Mechanistic insight into substrate processing and allosteric inhibition of human p97

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.

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Structural insights into the activation of human calcium-sensing receptor

10.1101/2021.03.30.437720 ◽

2021 ◽

Author(s):

Xiaochen Chen ◽

Lu Wang ◽

Zhanyu Ding ◽

Qianqian Cui ◽

Li Han ◽

...

Keyword(s):

Conformational Changes ◽

Large Scale ◽

Transmembrane Domains ◽

Structural Basis ◽

Calcium Sensing Receptor ◽

Calcium Sensing ◽

Cryo Electron Microscopy ◽

Activation Mechanisms ◽

G Protein Coupled ◽

Structural Insights

AbstractHuman calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that maintains Ca2+ homeostasis in serum. Here, we present the cryo-electron microscopy structures of the CaSR in the inactive and active states. Complemented with previously reported crystal structures of CaSR extracellular domains, it suggests that there are three distinct conformations: inactive, intermediate and active state during the activation. We used a negative allosteric nanobody to stabilize the CaSR in the fully inactive state and found a new binding site for Ca2+ ion that acts as a composite agonist with L-amino acid to stabilize the closure of active Venus flytraps. Our data shows that the agonist binding leads to the compaction of the dimer, the proximity of the cysteine-rich domains, the large-scale transitions of 7-transmembrane domains, and the inter-and intrasubunit conformational changes of 7-transmembrane domains to accommodate the downstream transducers. Our results reveal the structural basis for activation mechanisms of the CaSR.

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Structural insights on TRPV5 gating by endogenous modulators

10.1101/338798 ◽

2018 ◽

Cited By ~ 1

Author(s):

Taylor E.T. Hughes ◽

Ruth A. Pumroy ◽

Aysenur Torun Yazici ◽

Marina A. Kasimova ◽

Edwin C. Fluck ◽

...

Keyword(s):

Transient Receptor Potential ◽

Receptor Potential ◽

Transient Receptor Potential Channel ◽

Cryo Electron Microscopy ◽

Calcium Reabsorption ◽

Conformational Rearrangements ◽

Transient Receptor ◽

Endogenous Modulators ◽

Structural Insights ◽

Insight Into

ABSTRACTTRPV5 is a transient receptor potential channel involved in calcium reabsorption. Here we investigate the interaction of two endogenous modulators with TRPV5. Both phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and calmodulin (CaM) have been shown to directly bind to TRPV5 and activate or inactivate the channel, respectively. Using cryo-electron microscopy (cryo-EM), we determined TRPV5 structures in the presence of dioctanoyl PI(4,5)P2 and CaM. The PI(4,5)P2 structure revealed a novel binding site between the N-linker, S4-S5 linker and S6 helix of TRPV5. These interactions with PI(4,5)P2 induce conformational rearrangements in the lower gate, opening the channel. The CaM structure revealed two TRPV5 C-terminal peptides anchoring a single CaM molecule and that calcium inhibition is mediated through a cation-π interaction between Lys116 on the C-lobe of calcium-activated CaM and Trp583 at the intracellular gate of TRPV5. Overall, this investigation provides insight into the endogenous modulation of TRPV5, which has the potential to guide drug discovery.

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

Nature Communications ◽

10.1038/s41467-020-20359-x ◽

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.

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Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2114994119 ◽

2022 ◽

Vol 119 (2) ◽

pp. e2114994119

Author(s):

Benjamin J. LaFrance ◽

Johanna Roostalu ◽

Gil Henkin ◽

Basil J. Greber ◽

Rui Zhang ◽

...

Keyword(s):

Electron Microscopy ◽

Conformational Changes ◽

Total Internal Reflection ◽

Dynamic Instability ◽

Wild Type ◽

Gtp Hydrolysis ◽

Cryo Electron Microscopy ◽

Catalytically Active ◽

The Stability ◽

Insight Into

Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis–deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.

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Structural insights into integrin α5β1 opening by fibronectin ligand

Science Advances ◽

10.1126/sciadv.abe9716 ◽

2021 ◽

Vol 7 (19) ◽

pp. eabe9716

Author(s):

Stephanie Schumacher ◽

Dirk Dedden ◽

Roberto Vazquez Nunez ◽

Kyoko Matoba ◽

Junichi Takagi ◽

...

Keyword(s):

Ligand Binding ◽

Conformational Changes ◽

Metal Ion ◽

Manganese Ions ◽

Integrin Α5β1 ◽

Fibronectin Receptor ◽

Cryo Electron Microscopy ◽

Cell Tissue ◽

Structural Insights ◽

Adhesion Site

Integrin α5β1 is a major fibronectin receptor critical for cell migration. Upon complex formation, fibronectin and α5β1 undergo conformational changes. While this is key for cell-tissue connections, its mechanism is unknown. Here, we report cryo–electron microscopy structures of native human α5β1 with fibronectin to 3.1-angstrom resolution, and in its resting state to 4.6-angstrom resolution. The α5β1-fibronectin complex revealed simultaneous interactions at the arginine-glycine-aspartate loop, the synergy site, and a newly identified binding site proximal to adjacent to metal ion–dependent adhesion site, inducing the translocation of helix α1 to secure integrin opening. Resting α5β1 adopts an incompletely bent conformation, challenging the model of integrin sharp bending inhibiting ligand binding. Our biochemical and structural analyses showed that affinity of α5β1 for fibronectin is increased with manganese ions (Mn2+) while adopting the half-bent conformation, indicating that ligand-binding affinity does not depend on conformation, and α5β1 opening is induced by ligand-binding.

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Structural Insights into the BRAF Monomer-to-dimer Transition Mediated by RAS Binding

10.1101/2021.09.30.462573 ◽

2021 ◽

Author(s):

Juliana Andrea Martinez Fiesco ◽

David E Durrant ◽

Deborah K Morrison ◽

Ping Zhang

Keyword(s):

Electron Microscopy ◽

Conformational Changes ◽

Dynamic Process ◽

Mammalian Cells ◽

Dual Function ◽

Unresolved Issue ◽

Raf Kinase ◽

Cryo Electron Microscopy ◽

Terminal Site ◽

Structural Insights

An unresolved issue in RAF kinase signaling is how binding of autoinhibited RAF monomers to activated RAS initiates the conformational changes required to form active RAF dimers. Here, we present cryo-electron microscopy structures of full-length BRAF complexes derived from mammalian cells: autoinhibited monomeric BRAF:14-3-32:MEK and BRAF:14-3-32 complexes and an inhibitor-bound, dimeric BRAF2:14-3-32 complex, at 3.7, 4.1, and 3.9 Å resolution, respectively. The RAS binding domain (RBD) of BRAF is resolved in the autoinhibited structures, and we find that neither MEK nor ATP binding is required to stabilize the autoinhibited complexes. Notably, the RBD was found to interact extensively with the 14-3-3 protomer bound to the BRAF C-terminal site. Moreover, through structure-guided mutational studies, our findings indicate that RAS-RAF binding is a dynamic process and that RBD residues at the 14-3-3 interface have a dual function, first stabilizing RBD orientation in the autoinhibited state and then contributing to full RAS contact.

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Structural insight into substrate and inhibitor discrimination by human P-glycoprotein

Science ◽

10.1126/science.aav7102 ◽

2019 ◽

Vol 363 (6428) ◽

pp. 753-756 ◽

Cited By ~ 98

Author(s):

Amer Alam ◽

Julia Kowal ◽

Eugenia Broude ◽

Igor Roninson ◽

Kaspar P. Locher

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Binding Pocket ◽

Drug Binding ◽

Therapeutic Drug ◽

Binding Domains ◽

Drug Binding Site ◽

Cryo Electron Microscopy ◽

P Glycoprotein ◽

Insight Into

ABCB1, also known as P-glycoprotein, actively extrudes xenobiotic compounds across the plasma membrane of diverse cells, which contributes to cellular drug resistance and interferes with therapeutic drug delivery. We determined the 3.5-angstrom cryo–electron microscopy structure of substrate-bound human ABCB1 reconstituted in lipidic nanodiscs, revealing a single molecule of the chemotherapeutic compound paclitaxel (Taxol) bound in a central, occluded pocket. A second structure of inhibited, human-mouse chimeric ABCB1 revealed two molecules of zosuquidar occupying the same drug-binding pocket. Minor structural differences between substrate- and inhibitor-bound ABCB1 sites are amplified toward the nucleotide-binding domains (NBDs), revealing how the plasticity of the drug-binding site controls the dynamics of the adenosine triphosphate–hydrolyzing NBDs. Ordered cholesterol and phospholipid molecules suggest how the membrane modulates the conformational changes associated with drug binding and transport.

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Structural insights into the nucleic acid remodeling mechanisms of the yeast THO-Sub2 complex

eLife ◽

10.7554/elife.61467 ◽

2020 ◽

Vol 9 ◽

Author(s):

Sandra K Schuller ◽

Jan M Schuller ◽

J Rajan Prabu ◽

Marc Baumgärtner ◽

Fabien Bonneau ◽

...

Keyword(s):

Nucleic Acid ◽

Conformational Changes ◽

Molecular Mechanisms ◽

Cryo Electron Microscopy ◽

Messenger Ribonucleoprotein ◽

Rna:Dna Hybrid ◽

Structural Architecture ◽

Structural Insights ◽

Resolution Structure ◽

Active Genes

The yeast THO complex is recruited to active genes and interacts with the RNA-dependent ATPase Sub2 to facilitate the formation of mature export-competent messenger ribonucleoprotein particles and to prevent the co-transcriptional formation of RNA:DNA-hybrid-containing structures. How THO-containing complexes function at the mechanistic level is unclear. Here, we elucidated a 3.4 Å resolution structure of Saccharomyces cerevisiae THO-Sub2 by cryo-electron microscopy. THO subunits Tho2 and Hpr1 intertwine to form a platform that is bound by Mft1, Thp2, and Tex1. The resulting complex homodimerizes in an asymmetric fashion, with a Sub2 molecule attached to each protomer. The homodimerization interfaces serve as a fulcrum for a seesaw-like movement concomitant with conformational changes of the Sub2 ATPase. The overall structural architecture and topology suggest the molecular mechanisms of nucleic acid remodeling during mRNA biogenesis.

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Specific lid-base contacts in the 26S proteasome control the conformational switching required for substrate engagement and degradation

10.1101/687921 ◽

2019 ◽

Cited By ~ 1

Author(s):

Eric R. Greene ◽

Ellen A. Goodall ◽

Andres H. de la Peña ◽

Mary E. Matyskiela ◽

Gabriel C. Lander ◽

...

Keyword(s):

Conformational Changes ◽

26S Proteasome ◽

Conformational Switching ◽

Atpase Subunit ◽

Aaa Atpase ◽

Cellular Processes ◽

Substrate Processing ◽

And Control ◽

Early Degradation ◽

Insight Into

AbstractThe 26S proteasome is essential for protein homeostasis and the regulation of vital cellular processes through ATP-dependent degradation of ubiquitinated substrates. To accomplish the multi-step reaction of protein degradation, the proteasome’s regulatory particle, consisting of the lid and base subcomplexes, undergoes major conformational changes whose origin and control are largely unknown. Investigating the Saccharomyces cerevisiae 26S proteasome, we found that peripheral interactions between the lid subunit Rpn5 and the base AAA+ ATPase ring play critical roles in stabilizing the substrate-engagement-competent state and coordinating the conformational switch to processing states after a substrate has been engaged. Disrupting these interactions perturbs the conformational equilibrium and interferes with degradation initiation, while later steps of substrate processing remain unaffected. Similar defects in the early degradation steps are also observed when eliminating hydrolysis in the ATPase subunit Rpt6, whose nucleotide state seems to control conformational transitions of the proteasome. These results provide important insight into the network of interactions that coordinate conformational changes with various stages of proteasomal degradation, and how modulators of conformational equilibria may influence substrate turnover.

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Atomic structure of the 26S proteasome lid reveals the mechanism of deubiquitinase inhibition

eLife ◽

10.7554/elife.13027 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 66

Author(s):

Corey M Dambacher ◽

Evan J Worden ◽

Mark A Herzik ◽

Andreas Martin ◽

Gabriel C Lander

Keyword(s):

Electron Microscopy ◽

Atomic Structure ◽

26S Proteasome ◽

Atomic Model ◽

Cellular Proteins ◽

Cryo Electron Microscopy ◽

Substrate Degradation ◽

Conformational Rearrangements ◽

Substrate Processing ◽

Insight Into

The 26S proteasome is responsible for the selective, ATP-dependent degradation of polyubiquitinated cellular proteins. Removal of ubiquitin chains from targeted substrates at the proteasome is a prerequisite for substrate processing and is accomplished by Rpn11, a deubiquitinase within the ‘lid’ sub-complex. Prior to the lid’s incorporation into the proteasome, Rpn11 deubiquitinase activity is inhibited to prevent unwarranted deubiquitination of polyubiquitinated proteins. Here we present the atomic model of the isolated lid sub-complex, as determined by cryo-electron microscopy at 3.5 Å resolution, revealing how Rpn11 is inhibited through its interaction with a neighboring lid subunit, Rpn5. Through mutagenesis of specific residues, we describe the network of interactions that are required to stabilize this inhibited state. These results provide significant insight into the intricate mechanisms of proteasome assembly, outlining the substantial conformational rearrangements that occur during incorporation of the lid into the 26S holoenzyme, which ultimately activates the deubiquitinase for substrate degradation.

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