of methods to assess density fits in intermediate-to-low resolution maps, both globally and locally. It also provides procedures for single fit assessment, ensemble generation of fits, clustering, multiple and consensus scoring, as well as plots and output files for visualisation purposes to help the user in analysing rigid and flexible fits. The modular nature of TEMPy helps the integration of scoring and assessment of fits into large pipelines, making it a tool for both novice and expert structural biologists

MED27, SLC6A7, and MPPE1 variants in a complex neurodevelopmental disorder with severe dystonia

Background: Despite advances in next generation sequencing technologies, the identification of variants of uncertain significance (VUS) can often hinder definitive diagnosis in patients with complex neurodevelopmental disorders. Objective: The objective of this study was to identify and characterize the underlying cause of disease in a family with two children with severe developmental delay associated with generalized dystonia and episodic status dystonicus, chorea, epilepsy, and cataracts. Methods: Candidate genes identified by autozygosity mapping and whole-exome sequencing were characterized using cellular and vertebrate model systems. Results: Homozygous variants were found in three candidate genes: MED27, SLC6A7, and MPPE1. Although the patients had features of MED27-related disorder, the SLC6A7 and MPPE1 variants were functionally investigated. SLC6A7 variant in vitro overexpression caused decreased proline transport as a result of reduced cell-surface expression, and zebrafish knockdown of slc6a7 exhibited developmental delay and fragile motor neuron morphology that could not be rescued by L-proline transporter-G396S RNA. Lastly, patient fibroblasts displayed reduced cell-surface expression of glycophosphatidylinositol-anchored proteins linked to MPPE1 dysfunction. Conclusions: We report a family harboring a homozygous MED27 variant with additional loss-of-function SLC6A7 and MPPE1 gene variants, which potentially contribute to a blended phenotype caused by multilocus pathogenic variants. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

Chromatin alternates between A and B compartments at kilobase scale for subgenic organization

Nuclear compartments are prominent features of 3D chromatin organization, but sequencing depth limitations have impeded investigation at ultra fine-scale. CTCF loops are generally studied at a finer scale, but the impact of looping on proximal interactions remains enigmatic. Here, we critically examine nuclear compartments and CTCF loop-proximal interactions using a combination of in situ Hi-C at unparalleled depth, algorithm development, and biophysical modeling. Producing a large Hi-C map with 33 billion contacts in conjunction with an algorithm for performing principal component analysis on sparse, super massive matrices (POSSUMM), we resolve compartments to 500 bp. Our results demonstrate that essentially all active promoters and distal enhancers localize in the A compartment, even when flanking sequences do not. Furthermore, we find that the TSS and TTS of paused genes are often segregated into separate compartments. We then identify diffuse interactions that radiate from CTCF loop anchors, which correlate with strong enhancer-promoter interactions and proximal transcription. We also find that these diffuse interactions depend on CTCF's RNA binding domains. In this work, we demonstrate features of fine-scale chromatin organization consistent with a revised model in which compartments are more precise than commonly thought while CTCF loops are more protracted

Three-dimensional genome organization via triplex-forming RNAs

An increasing number of long noncoding RNAs (lncRNAs) have been proposed to act as nuclear organization factors during interphase. Direct RNA-DNA interactions can be achieved by the formation of triplex helix structures where a single-stranded RNA molecule hybridizes by complementarity into the major groove of double-stranded DNA. However, whether and how these direct RNA-DNA associations influence genome structure in interphase chromosomes remain poorly understood. Here we theorize that RNA organizes the genome in space via a triplex-forming mechanism. To test this theory, we apply a computational modeling approach of chromosomes that combines restraint-based modeling with polymer physics. Our models suggest that colocalization of triplex hotspots targeted by lncRNAs could contribute to large-scale chromosome compartmentalization cooperating, rather than competing, with architectural transcription factors such as CTCF.This work was supported by the European Research Council under the 7th Framework Program FP7/2007-2013 (ERC grant agreement no. 609989 to M.A.M.-R.) and the Spanish Ministerio de Ciencia, Innovación y Universidades through nos. IJCI-2015-23352 to I.F. and BFU2017-85926-P and PID2020-115696RB-I00 to M.A.M.-R. CRG acknowledges support from ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208 and the CERCA Program/Generalitat de Catalunya, as well as support from the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III and the EMBL partnership, the Generalitat de Catalunya through Departament de Salut and Departament d’Empresa i Coneixement, and cofinancing with funds from the European Regional Development Fund by the Spanish Ministry of Science and Innovation corresponding to the Programa Opertaivo FEDER Plurirregional de España 2014–2020 and by the Secretaria d’Universitats i Recerca, Departament d’Empresa i Coneixement of the Generalitat de Catalunya corresponding to the program Operatiu FEDER Catalunya 2014–2020 and the NIH (to C.T. Wu no. R01HD091797 for supporting I.F.

Hierarchical chromatin organization detected by TADpole

Mètodes computacionals; GenòmicaMétodos computacionales; GenómicaComputational Methods; GenomicsThe rapid development of Chromosome Conformation Capture (3C-based techniques), as well as imaging together with bioinformatics analyses, has been fundamental for unveiling that chromosomes are organized into the so-called topologically associating domains or TADs. While TADs appear as nested patterns in the 3C-based interaction matrices, the vast majority of available TAD callers are based on the hypothesis that TADs are individual and unrelated chromatin structures. Here we introduce TADpole, a computational tool designed to identify and analyze the entire hierarchy of TADs in intra-chromosomal interaction matrices. TADpole combines principal component analysis and constrained hierarchical clustering to provide a set of significant hierarchical chromatin levels in a genomic region of interest. TADpole is robust to data resolution, normalization strategy and sequencing depth. Domain borders defined by TADpole are enriched in main architectural proteins (CTCF and cohesin complex subunits) and in the histone mark H3K4me3, while their domain bodies, depending on their activation-state, are enriched in either H3K36me3 or H3K27me3, highlighting that TADpole is able to distinguish functional TAD units. Additionally, we demonstrate that TADpole's hierarchical annotation, together with the new DiffT score, allows for detecting significant topological differences on Capture Hi-C maps between wild-type and genetically engineered mouse.European Research Council under the Seventh Framework Program FP7/2007-2013 [609989, in part]; European Union's Horizon 2020 Research and Innovation Programme [676556]; Spanish Ministry of Science and Innovation [BFU2013-47736-P, BFU2017-85926-P to M.A.M-R., IJCI-2015-23352 to I.F., BES-2014-070327 to P.S-V.]; ‘Centro de Excelencia Severo Ochoa 2013–2017’, SEV-2012-0208; CERCA Programme/Generalitat de Catalunya (to C.R.G.). Funding for open access charge: European Research Council under the Seventh Framework Program FP7/2007-2013 [609989]. We also acknowledge the support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208, the CERCA Programme/Generalitat de Catalunya, Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III, the Generalitat de Catalunya through Departament de Salut and Departament d’Empresa i Coneixement and the Co-financing by the Spanish Ministry of Science and Innovation with funds from the European Regional Development Fund (ERDF) corresponding to the 2014-2020 Smart Growth Operating Program to the CRG

Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling

Chromosome organization is crucial for genome function. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization. Using the super-resolution microscopy methods of OligoSTORM and OligoDNA-PAINT, we trace 8 megabases of human chromosome 19, visualizing structures ranging in size from a few kilobases to over a megabase. Focusing on chromosomal regions that contribute to compartments, we discover distinct structures that, in spite of considerable variability, can predict whether such regions correspond to active (A-type) or inactive (B-type) compartments. Imaging through the depths of entire nuclei, we capture pairs of homologous regions in diploid cells, obtaining evidence that maternal and paternal homologous regions can be differentially organized. Finally, using restraint-based modeling to integrate imaging and Hi-C data, we implement a method-integrative modeling of genomic regions (IMGR)-to increase the genomic resolution of our traces to 10 kb.This work was supported by funds from Ministerio de Ciencia, Innovación y Universidades of Spain (http://www.ciencia.gob.es/) (IJCI-2015-23352) to IF, Damon Runyon Cancer Research Foundation (https://www.damonrunyon.org/) and Howard Hughes Medical Institute (https://www.hhmi.org/) to BJB, Uehara Memorial Foundation Research (https://www.taisho-holdings.co.jp/en/environment/social/sciences/) to HMS, William Randolph Hearst Foundation (https://www.hearstfdn.org/) to RBM, EMBO (Long-Term fellowship) (https://www.embo.org/) to JE, NSF (Center for Theoretical Biological Physics, Rice University) (https://www.nsf.gov/) to MDP and JNO, NSF (CCF-1054898, CCF-1317291) (https://www.nsf.gov/), NIH (1R01EB018659-01, 1-U01- MH106011-01) (https://www.nih.gov/), and Office of Naval Research (N00014-13-1-0593, N00014-14-1-0610, N00014-16-1-2182, N00014-16-1- 2410) (https://www.onr.navy.mil/) to PY, NIH (1DP2OD008540, U01HL130010, UM1HG009375, 4DP2OD008540) (https://www.nih.gov/), NSF (PHY-1427654) (https://www.nsf.gov/), USDA (2017-05741) (https://www.usda.gov/), Welch Foundation (Q-1866) (http://www.welch1.org/), NVIDIA (https://www.nvidia.com/en-us/), IBM (https://www.ibm.com/us-en/?lnk=m), Google (https://www.google.com/), Cancer Prevention Research Institute of Texas (R1304) (http://www.cprit.state.tx.us/), and McNair Medical Institute (http://www.mcnairfoundation.org/what-we-fund/mcnair-medical-institute/) to E.L.A., Horizon 2020 Research and Innovation Programme (676556) (https://ec.europa.eu/programmes/horizon2020/en/), European Research Council (609989) (https://erc.europa.eu/), Ministerio de Ciencia, Innovación y Universidades of Spain (BFU2017-85926-P) (http://www.ciencia.gob.es/), CERCA, and AGAUR Programme of the Generalitat de Catalunya and Centros de Excelencia Severo Ochoa (SEV-2012-0208) (http://www.ciencia.gob.es/portal/site/MICINN/menuitem.7eeac5cd345b4f34f09dfd1001432ea0/?vgnextoid=cba733a6368c2310VgnVCM1000001d04140aRCRD) to M.A.M-R., and NIH (5DP1GM106412, R01HD091797, R01GM123289) (https://www.nih.gov/) to C-tW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Hierarchical chromatin organization detected by TADpole

The rapid development of Chromosome Conformation Capture (3C-based techniques), as well as imaging together with bioinformatics analyses, has been fundamental for unveiling that chromosomes are organized into the so-called topologically associating domains or TADs. While TADs appear as nested patterns in the 3C-based interaction matrices, the vast majority of available TAD callers are based on the hypothesis that TADs are individual and unrelated chromatin structures. Here we introduce TADpole, a computational tool designed to identify and analyze the entire hierarchy of TADs in intra-chromosomal interaction matrices. TADpole combines principal component analysis and constrained hierarchical clustering to provide a set of significant hierarchical chromatin levels in a genomic region of interest. TADpole is robust to data resolution, normalization strategy and sequencing depth. Domain borders defined by TADpole are enriched in main architectural proteins (CTCF and cohesin complex subunits) and in the histone mark H3K4me3, while their domain bodies, depending on their activation-state, are enriched in either H3K36me3 or H3K27me3, highlighting that TADpole is able to distinguish functional TAD units. Additionally, we demonstrate that TADpole's hierarchical annotation, together with the new DiffT score, allows for detecting significant topological differences on Capture Hi-C maps between wild-type and genetically engineered mouse.European Research Council under the Seventh Framework Program FP7/2007-2013 [609989, in part]; European Union's Horizon 2020 Research and Innovation Programme [676556]; Spanish Ministry of Science and Innovation [BFU2013-47736-P, BFU2017-85926-P to M.A.M-R., IJCI-2015-23352 to I.F., BES-2014-070327 to P.S-V.]; ‘Centro de Excelencia Severo Ochoa 2013–2017’, SEV-2012-0208; CERCA Programme/Generalitat de Catalunya (to C.R.G.). Funding for open access charge: European Research Council under the Seventh Framework Program FP7/2007-2013 [609989]

3D reconstruction of genomic regions from sparse interaction data

Chromosome conformation capture (3C) technologies measure the interaction frequency between pairs of chromatin regions within the nucleus in a cell or a population of cells. Some of these 3C technologies retrieve interactions involving non-contiguous sets of loci, resulting in sparse interaction matrices. One of such 3C technologies is Promoter Capture Hi-C (pcHi-C) that is tailored to probe only interactions involving gene promoters. As such, pcHi-C provides sparse interaction matrices that are suitable to characterize short- and long-range enhancer-promoter interactions. Here, we introduce a new method to reconstruct the chromatin structural (3D) organization from sparse 3C-based datasets such as pcHi-C. Our method allows for data normalization, detection of significant interactions and reconstruction of the full 3D organization of the genomic region despite of the data sparseness. Specifically, it builds, with as low as the 2-3% of the data from the matrix, reliable 3D models of similar accuracy of those based on dense interaction matrices. Furthermore, the method is sensitive enough to detect cell-type-specific 3D organizational features such as the formation of different networks of active gene communities.This study makes use of data generated by the PCHI-C Consortium available in the EGA European Genome-Phenome Archive (National Institute for Health Research of England, UK Medical Research Council (MR/L007150/1) and UK Biotechnology and Biological Research Council (BB/J004480/1