scholarly journals Seesaw conformations of Npl4 in the human p97 complex and the inhibitory mechanism of a disulfiram derivative

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Man Pan ◽  
Qingyun Zheng ◽  
Yuanyuan Yu ◽  
Huasong Ai ◽  
Yuan Xie ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Atp Hydrolysis ◽  
Cellular Protein ◽  
Cancer Drug ◽  
Copper Transporter ◽  
Cryo Electron Microscopy ◽  
Zinc Finger Motifs ◽  
Structural Insights ◽  
Target Cancer

Abstractp97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis. Human p97 mutations are associated with several neurodegenerative diseases. Targeting p97 and its cofactors is a strategy for cancer drug development. Despite significant structural insights into the fungal homolog Cdc48, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) can bypass the copper transporter system and inhibit the function of p97 in the cytoplasm by releasing cupric ions under oxidative conditions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.

scholarly journals Seesaw Conformations of Npl4 in the Human p97 Complex and the Inhibitory Mechanism of a Disulfiram Derivative

2020 ◽  
Author(s):  
Man Pan ◽  
Qingyun Zheng ◽  
Yuanyuan Yu ◽  
Huasong Ai ◽  
Yuan Xie ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Atp Hydrolysis ◽  
Cellular Protein ◽  
Cancer Drug ◽  
Protein Homeostasis ◽  
Cryo Electron Microscopy ◽  
Zinc Finger Motifs ◽  
Structural Insights ◽  
Target Cancer

ABSTRACTp97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis1. Human p97 mutations are associated with several neurodegenerative diseases2,3. Targeting p97 and its cofactors is a strategy for cancer drug development4. Despite significant structural insights into the fungal homolog Cdc485–7, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p978, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that under oxidative conditions, the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) inhibits p97 function by releasing cupric ions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.

scholarly journals Functional analyses of the CIF1-CIF2 complex in Trypanosoma brucei identify the structural motifs required for complex formation and cytokinesis

2017 ◽  
Author(s):  
Huiqing Hu ◽  
Paul Majneri ◽  
Dielan Li ◽  
Yasuhiro Kurasawa ◽  
Tai An ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Coiled Coil ◽  
Longitudinal Axis ◽  
Zinc Finger Motif ◽  
Structural Motifs ◽  
Zinc Finger Motifs ◽  
Ef Hand ◽  
Functional Analyses ◽  
Attachment Zone ◽  
Structural Insights

ABSTRACTCytokinesis in trypanosome occurs uni-directionally along the longitudinal axis from the cell anterior towards the cell posterior and requires a trypanosome-specific CIF1-CIF2 protein complex. However, little is known about the contribution of the structural motifs in CIF1 and CIF2 to complex assembly and cytokinesis. Here, we demonstrated that the two zinc-finger motifs but not the coiled-coil motif in CIF1 are required for interaction with the EF-hand motifs in CIF2. We further showed that localization of CIF1 depends on the coiled-coil motif and the first zinc-finger motif and that localization of CIF2 depends on the EF-hand motifs. Deletion of the coiled-coil motif and mutation of either zinc-finger motifs in CIF1 disrupted cytokinesis. Further, mutation of either zinc-finger motif in CIF1 mis-localized CIF2 to the cytosol and destabilized CIF2, whereas deletion of the coiled-coil motif in CIF1 spread CIF2 over to the new flagellum attachment zone and stabilized CIF2. Together, these results uncovered the requirement of the coiled-coil motif and zinc-finger motifs for CIF1 function in cytokinesis and for CIF2 localization and stability, providing structural insights into the functional interplay between the two cytokinesis regulators.

scholarly journals Mechanistic insight into substrate processing and allosteric inhibition of human p97

2021 ◽  
Author(s):  
Man Pan ◽  
Yuanyuan Yu ◽  
Huasong Ai ◽  
Qingyun Zheng ◽  
Yuan Xie ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Cellular Protein ◽  
Power Stroke ◽  
Cryo Electron Microscopy ◽  
D2 Domain ◽  
The Central Nervous System ◽  
Degenerative Disorder ◽  
Substrate Processing ◽  
Structural Insights ◽  
Insight Into

ABSTRACTp97, also known as valosin-containing protein (VCP), processes ubiquitinated substrates and plays a central role in cellular protein homeostasis. Mutations in human p97 are associated with multisystem proteinopathy (MSP), a dominantly inherited degenerative disorder that can affect muscle, bone and the central nervous system. It is also a drug target for cancer therapy with various inhibitors developed over the past decade. Despite significant structural insights into the fungal homologue of p97, Cdc48, little is known about how human p97 processes its substrates and how the activity is allosterically affected by inhibitors. Here, we report a series of cryo-electron microscopy (cryo-EM) structures of substrate-engaged human p97 complex with resolutions ranging from 2.9 to 3.8 Å that captured “power stroke”-like motions of both the D1 and D2 ATPase rings of p97. The structures elucidated how the unfolded substrate is engaged in the pore at atomic level. Critical conformational changes of the inter-subunit signaling (ISS) motifs were revealed, providing molecular insights into substrate translocation. Furthermore, we also determined cryo-EM structures of human p97 in complex with NMS-873, the most potent p97 inhibitor, at a resolution of 2.4 Å. The structures showed that NMS-873 binds at a cryptic groove in the D2 domain and interacts with the ISS motif, preventing its conformational change, thus blocking substrate translocation allosterically. Finally, using NMS-873 at a substoichiometric concentration, we captured a series of intermediate states, suggesting how the cofactor Npl4 coordinates with the D1 ring of p97 to initiate the translocation.

scholarly journals Inhibition of red blood cell development by arsenic-induced disruption of GATA-1

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Xixi Zhou ◽  
Sebastian Medina ◽  
Alicia M. Bolt ◽  
Haikun Zhang ◽  
Guanghua Wan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Zinc Finger ◽  
Zinc Fingers ◽  
Arsenic Exposure ◽  
In Vivo Studies ◽  
Regulatory Factor ◽  
Zinc Finger Motifs ◽  
First Time

Abstract Anemia is a hematological disorder that adversely affects the health of millions of people worldwide. Although many variables influence the development and exacerbation of anemia, one major contributing factor is the impairment of erythropoiesis. Normal erythropoiesis is highly regulated by the zinc finger transcription factor GATA-1. Disruption of the zinc finger motifs in GATA-1, such as produced by germline mutations, compromises the function of this critical transcription factor and causes dyserythropoietic anemia. Herein, we utilize a combination of in vitro and in vivo studies to provide evidence that arsenic, a widespread environmental toxicant, inhibits erythropoiesis likely through replacing zinc within the zinc fingers of the critical transcription factor GATA-1. We found that arsenic interacts with the N- and C-terminal zinc finger motifs of GATA-1, causing zinc loss and inhibition of DNA and protein binding activities, leading to dyserythropoiesis and an imbalance of hematopoietic differentiation. For the first time, we show that exposures to a prevalent environmental contaminant compromises the function of a key regulatory factor in erythropoiesis, producing effects functionally similar to inherited GATA-1 mutations. These findings highlight a novel molecular mechanism by which arsenic exposure may cause anemia and provide critical insights into potential prevention and intervention for arsenic-related anemias.

scholarly journals Structural insights into the functional cycle of the ATPase module of the 26S proteasome

2017 ◽  
Vol 114 (6) ◽  
pp. 1305-1310 ◽  
Author(s):  
Marc Wehmer ◽  
Till Rudack ◽  
Florian Beck ◽  
Antje Aufderheide ◽  
Günter Pfeifer ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
Core Particle ◽  
Nucleotide Analogs ◽  
The Core ◽  
Cryo Electron Microscopy ◽  
Intracellular Proteins ◽  
Ubiquitin Proteasome ◽  
Structural Insights

In eukaryotic cells, the ubiquitin–proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA+ ATPase module, which unfolds and translocates substrates into the CP. Applying single-particle cryo-electron microscopy (cryo-EM) and image classification to samples in the presence of different nucleotides and nucleotide analogs, we were able to observe four distinct conformational states (s1 to s4). The resolution of the four conformers allowed for the construction of atomic models of the AAA+ ATPase module as it progresses through the functional cycle. In a hitherto unobserved state (s4), the gate controlling access to the CP is open. The structures described in this study allow us to put forward a model for the 26S functional cycle driven by ATP hydrolysis.

Zinc-finger motifs expressed in E. coli and folded in vitro direct specific binding to DNA

Nature ◽  
1988 ◽  
Vol 332 (6161) ◽  
pp. 284-286 ◽  
Author(s):  
Kiyoshi Nagai ◽  
Yukinobu Nakaseko ◽  
Kim Nasmyth ◽  
Daniela Rhodes
Keyword(s):  
Zinc Finger ◽  
Specific Binding ◽  
E Coli ◽  
Zinc Finger Motifs

Structure of nucleosome-bound human BAF complex

Science ◽  
2020 ◽  
Vol 367 (6480) ◽  
pp. 875-881 ◽  
Author(s):  
Shuang He ◽  
Zihan Wu ◽  
Yuan Tian ◽  
Zishuo Yu ◽  
Jiali Yu ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Chromatin Remodelers ◽  
Baf Complex ◽  
Cryo Electron Microscopy ◽  
Nucleosomal Dna ◽  
Α Helix ◽  
Subunit Organization ◽  
Structural Insights ◽  
Base Module ◽  
Positively Charged

Mammalian SWI/SNF family chromatin remodelers, BRG1/BRM-associated factor (BAF) and polybromo-associated BAF (PBAF), regulate chromatin structure and transcription, and their mutations are linked to cancers. The 3.7-angstrom-resolution cryo–electron microscopy structure of human BAF bound to the nucleosome reveals that the nucleosome is sandwiched by the base and the adenosine triphosphatase (ATPase) modules, which are bridged by the actin-related protein (ARP) module. The ATPase motor is positioned proximal to nucleosomal DNA and, upon ATP hydrolysis, engages with and pumps DNA along the nucleosome. The C-terminal α helix of SMARCB1, enriched in positively charged residues frequently mutated in cancers, mediates interactions with an acidic patch of the nucleosome. AT-rich interactive domain-containing protein 1A (ARID1A) and the SWI/SNF complex subunit SMARCC serve as a structural core and scaffold in the base module organization, respectively. Our study provides structural insights into subunit organization and nucleosome recognition of human BAF complex.

scholarly journals Importin α1 is involved in the nuclear localization of Zac1 and the induction of p21WAF1/CIP1 by Zac1

2007 ◽  
Vol 402 (2) ◽  
pp. 359-366 ◽  
Author(s):  
Shih-Ming Huang ◽  
Sheng-Ping Huang ◽  
Sung-Ling Wang ◽  
Pei-Yao Liu
Keyword(s):  
Zinc Finger ◽  
Nuclear Localization ◽  
Green Fluorescence Protein ◽  
Glutathione S Transferase ◽  
Transactivation Activity ◽  
Zinc Finger Motifs ◽  
Gene And Protein Expression ◽  
Dna Elements ◽  
Localization Signal

Zac1, a novel seven-zinc-finger transcription factor, preferentially binds GC-rich DNA elements and has intrinsic transactivation activity. To date, the NLS (nuclear localization signal) of Zac1 has not been empirically determined. We generated a series of EGFP (enhanced green fluorescence protein)-tagged deletion mutants of Zac1 and examined their subcellular localization, from which we defined two NLSs within the DNA-binding (or zinc-finger) domain. Fusion proteins consisting of the two EGFP-tagged zinc-finger clusters (zinc finger motifs 1–3 and 4–7) were located exclusively in the nucleus, demonstrating that each of the zinc-finger clusters is sufficient for nuclear localization. Physical interactions between these two zinc-finger clusters and importin α1 were demonstrated using an in vitro glutathione S-transferase pull-down assay. Finally, our results indicate that the association of Zac1 with importin α1 is also involved in regulating the transactivation activity of Zac1 on the p21WAF1/CIP1 gene and protein expression.

scholarly journals RoXaN, a Novel Cellular Protein Containing TPR, LD, and Zinc Finger Motifs, Forms a Ternary Complex with Eukaryotic Initiation Factor 4G and Rotavirus NSP3

2004 ◽  
Vol 78 (8) ◽  
pp. 3851-3862 ◽  
Author(s):  
Damien Vitour ◽  
Pierre Lindenbaum ◽  
Patrice Vende ◽  
Michelle M. Becker ◽  
Didier Poncet
Keyword(s):  
Amino Acids ◽  
Zinc Finger ◽  
Protein Interactions ◽  
Ternary Complex ◽  
Consensus Sequence ◽  
Cellular Protein ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Amino Terminal ◽  
Zinc Finger Motifs

ABSTRACT Rotavirus mRNAs are capped but not polyadenylated, and viral proteins are translated by the cellular translation machinery. This is accomplished through the action of the viral nonstructural protein NSP3, which specifically binds the 3′ consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G I. To further our understanding of the role of NSP3 in rotavirus replication, we looked for other cellular proteins capable of interacting with this viral protein. Using the yeast two-hybrid assay, we identified a novel cellular protein-binding partner for rotavirus NSP3. This 110-kDa cellular protein, named RoXaN (rotavirus X protein associated with NSP3), contains a minimum of three regions predicted to be involved in protein-protein or nucleic acid-protein interactions. A tetratricopeptide repeat region, a protein-protein interaction domain most often found in multiprotein complexes, is present in the amino-terminal region. In the carboxy terminus, at least five zinc finger motifs are observed, further suggesting the capacity of RoXaN to bind other proteins or nucleic acids. Between these two regions exists a paxillin leucine-aspartate repeat (LD) motif which is involved in protein-protein interactions. RoXaN is capable of interacting with NSP3 in vivo and during rotavirus infection. Domains of interaction were mapped and correspond to the dimerization domain of NSP3 (amino acids 163 to 237) and the LD domain of RoXaN (amino acids 244 to 341). The interaction between NSP3 and RoXaN does not impair the interaction between NSP3 and eIF4G I, and a ternary complex made of NSP3, RoXaN, and eIF4G I can be detected in rotavirus-infected cells, implicating RoXaN in translation regulation.

CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex

1990 ◽  
Vol 48 (1) ◽  
pp. 248-249
Author(s):  
John Trinickt ◽  
Howard White
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Myosin Head ◽  
Bound State ◽  
Cross Linking ◽  
Weakly Bound ◽  
Cryo Electron Microscopy ◽  
Myosin Subfragment 1 ◽  
Attached State ◽  
High Fraction

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

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