scholarly journals Structural basis of ALC1/CHD1L autoinhibition and the mechanism of activation by the nucleosome

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Li Wang ◽  
Kangjing Chen ◽  
Zhucheng Chen
Keyword(s):  
Crystal Structure ◽  
Liver Cancer ◽  
Chromatin Remodeling ◽  
Structural Basis ◽  
Chromatin Remodeler ◽  
Macro Domain ◽  
Regulation Mechanisms ◽  
Shed Light ◽  
The Way

AbstractChromatin remodeler ALC1 (amplification in liver cancer 1) is crucial for repairing damaged DNA. It is autoinhibited and activated by nucleosomal epitopes. However, the mechanisms by which ALC1 is regulated remain unclear. Here we report the crystal structure of human ALC1 and the cryoEM structure bound to the nucleosome. The structure shows the macro domain of ALC1 binds to lobe 2 of the ATPase motor, sequestering two elements for nucleosome recognition, explaining the autoinhibition mechanism of the enzyme. The H4 tail competes with the macro domain for lobe 2-binding, explaining the requirement for this nucleosomal epitope for ALC1 activation. A dual-arginine-anchor motif of ALC1 recognizes the acidic pocket of the nucleosome, which is critical for chromatin remodeling in vitro. Together, our findings illustrate the structures of ALC1 and shed light on its regulation mechanisms, paving the way for the discovery of drugs targeting ALC1 for the treatment of cancer.

scholarly journals Structural basis of ALC1/CHD1L autoinhibition and the mechanism of activation by the nucleosome

2021 ◽  
Author(s):  
zhucheng chen ◽  
li wang ◽  
kangjing chen
Keyword(s):  
Crystal Structure ◽  
Liver Cancer ◽  
Chromatin Remodeling ◽  
Structural Basis ◽  
Chromatin Remodeler ◽  
Macro Domain ◽  
Regulation Mechanisms ◽  
Shed Light ◽  
The Way

Chromatin remodeler ALC1 (amplification in liver cancer 1) is crucial for repairing damaged DNA. It is autoinhibited and activated by nucleosomal epitopes. However, the mechanisms by which ALC1 is regulated remain unclear. Here we report the crystal structure of human ALC1 and the cryoEM structure bound to the nucleosome. The structure shows the macro domain of ALC1 binds to lobe 2 of the ATPase motor, sequestering two elements for nucleosome recognition, explaining the autoinhibition mechanism of the enzyme. The H4 tail competes with the macro domain for lobe 2-binding, explaining the requirement for this nucleosomal epitope for ALC1 activation. A dual-arginine-anchor motif of ALC1 recognizes the acidic pocket of the nucleosome, which is critical for chromatin remodeling in vitro. Together, our findings illustrate the structures of ALC1 and shed light on its regulation mechanisms, paving the way for the discovery of drugs targeting ALC1 for the treatment of cancer.

scholarly journals Structural basis of Naa20 activity towards a canonical NatB substrate

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Dominik Layer ◽  
Jürgen Kopp ◽  
Miriam Fontanillo ◽  
Maja Köhn ◽  
Karine Lapouge ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Drug Development ◽  
Catalytic Mechanism ◽  
Peptide Binding ◽  
Competitive Inhibitor ◽  
Structural Basis ◽  
Substrate Affinity ◽  
Protein Homeostasis ◽  
Auxiliary Subunit

AbstractN-terminal acetylation is one of the most common protein modifications in eukaryotes and is carried out by N-terminal acetyltransferases (NATs). It plays important roles in protein homeostasis, localization, and interactions and is linked to various human diseases. NatB, one of the major co-translationally active NATs, is composed of the catalytic subunit Naa20 and the auxiliary subunit Naa25, and acetylates about 20% of the proteome. Here we show that NatB substrate specificity and catalytic mechanism are conserved among eukaryotes, and that Naa20 alone is able to acetylate NatB substrates in vitro. We show that Naa25 increases the Naa20 substrate affinity, and identify residues important for peptide binding and acetylation activity. We present the first Naa20 crystal structure in complex with the competitive inhibitor CoA-Ac-MDEL. Our findings demonstrate how Naa20 binds its substrates in the absence of Naa25 and support prospective endeavors to derive specific NAT inhibitors for drug development.

scholarly journals Distinct Subregions of Swi1 Manifest Striking Differences in Prion Transmission and SWI/SNF Function

2010 ◽  
Vol 30 (19) ◽  
pp. 4644-4655 ◽  
Author(s):  
Zhiqiang Du ◽  
Emily T. Crow ◽  
Hyun Seok Kang ◽  
Liming Li
Keyword(s):  
Chromatin Remodeling ◽  
De Novo ◽  
Amino Acid Residues ◽  
Coding Region ◽  
Conformational Switch ◽  
Amyloid Fibers ◽  
Aggregation Pattern ◽  
Prion Transmission ◽  
Shed Light

ABSTRACT We have recently reported that the yeast chromatin-remodeling factor Swi1 can exist as a prion, [SWI +], demonstrating a link between prionogenesis and global transcriptional regulation. To shed light on how the Swi1 conformational switch influences Swi1 function and to define the sequence and structural requirements for [SWI +] formation and propagation, we functionally dissected the Swi1 molecule. We show here that the [SWI +] prion features are solely attributable to the first 327 amino acid residues (N), a region that is asparagine rich. N was aggregated in [SWI+ ] cells but diffuse in [swi− ] cells; chromosomal deletion of the N-coding region resulted in [SWI +] loss, and recombinant N peptide was able to form infectious amyloid fibers in vitro, enabling [SWI +] de novo formation through a simple transformation. Although the glutamine-rich middle region (Q) was not sufficient to aggregate in [SWI +] cells or essential for SWI/SNF function, it significantly modified the Swi1 aggregation pattern and Swi1 function. We also show that excessive Swi1 incurred Li+/Na+ sensitivity and that the N/Q regions are important for this gain of sensitivity. Taken together, our results provide the final proof of “protein-only” transmission of [SWI +] and demonstrate that the widely distributed “dispensable” glutamine/asparagine-rich regions/motifs might have important and divergent biological functions.

scholarly journals Structural analysis reveals features of the spindle checkpoint kinase Bub1–kinetochore subunit Knl1 interaction

2012 ◽  
Vol 196 (4) ◽  
pp. 451-467 ◽  
Author(s):  
Veronica Krenn ◽  
Annemarie Wehenkel ◽  
Xiaozheng Li ◽  
Stefano Santaguida ◽  
Andrea Musacchio
Keyword(s):  
Crystal Structure ◽  
Structural Analysis ◽  
Convex Surface ◽  
Point Mutations ◽  
Tetratricopeptide Repeats ◽  
Checkpoint Protein ◽  
Checkpoint Kinases ◽  
Shed Light

The function of the essential checkpoint kinases Bub1 and BubR1 requires their recruitment to mitotic kinetochores. Kinetochore recruitment of Bub1 and BubR1 is proposed to rely on the interaction of the tetratricopeptide repeats (TPRs) of Bub1 and BubR1 with two KI motifs in the outer kinetochore protein Knl1. We determined the crystal structure of the Bub1 TPRs in complex with the cognate Knl1 KI motif and compared it with the structure of the equivalent BubR1TPR–KI motif complex. The interaction developed along the convex surface of the TPR assembly. Point mutations on this surface impaired the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo but did not cause significant displacement of Bub1 and BubR1 from kinetochores. Conversely, a 62-residue segment of Bub1 that includes a binding domain for the checkpoint protein Bub3 and is C terminal to the TPRs was necessary and largely sufficient for kinetochore recruitment of Bub1. These results shed light on the determinants of kinetochore recruitment of Bub1.

scholarly journals Structural Basis of VSIG3: The Ligand for VISTA

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaoxue Xie ◽  
Caiping Chen ◽  
Wenting Chen ◽  
Jingwei Jiang ◽  
Lanlan Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Biological Activities ◽  
Protein Docking ◽  
Structural Basis ◽  
Cell Functions ◽  
B7 Family

B7 family members and their receptors play key roles in regulating T cell responses, and constitute very attractive targets for developing immunotherapeutic drugs. V-Set and Immunoglobulin domain containing 3 (VSIG3), a ligand for the novel B7 family immune checkpoint V-domain immunoglobulin suppressor of T cell activation (VISTA), can significantly inhibit T cell functions. Inhibitors targeting the VISTA/VSIG3 pathway are of great significance in tumor immunology. Here, we show the crystal structure of the extracellular domain (ECD) of the human VSIG3 protein at 2.64 angstrom resolution, and we produce recombinant human VSIG-3 ECD in both CHO cells and E. coli. Furthermore, we demonstrated the interaction of VISTA and VSIG3 by coimmunoprecipitation (Co-IP). Based on protein-protein docking for VISTA and VSIG3, we report a small molecule inhibitor of VSIG3 K284-3046 and evaluate its biological activities in vitro. This study was the first to reveal the crystal structure of VSIG3, and provides the structural basis for designing antibodies or compounds for the unique VSIG3/VISTA coinhibitory pathway in the treatment of cancers, autoimmune diseases and may be beneficial of designing vaccines.

scholarly journals Structural basis for histone variant H3tK27me3 recognition by PHF1 and PHF19

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Cheng Dong ◽  
Reiko Nakagawa ◽  
Kyohei Oyama ◽  
Yusuke Yamamoto ◽  
Weilian Zhang ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Structural Basis ◽  
Complex Structures ◽  
Polycomb Repressive Complex 2 ◽  
Tudor Domain ◽  
Histone H3k27 ◽  
Critical Components ◽  
Preferential Recognition ◽  
Shed Light

The Polycomb repressive complex 2 (PRC2) is a multicomponent histone H3K27 methyltransferase complex, best known for silencing the Hox genes during embryonic development. The Polycomb-like proteins PHF1, MTF2, and PHF19 are critical components of PRC2 by stimulating its catalytic activity in embryonic stem cells. The Tudor domains of PHF1/19 have been previously shown to be readers of H3K36me3 in vitro. However, some other studies suggest that PHF1 and PHF19 co-localize with the H3K27me3 mark but not H3K36me3 in cells. Here, we provide further evidence that PHF1 co-localizes with H3t in testis and its Tudor domain preferentially binds to H3tK27me3 over canonical H3K27me3 in vitro. Our complex structures of the Tudor domains of PHF1 and PHF19 with H3tK27me3 shed light on the molecular basis for preferential recognition of H3tK27me3 by PHF1 and PHF19 over canonical H3K27me3, implicating that H3tK27me3 might be a physiological ligand of PHF1/19.

scholarly journals Smarca5 mediated epigenetic programming facilitates fetal HSPC development in vertebrates

Blood ◽  
2020 ◽  
Author(s):  
Yanyan Ding ◽  
Wen Wang ◽  
Dongyuan Ma ◽  
Guixian Liang ◽  
Zhixin Kang ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Chromatin Accessibility ◽  
Rna Seq ◽  
Chromatin Remodeler ◽  
Dynamic Changes ◽  
Functional Assays ◽  
Epigenetic Programming

Nascent HSPCs acquire definitive hematopoietic characteristics only when they develop into fetal HSPCs; however, the mechanisms underlying fetal HSPC development are poorly understood. Here, we profiled the chromatin accessibility and transcriptional features of zebrafish nascent- and fetal HSPCs using ATAC-seq and RNA-seq and revealed dynamic changes during HSPC transition. Functional assays demonstrated that chromatin remodeler-mediated epigenetic programming facilitates fetal HSPC development in vertebrates. Systematical screening of chromatin remodeler-related genes identified that smarca5 is responsible for the maintenance of chromatin accessibility at promoters of hematopoiesis-related genes in fetal HSPCs. Mechanistically, Smarca5 interacts with Nucleolin to promote chromatin remodeling, thereby facilitating genomic binding of transcription factors to regulate expression of hematopoietic regulators such as bcl11ab. Our results unravel a new role of epigenetic regulation and reveal that Smarca5-mediated epigenetic programming is responsible for fetal HSPC development, which will provide new insights into the generation of functional HSPCs both in vivo and in vitro.

scholarly journals Phytophthora sojae effector Avr1d functions as E2 competitor and inhibits ubiquitination activity of GmPUB13 to facilitate infection

2020 ◽  
Author(s):  
Yachun Lin ◽  
Qinli Hu ◽  
Jia Zhou ◽  
Weixiao Yin ◽  
Deqiang Yao ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Ubiquitin Ligase ◽  
E3 Ligase ◽  
Phytophthora Sojae ◽  
Structural Basis ◽  
Susceptibility Factor ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitination System

AbstractOomycete pathogens such as Phytophthora secrete a repertoire of effectors to host cells to manipulate host immunity and benefit infection. In this study, we found that an RxLR effector, Avr1d, promoted Phytophthora sojae infection in soybean hairy-roots. Using a yeast two-hybrid screen, we identified the soybean E3 ubiquitin ligase GmPUB13 as a host target for Avr1d. By co-immunoprecipitation, gel infiltration and ITC assays, we confirmed that Avr1d interacts with GmPUB13 both in vivo and in vitro. Furthermore, we found that Avr1d inhibits the E3 ligase activity of GmPUB13. The crystal structure of Avr1d in complex with GmPUB13 was solved and revealed that Avr1d occupies the binding site for E2 ubiquitin conjugating enzyme on GmPUB13. In line with this, Avr1d competed with E2 ubiquitin conjugating enzymes for GmPUB13 binding in vitro, thereby decreasing the E3 ligase activity of GmPUB13. Meanwhile, we found that inactivation of the ubiquitin ligase activity of GmPUB13 stabilized GmPUB13 by blocking GmPUB13 degradation. Silencing of GmPUB13 in soybean hairy-roots decreased P. sojae infection, suggesting that GmPUB13 acts as a susceptibility factor, negatively regulating soybean resistance against P. sojae. Altogether, this study highlights a novel virulence mechanism of Phytophthora effectors, by which Avr1d competes with E2 for GmPUB13 binding to repress the GmPUB13 E3 ligase activity and thereby stabilizing the susceptibility factor GmPUB13 to facilitate Phytophthora infection. This is the first study to unravel the structural basis for modulation of host targets by Phytophthora effectors and will be instrumental for boosting plant resistance breeding.Significance StatementUbiquitination acts as a crucial regulator in plant immunity. Accordingly, microbial pathogens secrete effectors to hijak host ubiquitination system. However, the molecular mechanisms by which microbial effectors modulate host ubiquitination system are not yet clear. Here, we found that the Phytophthora sojae effector Avr1d physically binds to the U-box type E3 ligase GmPUB13, a susceptibility factor in soybean. The crystal structure of Avr1d in complex with GmPUB13 revealed that Avr1d occupies the binding site in GmPUB13 for the E2 ubiquitin conjugating enzyme and competes with E2 for physical binding to GmPUB13. Avr1d stabilized GmPUB13 by suppressing the self-ubiquitination activity of GmPUB13 and thereby promoting Phytophthora infection. This study provides structural basis for modulation of host targets by Phytophthora effectors.

scholarly journals Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Janine Weber ◽  
Han Bao ◽  
Christoph Hartlmüller ◽  
Zhiqin Wang ◽  
Almut Windhager ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nucleic Acid ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Point Mutations ◽  
Structural Basis ◽  
Binding Modes ◽  
Nucleic Acid Binding

The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.

scholarly journals Structural basis for histone variant H3tK27me3 recognition by PHF1 and PHF19

2020 ◽  
Author(s):  
Cheng Dong ◽  
Reiko Nakagawa ◽  
Kyohei Oyama ◽  
Yusuke Yamamoto ◽  
Weilian Zhang ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Structural Basis ◽  
Complex Structures ◽  
Polycomb Repressive Complex 2 ◽  
Histone H3k27 ◽  
Critical Components ◽  
Preferential Recognition ◽  
Shed Light

AbstractThe PRC2 (Polycomb repressive complex 2) complex is a multi-component histone H3K27 methyltransferase, best known for silencing Hox genes during embryonic development. The Polycomb-like proteins PHF1, MTF2 and PHF19 are critical components of PRC2 by stimulating its catalytic activity in embryonic stem (ES) cells. The Tudor domains of PHF1/19 have been previously shown to be readers of H3K36me3 in vitro. However, some other studies suggest that PHF1 and PHF19 co-localize with the H3K27me3 mark, but not H3K36me3 in cells. Here, we provide further evidence that PHF1 co-localizes with H3tK27 in testis, and its Tudor domain preferentially binds to H3tK27me3 over canonical H3K27me3 in vitro. Our complex structures of the Tudor domains of PHF1 and PHF19 with H3tK27me3 shed light on the molecular basis for preferential recognition of H3tK27me3 by PHF1 and PHF19 over canonical H3K27me3, implicating that H3tK27me3 might be a physiological ligand of PHF1/19.

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