Selected publications
E. Marklund*, E. Amselem*, K. Kipper, X. Zheng, M. Johansson, S. Deindl**, J. Elf**
Direct observation of rotation-coupled protein diffusion along DNA on the microsecond timescale
bioRxiv 2018, DOI: http://dx.doi.org/10.1101/401414
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 68 (2017), pp. 847–859
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 512 (2014), pp. 213-217
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 152 (2013), pp. 442-52
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 129 (2007), pp.735-746
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 123 (2005), pp. 849-860

S. Deindl, T.A. Kadlecek, X. Cao, J. Kuriyan and A. Weiss
Stability of an autoinhibitory interface in the structure of the tyrosine kinase ZAP-70 impacts T cell receptor response
PNAS 106 (2009), pp. 20699-704
Complete list of publications
E. Marklund*, E. Amselem*, K. Kipper, X. Zheng, M. Johansson, S. Deindl**, J. Elf**
Direct observation of rotation-coupled protein diffusion along DNA on the microsecond timescale
bioRxiv 2018, DOI: http://dx.doi.org/10.1101/401414
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, DOI: 10.1039/C8SC01502C
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 13 (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 68 (2017), pp. 847–859
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), Dec 29;5
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), Dec 19 [Epub ahead of print]
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), Feb 19;5
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 512 (2014), pp. 213-217
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 152 (2013), pp. 442-52
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 33 (2013), pp. 2188-201
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 20 (2013), pp. 222-9
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 17 (2010), pp. 264-72
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 137 (2009), pp. 1293-307
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 228 (2009), pp. 41-57
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.
S. Deindl, T.A. Kadlecek, X. Cao, J. Kuriyan, and A. Weiss
Stability of an autoinhibitory interface in the structure of the tyrosine kinase ZAP-70 impacts T cell receptor response
PNAS 106 (2009), pp. 20699-704
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.
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 273 (2006), pp. 682-694
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.
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 129 (2007), pp.735-746
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
Structure of the autoinhibited kinase domain of CaMKII and SAXS analysis of the holoenzyme
Cell 123 (2005), pp. 849-860
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 10 (2003), pp. 487-490
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
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), pp. 847–859
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), pp. 213-217
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), pp. 442-52
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), pp.735-746
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), pp. 849-860
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.
S. Deindl, T.A. Kadlecek, X. Cao, J. Kuriyan and A. Weiss
PNAS 106 (2009), pp. 20699-704
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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