Selected publications
A. Sabantsev, G. Mao, J. Aguirre Rivera, M. Panfilov, A. Arseniev, O. Ho, M. Khodorkovskiy, S. Deindl
Spatiotemporally controlled generation of NTPs for single-molecule studies
Nature Chemical Biology 2022
see also the accompanying News & Views article Very fast nucleotides on demand

E. Marklund, G. Mao, J. Yan, S. Zikrin, E. Abdurakhmanov, S. Deindl*, J. Elf*
Sequence specificity in DNA binding is mainly governed by association
Science 2022
L. Bacic, G. Gaullier, A. Sabantsev, L. C. Lehmann, K. Brackmann, D. Dimakou, M. Halic, G. Hewitt, S. J. Boulton, S. Deindl
Structure and dynamics of the chromatin remodeler ALC1 bound to a PARylated nucleosome
eLife 2021
I. Regadas, O. Dahlberg, R. Vaid, O. Ho, S. Belikov, G. Dixit, S. Deindl, J. Wen, M. Mannervik
A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation
Molecular Cell 2021
G. Hewitt, V. Borel, S. Segura-Bayona, T. Takaki, P. Ruis, R. Bellelli, L. C. Lehmann, L. Sommerova, A. Vancevska, A. Tomas-Loba, K. Zhu, C. Cooper, K. Fugger, H. Patel, R. Goldstone, D. Schneider-Luftman, E. Herbert, G. Stamp, R. Brough, S. Pettitt, C. J. Lord, S. C. West, I. Ahel, D. Ahel, J. R. Chapman, S. Deindl , S. J. Boulton
Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD
Molecular Cell 2021
E. Marklund, B. van Oosten, G. Mao, E. Amselem, K. Kipper, A. Sabantsev, A. Emmerich, D. Globisch, X. Zheng, L. C. Lehmann, O. Berg, M. Johansson, J. Elf, S. Deindl
DNA surface exploration and operator bypassing during target search
Nature 2020
L. Bacic, A. Sabantsev, and S. Deindl
Recent advances in single-molecule fluorescence microscopy render structural biology dynamic
Current Opinion in Structural Biology 2020
G. Bowman and S. Deindl
Remodeling the genome with DNA twists
Science 2019
A. Sabantsev, R.F. Levendosky, X. Zhuang, G.D. Bowman and S. Deindl
Direct observation of coordinated DNA movements on the nucleosome during chromatin remodelling
Nature Communications 2019

L.C. Lehmann, G. Hewitt, S. Aibara, A. Leitner, E. Marklund, S.L. Maslen, V. Maturi, Y. Chen, D. van der Spoel, J.M. Skehel, A. Moustakas, S. J. Boulton and S. Deindl
Mechanistic Insights into Autoinhibition of the Oncogenic Chromatin Remodeler ALC1
Molecular Cell 2017
W.L. Hwang*, S. Deindl*, B.T. Harada and X. Zhuang
*equal contribution

Histone H4 tail mediates allosteric regulation of nucleosome remodelling by linker DNA
Nature 2014
S. Deindl, W.L. Hwang, S.K. Hota, T.R. Blosser, P. Prasad, B. Bartholomew and X. Zhuang
ISWI Remodelers Slide Nucleosomes with Coordinated Multi-Base-Pair Entry Steps and Single-Base-Pair Exit Steps
Cell 2013
S. Deindl, T.A. Kadlecek, T. Brdicka, X. Cao, A. Weiss and J. Kuriyan
Structural basis for the inhibition of tyrosine kinase activity of ZAP-70

Cell 2007
O.S. Rosenberg, S. Deindl, R.J. Sung, A.C. Nairn, J. Kuriyan
Structure of the Autoinhibited Kinase Domain of CaMKII and SAXS Analysis of the Holoenzyme
Cell 2005

Complete list of publications
L. Bacic, G. Gaullier, J. Mohapatra, G. Mao, K. Brackmann, M. Panfilov, G. Liszczak, A. Sabantsev, and S. Deindl
Asymmetric nucleosome PARylation at DNA breaks mediates directional nucleosome sliding by ALC1
Nature Communications, accepted
A. Sabantsev, G. Mao, J. Aguirre Rivera, M. Panfilov, A. Arseniev, O. Ho, M. Khodorkovskiy, S. Deindl
Spatiotemporally controlled generation of NTPs for single-molecule studies
Nature Chemical Biology 2022
see also the accompanying News & Views article Very fast nucleotides on demand
Many essential processes in the cell depend on proteins that use nucleoside triphosphates (NTPs). Methods that directly monitor the often-complex dynamics of these proteins at the single-molecule level have helped to uncover their mechanisms of action. However, the measurement throughput is typically limited for NTP-utilizing reactions, and the quantitative dissection of complex dynamics over multiple sequential turnovers remains challenging. Here we present a method for controlling NTP-driven reactions in single-molecule experiments via the local generation of NTPs (LAGOON) that markedly increases the measurement throughput and enables single-turnover observations. We demonstrate the effectiveness of LAGOON in single-molecule fluorescence and force spectroscopy assays by monitoring DNA unwinding, nucleosome sliding and RNA polymerase elongation. LAGOON can be readily integrated with many single-molecule techniques, and we anticipate that it will facilitate studies of a wide range of crucial NTP-driven processes.
E. Marklund, G. Mao, J. Yan, S. Zikrin, E. Abdurakhmanov, S. Deindl*, J. Elf*
Sequence specificity in DNA binding is mainly governed by association
Science 2022
Sequence-specific binding of proteins to DNA is essential for accessing genetic information. We derive a model that predicts an anticorrelation between the macroscopic association and dissociation rates of DNA binding proteins. We tested the model for thousands of different lac operator sequences with a protein binding microarray and by observing kinetics for individual lac repressor molecules in single-molecule experiments. We found that sequence specificity is mainly governed by the efficiency with which the protein recognizes different targets. The variation in probability of recognizing different targets is at least 1.7 times as large as the variation in microscopic dissociation rates. Modulating the rate of binding instead of the rate of dissociation effectively reduces the risk of the protein being retained on nontarget sequences while searching.
L. Bacic, G. Gaullier, A. Sabantsev, L. C. Lehmann, K. Brackmann, D. Dimakou, M. Halic, G. Hewitt, S. J. Boulton, S. Deindl
Structure and dynamics of the chromatin remodeler ALC1 bound to a PARylated nucleosome
eLife 2021
The chromatin remodeler ALC1 is recruited to and activated by DNA damage-induced poly(ADP-ribose) (PAR) chains deposited by PARP1/PARP2/HPF1 upon detection of DNA lesions. ALC1 has emerged as a candidate drug target for cancer therapy as its loss confers synthetic lethality in homologous recombination-deficient cells. However, structure-based drug design and molecular analysis of ALC1 have been hindered by the requirement for PARylation and the highly heterogeneous nature of this post-translational modification. Here, we reconstituted an ALC1 and PARylated nucleosome complex modified in vitro using PARP2 and HPF1. This complex was amenable to cryo-EM structure determination without cross-linking, which enabled visualization of several intermediate states of ALC1 from the recognition of the PARylated nucleosome to the tight binding and activation of the remodeler. Functional biochemical assays with PARylated nucleosomes highlight the importance of nucleosomal epitopes for productive remodeling and suggest that ALC1 preferentially slides nucleosomes away from DNA breaks.
B. Luescher, I. Ahel, M. Altmeyer, A. Ashworth, P. Bai, P. Chang, M. Cohen, D. Corda, F. Dantzer, M. D. Daugherty, T. M. Dawson, V. L. Dawson, S. Deindl, A. R. Fehr, K. L. H. Feijs, D. V. Filippov, J-P. Gagné, G. Grimaldi, S. Guettler, N. C. Hoch, M. O. Hottiger, P. Korn, W. L. Kraus, A. Ladurner, L. Lehtiö, A. K. L. Leung, C. J. Lord, A. Mangerich, I. Matic, J. Matthews, G-L. Moldovan, J. Moss, G. Natoli, M. L. Nielsen, M. Niepel, F. Nolte, J. Pascal, B. M. Paschal, K. Pawlowski, G. G. Poirier, S. Smith, G. Timinszky, Z-Q. Wang, J. Yélamos, X. Yu, R. Zaja, M. Ziegler
ADP-ribosyltransferases, an update on function and nomenclature

FEBS Journal 2021
ADP-ribosylation, a modification of proteins, nucleic acids and metabolites, confers broad functions, including roles in stress responses elicited for example by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases, which transfer ADP-ribose from NAD+ onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ADP-ribosyltransferases are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as anti-viral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ADP-ribosyltransferases and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ADP-ribosyltransferases that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ADP-ribosyltransferases to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.
S. Deindl, J. Elf
More Than Just Letters and Chemistry: Genomics Goes Mechanics

Trends in Biochemical Sciences 2021
Although ubiquitously thought of as a simple string of letters, DNA exhibits complex physicochemical properties. As a result, DNA can store information beyond the extensively studied explicit genetic message. The mechanical code of DNA has not been studied systematically in a genome-wide context until recent groundbreaking work by Basu et al.
I. Regadas, O. Dahlberg, R. Vaid, O. Ho, S. Belikov, G. Dixit, S. Deindl, J. Wen, M. Mannervik
A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation

Molecular Cell 2021
Organismal development and cell differentiation critically depend on chromatin state transitions. However, certain developmentally regulated genes lack histone 3 lysine 9 and 27 acetylation (H3K9ac and H3K27ac, respectively) and histone 3 lysine 4 (H3K4) methylation, histone modifications common to most active genes. Here we describe a chromatin state featuring unique histone 3 lysine 14 acetylation (H3K14ac) peaks in key tissue-specific genes in Drosophila and human cells. Replacing H3K14 in Drosophila demonstrates that H3K14 is essential for expression of genes devoid of canonical histone modifications in the embryonic gut and larval wing imaginal disc, causing lethality and defective wing patterning. We find that the SWI/SNF protein Brahma (Brm) recognizes H3K14ac, that brm acts in the same genetic pathway as H3K14R, and that chromatin accessibility at H3K14ac-unique genes is decreased in H3K14R mutants. Our results show that acetylation of a single lysine is essential at genes devoid of canonical histone marks and uncover an important requirement for H3K14 in tissue-specific gene regulation.
G. Hewitt, V. Borel, S. Segura-Bayona, T. Takaki, P. Ruis, R. Bellelli, L. C. Lehmann, L. Sommerova, A. Vancevska, A. Tomas-Loba, K. Zhu, C. Cooper, K. Fugger, H. Patel, R. Goldstone, D. Schneider-Luftman, E. Herbert, G. Stamp, R. Brough, S. Pettitt, C. J. Lord, S. C. West, I. Ahel, D. Ahel, J. R. Chapman, S. Deindl , S. J. Boulton
Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD
Molecular Cell 2021
Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.
L. C. Lehmann, L. Bacic, G. Hewitt, K. Brackmann, A. Sabantsev, G. Gaullier, S. Pytharopoulou, G. Degliesposti, H. Okkenhaug, S. Tan, A. Costa, J. M. Skehel, S. J. Boulton, S. Deindl
Mechanistic Insights into Regulation of the ALC1 Remodeler by the Nucleosome Acidic Patch
Cell Reports 2020
Upon DNA damage, the ALC1/CHD1L nucleosome remodeling enzyme (remodeler) is activated by binding to poly(ADP-ribose). How activated ALC1 recognizes the nucleosome, as well as how this recognition is coupled to remodeling, is unknown. Here, we show that remodeling by ALC1 requires a wild-type acidic patch on the entry side of the nucleosome. The cryo-electron microscopy structure of a nucleosome-ALC1 linker complex reveals a regulatory linker segment that binds to the acidic patch. Mutations within this interface alter the dynamics of ALC1 recruitment to DNA damage and impede the ATPase and remodeling activities of ALC1. Full activation requires acidic patch-linker segment interactions that tether the remodeler to the nucleosome and couple ATP hydrolysis to nucleosome mobilization. Upon DNA damage, such a requirement may be used to modulate ALC1 activity via changes in the nucleosome acidic patches.
L. Bacic, A. Sabantsev, and S. Deindl
Recent advances in single-molecule fluorescence microscopy render structural biology dynamic
Current Opinion in Structural Biology 2020
Single-molecule fluorescence microscopy has long been appreciated as a powerful tool to study the structural dynamics that enable biological function of macromolecules. Recent years have witnessed the development of more complex single-molecule fluorescence techniques as well as powerful combinations with structural approaches to obtain mechanistic insights into the workings of various molecular machines and protein complexes. In this review, we highlight these developments that together bring us one step closer to a dynamic understanding of biological processes in atomic details.
E. Marklund, B. van Oosten, G. Mao, E. Amselem, K. Kipper, A. Sabantsev, A. Emmerich, D. Globisch, X. Zheng, L. C. Lehmann, O. Berg, M. Johansson, J. Elf, S. Deindl
DNA surface exploration and operator bypassing during target search
Nature 2020
Many proteins that bind specific DNA sequences search the genome by combining three-dimensional diffusion with one-dimensional sliding on nonspecific DNA. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore the DNA surface during the one-dimensional phase of target search. To track the rotation of sliding LacI molecules on the microsecond timescale, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT–FCS). The fluctuations in fluorescence signal are accurately described by rotation-coupled sliding, in which LacI traverses about 40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA; this suggests that the sliding protein frequently hops out of the DNA groove, which would result in the frequent bypassing of target sequences. We directly observe such bypassing using single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT–FCS data shows that LacI hops one or two grooves (10–20 bp) every 200–700 μs. Our data suggest a trade-off between speed and accuracy during sliding: the weak nature of nonspecific protein–DNA interactions underlies operator bypassing, but also speeds up sliding. We anticipate that SMCT–FCS, which monitors rotational diffusion on the microsecond timescale while tracking individual molecules with millisecond resolution, will be applicable to the real-time investigation of many other biological interactions and will effectively extend the accessible time regime for observing these interactions by two orders of magnitude.
G. Bowman and S. Deindl
Remodeling the genome with DNA twists
Science 2019
In complex organisms such as humans, a single genetic blueprint can give rise to a multitude of different cell types, from nerve to liver to muscle. Such cellular diversity relies on restricting which portions of genomic DNA are accessible and therefore can be read by cellular machinery. Ultimately, access to DNA depends on placement of a repetitive, spool-like structure called the nucleosome, the basic packaging unit of chromosomes. The nucleosome occludes two tight loops of DNA and thus represents a fundamentally repressive element. When and where nucleosomes are positioned can affect complex transcriptional programs, and therefore disruptions in the factors responsible for nucleosome positioning often result in cancers and multisystem developmental diseases. Although the mechanism of shifting nucleosomes along DNA has long proved elusive, a recent flurry of structural, biophysical, and biochemical work has revealed a core mechanistic framework explaining how nucleosomes are actively repositioned throughout the genome.
In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 tisB mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In tisB and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking–immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient tisB mRNAs, we provide a thorough characterization of the tisB standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30∆S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional tisB standby site consists of the expected single-stranded region, but surprisingly, also a 5′-end stem-loop structure. Removal of the latter by 5′-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 tisB mRNA (∼100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.
A. Sabantsev, R.F. Levendosky, X. Zhuang, G.D. Bowman and S. Deindl
Direct observation of coordinated DNA movements on the nucleosome during chromatin remodelling
Nature Communications 2019
ATP-dependent chromatin remodelling enzymes (remodellers) regulate DNA accessibility in eukaryotic genomes. Many remodellers reposition (slide) nucleosomes, however, how DNA is propagated around the histone octamer during this process is unclear. Here we examine the real-time coordination of remodeller-induced DNA movements on both sides of the nucleosome using three-colour single-molecule FRET. During sliding by Chd1 and SNF2h remodellers, DNA is shifted discontinuously, with movement of entry-side DNA preceding that of exit-side DNA. The temporal delay between these movements implies a single rate-limiting step dependent on ATP binding and transient absorption or buffering of at least one base pair. High-resolution cross-linking experiments show that sliding can be achieved by buffering as few as 3 bp between entry and exit sides of the nucleosome. We propose that DNA buffering ensures nucleosome stability during ATP-dependent remodelling, and provides a means for communication between remodellers acting on opposite sides of the nucleosome.
Q. Liao, M. Lüking , D.M. Krüger , S. Deindl , J. Elf , P.M. Kasson and S.C.L. Kamerlin
Long Time-Scale Atomistic Simulations of the Structure and Dynamics of Transcription Factor-DNA Recognition

J. Phys. Chem. B 2019
Recent years have witnessed an explosion of interest in computational studies of DNA binding proteins, including both coarse grained and atomistic simulations of transcription factor-DNA recognition, in order to understand how these transcription factors recognize their binding sites on the DNA with such exquisite specificity. The present study performs μs-timescale all-atom simulations of the dimeric form of the lactose repressor (LacI), both in the absence of any DNA, and in the presence of both specific and non-specific complexes, considering three different DNA sequences. We examine, specifically, the conformational differences between specific and non-specific protein-DNA interactions, as well as the behavior of the helix-turn-helix motif of LacI when interacting with the DNA. Our simulations suggest that stable LacI binding occurs primarily to bent A-form DNA, with a loss of LacI conformational entropy and optimization of correlated conformational equilibria across the protein. In addition, binding to the specific operator sequence involves a slightly larger number of stabilizing DNA-protein hydrogen bonds (in comparison to non-specific complexes), that may account for the experimentally observed specificity for this operator. In doing so, our simulations provide a detailed atomistic description of potential structural drivers for LacI selectivity.
E. Marklund*, E. Amselem*, K. Kipper, X. Zheng, M. Johansson, S. Deindl and J. Elf
Direct observation of rotation-coupled protein diffusion along DNA on the microsecond timescale
bioRxiv 2018
Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific regions of the DNA. It is however not known how sliding proteins are oriented with respect to DNA in order to recognize specific sequences. Here we measure the polarization of fluorescence emission from single fluorescently labeled lac repressor (LacI) molecules sliding on stretched DNA. Real-time feedback-coupled confocal single-particle tracking allows us to measure fluorescence correlation of the sliding molecules. We find that the fluctuations in the fluorescence signal on the μs timescale are accurately described by rotation-coupled sliding on DNA. On average, LacI moves ~50 base pairs per revolution, which is significantly longer than the 10.5 bp helical periodicity of DNA. Our data support a facilitated diffusion model where the transcription factor (TF) scans the DNA grooves for hydrogen bonding opportunities in a pre-aligned orientation with occasional slippage out of the groove.
C. Ballet, M. S. P. Correia, L. P. Conway, T. L. Locher, L.C. Lehmann, N. Garg, M. Vujasinovic, S. Deindl, J.-M. Löhr, and D. Globisch
New enzymatic and mass spectrometric methodology for the selective investigation of gut microbiota-derived metabolites
Chemical Science 2018
Gut microbiota significantly impact human physiology through metabolic interaction. Selective investigation of the co-metabolism of bacteria and their human host is a challenging task and methods for their analysis are limited. One class of metabolites associated with this co-metabolism are O-sulfated compounds. Herein, we describe the development of a new enzymatic assay for the selective mass spectrometric investigation of this phase II modification class. Analysis of human urine and fecal samples resulted in the detection of 206 sulfated metabolites, which is three times more than reported in the Human Metabolome Database. We confirmed the chemical structure of 36 sulfated metabolites including unknown and commonly reported microbiota-derived sulfated metabolites using synthesized internal standards and mass spectrometric fragmentation experiments. Our findings demonstrate that enzymatic sample pre-treatment combined with state-of-the-art metabolomics analysis represents a new and efficient strategy for the discovery of unknown microbiota-derived metabolites in human samples. Our described approach can be adapted for the targeted investigation of other metabolite classes as well as the discovery of biomarkers for diseases affected by microbiota.
K. Kipper, N. Eremina, E. Marklund, S. Tubasum, G. Mao, L. C. Lehmann, J. Elf, and S. Deindl
Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI
PLoS One 2018
The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis-based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.
L.C. Lehmann, G. Hewitt, S. Aibara, A. Leitner, E. Marklund, S.L. Maslen, V. Maturi, Y. Chen, D. van der Spoel, J.M. Skehel, A. Moustakas, S. J. Boulton, and S. Deindl
Mechanistic Insights into Autoinhibition of the Oncogenic Chromatin Remodeler ALC1
Molecular Cell 2017
Human ALC1 is an oncogene-encoded chromatin-remodeling enzyme required for DNA repair that possesses a poly(ADP-ribose) (PAR)-binding macro domain. Its engagement with PARylated PARP1 activates ALC1 at sites of DNA damage, but the underlying mechanism remains unclear. Here, we establish a dual role for the macro domain in autoinhibition of ALC1 ATPase activity and coupling to nucleosome mobilization. In the absence of DNA damage, an inactive conformation of the ATPase is maintained by juxtaposition of the macro domain against predominantly the C-terminal ATPase lobe through conserved electrostatic interactions. Mutations within this interface displace the macro domain, constitutively activate the ALC1 ATPase independent of PARylated PARP1, and alter the dynamics of ALC1 recruitment at DNA damage sites. Upon DNA damage, binding of PARylated PARP1 by the macro domain induces a conformational change that relieves autoinhibitory interactions with the ATPase motor, which selectively activates ALC1 remodeling upon recruitment to sites of DNA damage.
R. Levendosky, A. Sabantsev, S. Deindl, G. Bowman
The Chd1 chromatin remodeler shifts hexasomes unidirectionally
eLife 2016
Despite their canonical two-fold symmetry, nucleosomes in biological contexts are often asymmetric: functionalized with post-translational modifications (PTMs), substituted with histone variants, and even lacking H2A/H2B dimers. Here we show that the Widom 601 nucleosome positioning sequence can produce hexasomes in a specific orientation on DNA, which provide a useful tool for interrogating chromatin enzymes and allow for the generation of precisely defined asymmetry in nucleosomes. Using this methodology, we demonstrate that the Chd1 chromatin remodeler from Saccharomyces cerevisiae requires H2A/H2B on the entry side for sliding, and thus, unlike the back-and-forth sliding observed for nucleosomes, Chd1 shifts hexasomes unidirectionally. Chd1 takes part in chromatin reorganization surrounding transcribing RNA polymerase II (Pol II), and using asymmetric nucleosomes we show that ubiquitin-conjugated H2B on the entry side stimulates nucleosome sliding by Chd1. We speculate that biased nucleosome and hexasome sliding due to asymmetry contributes to the packing of arrays observed in vivo.
F. Saupe, M. Reichel, E. Huijbers, J. Femel, P. Markgren, E. Andersson, S. Deindl, H. Danielsson, L. Hellman, and A. Olsson
Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously
FASEB J 2016
With the aim to improve the efficacy of therapeutic vaccines that target self-antigens, we have developed a novel fusion protein vaccine on the basis of the C-terminal multimerizing end of the variable lymphocyte receptor B (VLRB), the Ig equivalent in jawless fishes. Recombinant vaccines were produced in Escherichia coli by fusing the VLRB sequence to 4 different cancer-associated target molecules. The anti-self-immune response generated in mice that were vaccinated with VLRB vaccines was compared with the response in mice that received vaccines that contained bacterial thioredoxin (TRX), previously identified as an efficient carrier. The anti-self-Abs were analyzed with respect to titers, binding properties, and duration of response. VLRB-vaccinated mice displayed a 2- to 10-fold increase in anti-self-Ab titers and a substantial decrease in Abs against the foreign part of the fusion protein compared with the response in TRX-vaccinated mice (P < 0.01). VLRB-generated Ab response had duration similar to the corresponding TRX-generated Abs, but displayed a higher diversity in binding characteristics. Of importance, VLRB vaccines could sustain an immune response against several targets simultaneously. VLRB vaccines fulfill several key criteria for an efficient therapeutic vaccine that targets self-antigens as a result of its small size, its multimerizing capacity, and nonexposed foreign sequences in the fusion protein.
B.T. Harada, W.L. Hwang, S. Deindl, N. Chatterjee, B. Bartholomew, X. Zhuang
Stepwise nucleosome translocation by RSC remodeling complexes
eLife 2016
The SWI/SNF-family remodelers regulate chromatin structure by coupling the free energy from ATP hydrolysis to the repositioning and restructuring of nucleosomes, but how the ATPase activity of these enzymes drives the motion of DNA across the nucleosome remains unclear. Here, we used single-molecule FRET to monitor the remodeling of mononucleosomes by the yeast SWI/SNF remodeler, RSC. We observed that RSC primarily translocates DNA around the nucleosome without substantial displacement of the H2A-H2B dimer. At the sites where DNA enters and exits the nucleosome, the DNA moves largely along or near its canonical wrapping path. The translocation of DNA occurs in a stepwise manner, and at both sites where DNA enters and exits the nucleosome, the step size distributions exhibit a peak at approximately 1-2 bp. These results suggest that the movement of DNA across the nucleosome is likely coupled directly to DNA translocation by the ATPase at its binding site inside the nucleosome.
W.L. Hwang*, S. Deindl*, B.T. Harada, and X. Zhuang
Histone H4 tail mediates allosteric regulation of nucleosome remodelling by linker DNA
*equal contribution
Nature 2014
Imitation switch (ISWI)-family remodelling enzymes regulate access to genomic DNA by mobilizing nucleosomes1. These ATP-dependent chromatin remodellers promote heterochromatin formation and transcriptional silencing1 by generating regularly spaced nucleosome arrays2, 3, 4, 5. The nucleosome-spacing activity arises from the dependence of nucleosome translocation on the length of extranucleosomal linker DNA6, 7, 8, 9, 10, but the underlying mechanism remains unclear. Here we study nucleosome remodelling by human ATP-dependent chromatin assembly and remodelling factor (ACF), an ISWI enzyme comprising a catalytic subunit, Snf2h, and an accessory subunit, Acf1 (refs 2, 11, 12, 13). We find that ACF senses linker DNA length through an interplay between its accessory and catalytic subunits mediated by the histone H4 tail of the nucleosome. Mutation of AutoN, an auto-inhibitory domain within Snf2h that bears sequence homology to the H4 tail14, abolishes the linker-length sensitivity in remodelling. Addition of exogenous H4-tail peptide or deletion of the nucleosomal H4 tail also diminishes the linker-length sensitivity. Moreover, Acf1 binds both the H4-tail peptide and DNA in an amino (N)-terminal domain dependent manner, and in the ACF-bound nucleosome, lengthening the linker DNA reduces the Acf1-H4 tail proximity. Deletion of the N-terminal portion of Acf1 (or its homologue in yeast) abolishes linker-length sensitivity in remodelling and leads to severe growth defects in vivo. Taken together, our results suggest a mechanism for nucleosome spacing where linker DNA sensing by Acf1 is allosterically transmitted to Snf2h through the H4 tail of the nucleosome. For nucleosomes with short linker DNA, Acf1 preferentially binds to the H4 tail, allowing AutoN to inhibit the ATPase activity of Snf2h. As the linker DNA lengthens, Acf1 shifts its binding preference to the linker DNA, freeing the H4 tail to compete AutoN off the ATPase and thereby activating ACF.
S. Deindl, W.L. Hwang, S.K. Hota, T.R. Blosser, P. Prasad, B. Bartholomew, and X. Zhuang
ISWI remodelers slide nucleosomes with coordinated multi-base-pair entry steps and single-base-pair exit steps
Cell 2013
ISWI-family enzymes remodel chromatin by sliding nucleosomes along DNA, but the nucleosome translocation mechanism remains unclear. Here we use single-molecule FRET to probe nucleosome translocation by ISWI-family remodelers. Distinct ISWI-family members translocate nucleosomes with a similar stepping pattern maintained by the catalytic subunit of the enzyme. Nucleosome remodeling begins with a 7 bp step of DNA translocation followed by 3 bp subsequent steps toward the exit side of nucleosomes. These multi-bp, compound steps are comprised of 1 bp substeps. DNA movement on the entry side of the nucleosome occurs only after 7 bp of exit-side translocation, and each entry-side step draws in a 3 bp equivalent of DNA that allows three additional base pairs to be moved to the exit side. Our results suggest a remodeling mechanism with well-defined coordination at different nucleosomal sites featuring DNA translocation toward the exit side in 1 bp steps preceding multi-bp steps of DNA movement on the entry side.
Q. Yan, T. Barros, P.R. Visperas, S. Deindl, T.A. Kadlecek, A. Weiss, and J. Kuriyan
Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker
Molecular and Cellular Biology 2013
Serial activation of the tyrosine kinases Lck and ZAP-70 initiates signaling downstream of the T cell receptor. We previously reported the structure of an autoinhibited ZAP-70 variant in which two regulatory tyrosine residues (315 and 319) in the SH2-kinase linker were replaced by phenylalanine. We now present a crystal structure of ZAP-70 in which Tyr 315 and Tyr 319 are not mutated, leading to the recognition of a five-residue sequence register error in the SH2-kinase linker of the original crystallographic model. The revised model identifies distinct roles for these two tyrosines. As seen in a recently reported structure of the related tyrosine kinase Syk, Tyr 315 of ZAP-70 is part of a hydrophobic interface between the regulatory apparatus and the kinase domain, and the integrity of this interface would be lost upon engagement of doubly phosphorylated peptides by the SH2 domains. Tyr 319 is not necessarily dislodged by SH2 engagement, which activates ZAP-70 only ∼5-fold in vitro. In contrast, phosphorylation by Lck activates ZAP-70 ∼100-fold. This difference is due to the ability of Tyr 319 to suppress ZAP-70 activity even when the SH2 domains are dislodged from the kinase domain, providing stringent control of ZAP-70 activity downstream of Lck.
S.K. Hota, S.K. Bhardwaj, S. Deindl, Y. Lin, X. Zhuang, and B. Bartholomew
Nucleosome mobilization by ISW2 requires the concerted action of the ATPase and SLIDE domains
Nature Structural & Molecular Biology 2013
The ISWI family of ATP-dependent chromatin remodelers represses transcription by changing nucleosome positions. ISWI regulates nucleosome positioning by requiring a minimal length of extranucleosomal DNA for moving nucleosomes. ISW2 from Saccharomyces cerevisiae, a member of the ISWI family, has a conserved domain called SLIDE (SANT-like ISWI domain) that binds to extranucleosomal DNA ~19 base pairs from the edge of nucleosomes. Loss of SLIDE binding does not perturb binding of the ATPase domain or the initial movement of DNA inside of nucleosomes. Not only is extranucleosomal DNA required to help recruit ISW2, but also the interactions of the SLIDE domain with extranucleosomal DNA are functionally required to move nucleosomes.
Due to its ability to track distance changes within individual molecules or molecular complexes on the nanometer scale and in real time, single-molecule fluorescence resonance energy transfer (single-molecule FRET) is a powerful tool to tackle a wide range of important biological questions. Using our recently developed single-molecule FRET assay to monitor nucleosome translocation as an illustrative example, we describe here in detail how to set up, carry out, and analyze single-molecule FRET experiments that provide time-dependent information on biomolecular processes.
L.H. Chao, P. Pellicena, S. Deindl, L.A. Barclay, H. Schulman, and J. Kuriyan
Inter-subunit capture of regulatory segments is a component of cooperative CaMKII activation
Nature Structural & Molecular Biology 2010
The dodecameric holoenzyme of calcium–calmodulin-dependent protein kinase II (CaMKII) responds to high-frequency Ca2+ pulses to become Ca2+ independent. A simple coincidence-detector model for Ca2+-frequency dependency assumes noncooperative activation of kinase domains. We show that activation of CaMKII by Ca2+–calmodulin is cooperative, with a Hill coefficient of ~3.0, implying sequential kinase-domain activation beyond dimeric units. We present data for a model in which cooperative activation includes the intersubunit 'capture' of regulatory segments. Such a capture interaction is seen in a crystal structure that shows extensive contacts between the regulatory segment of one kinase and the catalytic domain of another. These interactions are mimicked by a natural inhibitor of CaMKII. Our results show that a simple coincidence-detection model cannot be operative and point to the importance of kinetic dissection of the frequency-response mechanism in future experiments.
N. Jura, N.F. Endres, K. Engel, S. Deindl, X. Zhang, and J. Kuriyan
Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment
Cell 2009
Signaling by the epidermal growth factor receptor requires an allosteric interaction between the kinase domains of two receptors, whereby one activates the other. We show that the intracellular juxtamembrane segment of the receptor, known to potentiate kinase activity, is able to dimerize the kinase domains. The C-terminal half of the juxtamembrane segment latches the activated kinase domain to the activator, and the N-terminal half of this segment further potentiates dimerization, most likely by forming an antiparallel helical dimer that engages the transmembrane helices of the activated receptor. Our data are consistent with a mechanism in which the extracellular domains block the intrinsic ability of the transmembrane and cytoplasmic domains to dimerize and activate, with ligand binding releasing this block. The formation of the activating juxtamembrane latch is prevented by the C-terminal tails in a structure of an inactive kinase domain dimer, suggesting how alternative dimers can prevent ligand-independent activation.
B.B. Au-Yeung, S. Deindl, H. Lih-Yun, E.H. Palacios, S.E. Levin, J. Kuriyan, and A. Weiss
The structure, regulation, and function of ZAP-70
Immunological Reviews 2009
The tyrosine ZAP-70 (ζ-associated protein of 70 kDa) kinase plays a critical role in activating many downstream signal transduction pathways in T cells following T-cell receptor (TCR) engagement. The importance of ZAP-70 is evidenced by the severe combined immunodeficiency that occurs in ZAP-70-deficient mice and humans. In this review, we describe recent analyses of the ZAP-70 crystal structure, revealing a complex regulatory mechanism of ZAP-70 activity, the differential requirements for ZAP-70 and spleen tyrosine kinase (SyK) in early T-cell development, as well as the role of ZAP-70 in chronic lymphocytic leukemia and autoimmunity. Thus, the critical importance of ZAP-70 in TCR signaling and its predominantly T-cell-restricted expression pattern make ZAP-70 an attractive drug target for the inhibition of pathological T-cell responses in disease.
The delivery of signals from the activated T cell antigen receptor (TCR) inside the cell relies on the protein tyrosine kinase ZAP-70 (ζ-associated protein of 70 kDa). A recent crystal structure of inactive full-length ZAP-70 suggests that a central interface formed by the docking of the two SH2 domains of ZAP-70 onto the kinase domain is crucial for suppressing catalytic activity. Here we validate the significance of this autoinhibitory interface for the regulation of ZAP-70 catalytic activity and the T cell response. For this purpose, we perform in vitro catalytic activity assays and binding experiments using ZAP-70 proteins purified from insect cells to examine activation of ZAP-70. Furthermore, we use cell lines stably expressing wild-type or mutant ZAP-70 to monitor proximal events in T cell signaling, including TCR-induced phosphorylation of ZAP-70 substrates, activation of the MAP kinase pathway, and intracellular Ca2+ levels. Taken together, our results directly correlate the stability of the autoinhibitory interface with the activation of these key events in the T cell response.
S. Deindl, T.A. Kadlecek, T. Brdicka, X. Cao, A. Weiss and J. Kuriyan
Structural basis for the inhibition of tyrosine kinase activity of ZAP-70
Cell 2007
ZAP-70, a cytoplasmic tyrosine kinase required for T cell antigen receptor signaling, is controlled by a regulatory segment that includes a tandem SH2 unit responsible for binding to immunoreceptor tyrosine-based activation motifs (ITAMs). The crystal structure of autoinhibited ZAP-70 reveals that the inactive kinase domain adopts a conformation similar to that of cyclin-dependent kinases and Src kinases. The autoinhibitory mechanism of ZAP-70 is, however, distinct and involves interactions between the regulatory segment and the hinge region of the kinase domain that reduce its flexibility. Two tyrosine residues in the SH2-kinase linker that activate ZAP-70 when phosphorylated are involved in aromatic-aromatic interactions that connect the linker to the kinase domain. These interactions are inconsistent with ITAM binding, suggesting that destabilization of this autoinhibited ZAP-70 conformation is the first step in kinase activation.
O.S. Rosenberg, S. Deindl, L.R. Comolli, A. Hoelz, K.H. Downing, A.C. Nairn, and J. Kuriyan
Oligomerization states of the association domain and the holoenyzme of Ca2+/CaM kinase II
FEBS J 2006
Ca2+/calmodulin activated protein kinase II (CaMKII) is an oligomeric protein kinase with a unique holoenyzme architecture. The subunits of CaMKII are bound together into the holoenzyme by the association domain, a C-terminal region of ≈ 140 residues in the CaMKII polypeptide. Single particle analyses of electron micrographs have suggested previously that the holoenyzme forms a dodecamer that contains two stacked 6-fold symmetric rings. In contrast, a recent crystal structure of the isolated association domain of mouse CaMKIIα has revealed a tetradecameric assembly with two stacked 7-fold symmetric rings. In this study, we have determined the crystal structure of theCaenorhabditis elegans CaMKII association domain and it too forms a tetradecamer. We also show by electron microscopy that in its fully assembled form the CaMKII holoenzyme is a dodecamer but without the kinase domains, either from expression of the isolated association domain in bacteria or following their removal by proteolysis, the association domains form a tetradecamer. We speculate that the holoenzyme is held in its 6-fold symmetric state by the interactions of the N-terminal ≈ 1–335 residues and that the removal of this region allows the association domain to convert into a more stable 7-fold symmetric form.
O.S. Rosenberg, S. Deindl, R.J. Sung, A.C. Nairn, J. Kuriyan
Structure of the autoinhibited kinase domain of CaMKII and SAXS analysis of the holoenzyme
Cell 2005
Ca2+/calmodulin-dependent protein kinase-II (CaMKII) is unique among protein kinases for its dodecameric assembly and its complex response to Ca2+. The crystal structure of the autoinhibited kinase domain of CaMKII, determined at 1.8 Å resolution, reveals an unexpected dimeric organization in which the calmodulin-responsive regulatory segments form a coiled-coil strut that blocks peptide and ATP binding to the otherwise intrinsically active kinase domains. A threonine residue in the regulatory segment, which when phosphorylated renders CaMKII calmodulin independent, is held apart from the catalytic sites by the organization of the dimer. This ensures a strict Ca2+ dependence for initial activation. The structure of the kinase dimer, when combined with small-angle X-ray scattering data for the holoenzyme, suggests that inactive CaMKII forms tightly packed autoinhibited assemblies that convert upon activation into clusters of loosely tethered and independent kinase domains.
M. Schütt, S.S. Krupka, A.G. Milbradt, S. Deindl, E.K. Sinner, D. Oesterhelt, C. Renner, L. Moroder
Photocontrol of cell adhesion processes: model studies with cyclic azobenzene-RGD peptides
Chemistry & Biology 2003
A photoresponsive integrin ligand was synthesized by backbone-cyclization of a heptapeptide containing the integrin binding motif Arg-Gly-Asp (RGD) with 4-(aminomethyl)phenylazobenzoic acid (AMPB). Surface plasmon enhanced fluorescence spectroscopy showed that binding of the azobenzene peptide to αvβ3 integrin depends on the photoisomeric state of the peptide chromophore. The higher affinity of the transisomer could be rationalized by comparing the NMR conformations of the cis and transisomers with the recently solved X-ray structure of a cyclic RGD-pentapeptide bound to integrin.
Selected publications
A. Sabantsev, G. Mao, J. Aguirre Rivera, M. Panfilov, A. Arseniev, O. Ho, M. Khodorkovskiy, S. Deindl
Nature Chemical Biology (2022)
Summary

Many essential processes in the cell depend on proteins that use nucleoside triphosphates (NTPs). Methods that directly monitor the often-complex dynamics of these proteins at the single-molecule level have helped to uncover their mechanisms of action. However, the measurement throughput is typically limited for NTP-utilizing reactions, and the quantitative dissection of complex dynamics over multiple sequential turnovers remains challenging. Here we present a method for controlling NTP-driven reactions in single-molecule experiments via the local generation of NTPs (LAGOON) that markedly increases the measurement throughput and enables single-turnover observations. We demonstrate the effectiveness of LAGOON in single-molecule fluorescence and force spectroscopy assays by monitoring DNA unwinding, nucleosome sliding and RNA polymerase elongation. LAGOON can be readily integrated with many single-molecule techniques, and we anticipate that it will facilitate studies of a wide range of crucial NTP-driven processes.

See also the accompanying News & Views article Very fast nucleotides on demand

E. Marklund, G. Mao, J. Yan, S. Zikrin, E. Abdurakhmanov, S. Deindl*, J. Elf*
Science (2022)
Summary

Sequence-specific binding of proteins to DNA is essential for accessing genetic information. We derive a model that predicts an anticorrelation between the macroscopic association and dissociation rates of DNA binding proteins. We tested the model for thousands of different lac operator sequences with a protein binding microarray and by observing kinetics for individual lac repressor molecules in single-molecule experiments. We found that sequence specificity is mainly governed by the efficiency with which the protein recognizes different targets. The variation in probability of recognizing different targets is at least 1.7 times as large as the variation in microscopic dissociation rates. Modulating the rate of binding instead of the rate of dissociation effectively reduces the risk of the protein being retained on nontarget sequences while searching.
L. Bacic, G. Gaullier, A. Sabantsev, L. C. Lehmann, K. Brackmann, D. Dimakou, M. Halic, G. Hewitt, S. J. Boulton, S. Deindl
eLife (2021)
Summary

The chromatin remodeler ALC1 is recruited to and activated by DNA damage-induced poly(ADP-ribose) (PAR) chains deposited by PARP1/PARP2/HPF1 upon detection of DNA lesions. ALC1 has emerged as a candidate drug target for cancer therapy as its loss confers synthetic lethality in homologous recombination-deficient cells. However, structure-based drug design and molecular analysis of ALC1 have been hindered by the requirement for PARylation and the highly heterogeneous nature of this post-translational modification. Here, we reconstituted an ALC1 and PARylated nucleosome complex modified in vitro using PARP2 and HPF1. This complex was amenable to cryo-EM structure determination without cross-linking, which enabled visualization of several intermediate states of ALC1 from the recognition of the PARylated nucleosome to the tight binding and activation of the remodeler. Functional biochemical assays with PARylated nucleosomes highlight the importance of nucleosomal epitopes for productive remodeling and suggest that ALC1 preferentially slides nucleosomes away from DNA breaks.
I. Regadas, O. Dahlberg, R. Vaid, O. Ho, S. Belikov, G. Dixit, S. Deindl, J. Wen, M. Mannervik
Molecular Cell (2021)
Summary

Organismal development and cell differentiation critically depend on chromatin state transitions. However, certain developmentally regulated genes lack histone 3 lysine 9 and 27 acetylation (H3K9ac and H3K27ac, respectively) and histone 3 lysine 4 (H3K4) methylation, histone modifications common to most active genes. Here we describe a chromatin state featuring unique histone 3 lysine 14 acetylation (H3K14ac) peaks in key tissue-specific genes in Drosophila and human cells. Replacing H3K14 in Drosophila demonstrates that H3K14 is essential for expression of genes devoid of canonical histone modifications in the embryonic gut and larval wing imaginal disc, causing lethality and defective wing patterning. We find that the SWI/SNF protein Brahma (Brm) recognizes H3K14ac, that brm acts in the same genetic pathway as H3K14R, and that chromatin accessibility at H3K14ac-unique genes is decreased in H3K14R mutants. Our results show that acetylation of a single lysine is essential at genes devoid of canonical histone marks and uncover an important requirement for H3K14 in tissue-specific gene regulation.

G. Hewitt, V. Borel, S. Segura-Bayona, T. Takaki, P. Ruis, R. Bellelli, L. C. Lehmann, L. Sommerova, A. Vancevska, A. Tomas-Loba, K. Zhu, C. Cooper, K. Fugger, H. Patel, R. Goldstone, D. Schneider-Luftman, E. Herbert, G. Stamp, R. Brough, S. Pettitt, C. J. Lord, S. C. West, I. Ahel, D. Ahel, J. R. Chapman, S. Deindl , S. J. Boulton
Molecular Cell (2021)
Summary

Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.

L. Bacic, A. Sabantsev, and S. Deindl
Current Opinion in Structural Biology (2020)
Summary

Single-molecule fluorescence microscopy has long been appreciated as a powerful tool to study the structural dynamics that enable biological function of macromolecules. Recent years have witnessed the development of more complex single-molecule fluorescence techniques as well as powerful combinations with structural approaches to obtain mechanistic insights into the workings of various molecular machines and protein complexes. In this review, we highlight these developments that together bring us one step closer to a dynamic understanding of biological processes in atomic details.
E. Marklund, B. van Oosten, G. Mao, E. Amselem, K. Kipper, A. Sabantsev, A. Emmerich, D. Globisch, X. Zheng, L. C. Lehmann, O. Berg, M. Johansson, J. Elf, S. Deindl
Nature (2020)
Summary

Many proteins that bind specific DNA sequences search the genome by combining three-dimensional diffusion with one-dimensional sliding on nonspecific DNA. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore the DNA surface during the one-dimensional phase of target search. To track the rotation of sliding LacI molecules on the microsecond timescale, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT–FCS). The fluctuations in fluorescence signal are accurately described by rotation-coupled sliding, in which LacI traverses about 40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA; this suggests that the sliding protein frequently hops out of the DNA groove, which would result in the frequent bypassing of target sequences. We directly observe such bypassing using single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT–FCS data shows that LacI hops one or two grooves (10–20 bp) every 200–700 μs. Our data suggest a trade-off between speed and accuracy during sliding: the weak nature of nonspecific protein–DNA interactions underlies operator bypassing, but also speeds up sliding. We anticipate that SMCT–FCS, which monitors rotational diffusion on the microsecond timescale while tracking individual molecules with millisecond resolution, will be applicable to the real-time investigation of many other biological interactions and will effectively extend the accessible time regime for observing these interactions by two orders of magnitude.
G. Bowman and S. Deindl
Science (2019)
Summary

In complex organisms such as humans, a single genetic blueprint can give rise to a multitude of different cell types, from nerve to liver to muscle. Such cellular diversity relies on restricting which portions of genomic DNA are accessible and therefore can be read by cellular machinery. Ultimately, access to DNA depends on placement of a repetitive, spool-like structure called the nucleosome, the basic packaging unit of chromosomes. The nucleosome occludes two tight loops of DNA and thus represents a fundamentally repressive element. When and where nucleosomes are positioned can affect complex transcriptional programs, and therefore disruptions in the factors responsible for nucleosome positioning often result in cancers and multisystem developmental diseases. Although the mechanism of shifting nucleosomes along DNA has long proved elusive, a recent flurry of structural, biophysical, and biochemical work has revealed a core mechanistic framework explaining how nucleosomes are actively repositioned throughout the genome.
Summary

In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 tisB mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In tisB and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking–immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient tisB mRNAs, we provide a thorough characterization of the tisB standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30∆S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional tisB standby site consists of the expected single-stranded region, but surprisingly, also a 5′-end stem-loop structure. Removal of the latter by 5′-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 tisB mRNA (∼100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.
A. Sabantsev, R.F. Levendosky, X. Zhuang, G.D. Bowman and S. Deindl
Nature Communications 10 (2019)
Summary

ATP-dependent chromatin remodelling enzymes (remodellers) regulate DNA accessibility in eukaryotic genomes. Many remodellers reposition (slide) nucleosomes, however, how DNA is propagated around the histone octamer during this process is unclear. Here we examine the real-time coordination of remodeller-induced DNA movements on both sides of the nucleosome using three-colour single-molecule FRET. During sliding by Chd1 and SNF2h remodellers, DNA is shifted discontinuously, with movement of entry-side DNA preceding that of exit-side DNA. The temporal delay between these movements implies a single rate-limiting step dependent on ATP binding and transient absorption or buffering of at least one base pair. High-resolution cross-linking experiments show that sliding can be achieved by buffering as few as 3 bp between entry and exit sides of the nucleosome. We propose that DNA buffering ensures nucleosome stability during ATP-dependent remodelling, and provides a means for communication between remodellers acting on opposite sides of the nucleosome.
L.C. Lehmann, G. Hewitt, S. Aibara, A. Leitner, E. Marklund, S.L. Maslen, V. Maturi, Y. Chen, D. van der Spoel, J.M. Skehel, A. Moustakas, S. J. Boulton and S. Deindl
Molecular Cell 68 (2017)
Summary

Human ALC1 is an oncogene-encoded chromatin-remodeling enzyme required for DNA repair that possesses a poly(ADP-ribose) (PAR)-binding macro domain. Its engagement with PARylated PARP1 activates ALC1 at sites of DNA damage, but the underlying mechanism remains unclear. Here, we establish a dual role for the macro domain in autoinhibition of ALC1 ATPase activity and coupling to nucleosome mobilization. In the absence of DNA damage, an inactive conformation of the ATPase is maintained by juxtaposition of the macro domain against predominantly the C-terminal ATPase lobe through conserved electrostatic interactions. Mutations within this interface displace the macro domain, constitutively activate the ALC1 ATPase independent of PARylated PARP1, and alter the dynamics of ALC1 recruitment at DNA damage sites. Upon DNA damage, binding of PARylated PARP1 by the macro domain induces a conformational change that relieves autoinhibitory interactions with the ATPase motor, which selectively activates ALC1 remodeling upon recruitment to sites of DNA damage.

W.L. Hwang*, S. Deindl*, B.T. Harada and X. Zhuang
*equal contribution
Nature 512 (2014)
Summary

Imitation switch (ISWI)-family remodelling enzymes regulate access to genomic DNA by mobilizing nucleosomes1. These ATP-dependent chromatin remodellers promote heterochromatin formation and transcriptional silencing1 by generating regularly spaced nucleosome arrays2, 3, 4, 5. The nucleosome-spacing activity arises from the dependence of nucleosome translocation on the length of extranucleosomal linker DNA6, 7, 8, 9, 10, but the underlying mechanism remains unclear. Here we study nucleosome remodelling by human ATP-dependent chromatin assembly and remodelling factor (ACF), an ISWI enzyme comprising a catalytic subunit, Snf2h, and an accessory subunit, Acf1 (refs 2, 11, 12, 13). We find that ACF senses linker DNA length through an interplay between its accessory and catalytic subunits mediated by the histone H4 tail of the nucleosome. Mutation of AutoN, an auto-inhibitory domain within Snf2h that bears sequence homology to the H4 tail14, abolishes the linker-length sensitivity in remodelling. Addition of exogenous H4-tail peptide or deletion of the nucleosomal H4 tail also diminishes the linker-length sensitivity. Moreover, Acf1 binds both the H4-tail peptide and DNA in an amino (N)-terminal domain dependent manner, and in the ACF-bound nucleosome, lengthening the linker DNA reduces the Acf1-H4 tail proximity. Deletion of the N-terminal portion of Acf1 (or its homologue in yeast) abolishes linker-length sensitivity in remodelling and leads to severe growth defects in vivo. Taken together, our results suggest a mechanism for nucleosome spacing where linker DNA sensing by Acf1 is allosterically transmitted to Snf2h through the H4 tail of the nucleosome. For nucleosomes with short linker DNA, Acf1 preferentially binds to the H4 tail, allowing AutoN to inhibit the ATPase activity of Snf2h. As the linker DNA lengthens, Acf1 shifts its binding preference to the linker DNA, freeing the H4 tail to compete AutoN off the ATPase and thereby activating ACF.
S. Deindl, W.L. Hwang, S.K. Hota, T.R. Blosser, P. Prasad, B. Bartholomew and X. Zhuang
Cell 152 (2013)
Summary

ISWI-family enzymes remodel chromatin by sliding nucleosomes along DNA, but the nucleosome translocation mechanism remains unclear. Here we use single-molecule FRET to probe nucleosome translocation by ISWI-family remodelers. Distinct ISWI-family members translocate nucleosomes with a similar stepping pattern maintained by the catalytic subunit of the enzyme. Nucleosome remodeling begins with a 7 bp step of DNA translocation followed by 3 bp subsequent steps toward the exit side of nucleosomes. These multi-bp, compound steps are comprised of 1 bp substeps. DNA movement on the entry side of the nucleosome occurs only after 7 bp of exit-side translocation, and each entry-side step draws in a 3 bp equivalent of DNA that allows three additional base pairs to be moved to the exit side. Our results suggest a remodeling mechanism with well-defined coordination at different nucleosomal sites featuring DNA translocation toward the exit side in 1 bp steps preceding multi-bp steps of DNA movement on the entry side.

S. Deindl, T.A. Kadlecek, T. Brdicka, X. Cao, A. Weiss and J. Kuriyan
Cell 129 (2007)
Summary

ZAP-70, a cytoplasmic tyrosine kinase required for T cell antigen receptor signaling, is controlled by a regulatory segment that includes a tandem SH2 unit responsible for binding to immunoreceptor tyrosine-based activation motifs (ITAMs). The crystal structure of autoinhibited ZAP-70 reveals that the inactive kinase domain adopts a conformation similar to that of cyclin-dependent kinases and Src kinases. The autoinhibitory mechanism of ZAP-70 is, however, distinct and involves interactions between the regulatory segment and the hinge region of the kinase domain that reduce its flexibility. Two tyrosine residues in the SH2-kinase linker that activate ZAP-70 when phosphorylated are involved in aromatic-aromatic interactions that connect the linker to the kinase domain. These interactions are inconsistent with ITAM binding, suggesting that destabilization of this autoinhibited ZAP-70 conformation is the first step in kinase activation.

O.S. Rosenberg, S. Deindl, R.J. Sung, A.C. Nairn, J. Kuriyan
Cell 123 (2005)
Summary

Ca2+/calmodulin-dependent protein kinase-II (CaMKII) is unique among protein kinases for its dodecameric assembly and its complex response to Ca2+. The crystal structure of the autoinhibited kinase domain of CaMKII, determined at 1.8 Å resolution, reveals an unexpected dimeric organization in which the calmodulin-responsive regulatory segments form a coiled-coil strut that blocks peptide and ATP binding to the otherwise intrinsically active kinase domains. A threonine residue in the regulatory segment, which when phosphorylated renders CaMKII calmodulin independent, is held apart from the catalytic sites by the organization of the dimer. This ensures a strict Ca2+ dependence for initial activation. The structure of the kinase dimer, when combined with small-angle X-ray scattering data for the holoenzyme, suggests that inactive CaMKII forms tightly packed autoinhibited assemblies that convert upon activation into clusters of loosely tethered and independent kinase domains.
Summary

The delivery of signals from the activated T cell antigen receptor (TCR) inside the cell relies on the protein tyrosine kinase ZAP-70 (ζ-associated protein of 70 kDa). A recent crystal structure of inactive full-length ZAP-70 suggests that a central interface formed by the docking of the two SH2 domains of ZAP-70 onto the kinase domain is crucial for suppressing catalytic activity. Here we validate the significance of this autoinhibitory interface for the regulation of ZAP-70 catalytic activity and the T cell response. For this purpose, we perform in vitro catalytic activity assays and binding experiments using ZAP-70 proteins purified from insect cells to examine activation of ZAP-70. Furthermore, we use cell lines stably expressing wild-type or mutant ZAP-70 to monitor proximal events in T cell signaling, including TCR-induced phosphorylation of ZAP-70 substrates, activation of the MAP kinase pathway, and intracellular Ca2+ levels. Taken together, our results directly correlate the stability of the autoinhibitory interface with the activation of these key events in the T cell response.
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