Cryo-EM structures reveal a multistep mechanism of Hsp90 activation by co-chaperone Aha1

AbstractHsp90 is a ubiquitous molecular chaperone that facilitates the folding and maturation of hundreds of cellular “client” proteins. The ATP-driven client maturation process is regulated by a large number of co-chaperones. Among them, Aha1 is the most potent activator of Hsp90 ATPase activity and thus dramatically affects Hsp90’s client proteins. To understand the Aha1 activation mechanism, we determined full-length Hsp90:Aha1 structures in six different states by cryo-electron microscopy, including nucleotide-free semi-closed, nucleotide-bound pre-hydrolysis, and semi-hydrolyzed states. Our structures demonstrate that the two Aha1 domains can each interact with Hsp90 in two different modes, uncovering a complex multistep activation mechanism. The results show that Aha1 accelerates the chemical step of ATP hydrolysis like a conventional enzyme, but most unusually, catalyzes the rate-limiting large-scale conformational changes of Hsp90 fundamentally required for ATP hydrolysis. Our work provides a structural framework to guide small molecule development targeting this critical modulator of client protein maturation.

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A structural framework for unidirectional transport by a bacterial ABC exporter

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2006526117 ◽

2020 ◽

Vol 117 (32) ◽

pp. 19228-19236

Author(s):

Chengcheng Fan ◽

Jens T. Kaiser ◽

Douglas C. Rees

Keyword(s):

Conformational Changes ◽

Iron Homeostasis ◽

Atp Hydrolysis ◽

Transmembrane Helix ◽

Reverse Direction ◽

Conformational States ◽

X Ray Crystallography ◽

Binding Domains ◽

Structural Framework ◽

Glutathione Derivatives

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog fromNovosphingobium aromaticivorans(NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying theNaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. AsNaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport byNaAtm1. One of the disulfide crosslinkedNaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

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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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Structure and activation mechanism of the hexameric plasma membrane H+-ATPase

Nature Communications ◽

10.1038/s41467-021-26782-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Peng Zhao ◽

Chaoran Zhao ◽

Dandan Chen ◽

Caihong Yun ◽

Huilin Li ◽

...

Keyword(s):

Plasma Membrane ◽

Conformational Changes ◽

Liquid Crystalline ◽

Atp Hydrolysis ◽

Proton Translocation ◽

Proton Pumping ◽

Antifungal Drugs ◽

Activation Mechanism ◽

Activation Process ◽

Α Helix

AbstractThe S. cerevisiae plasma membrane H+-ATPase, Pma1, is a P3A-type ATPase and the primary protein component of the membrane compartment of Pma1 (MCP). Like other plasma membrane H+-ATPases, Pma1 assembles and functions as a hexamer, a property unique to this subfamily among the larger family of P-type ATPases. It has been unclear how Pma1 organizes the yeast membrane into MCP microdomains, or why it is that Pma1 needs to assemble into a hexamer to establish the membrane electrochemical proton gradient. Here we report a high-resolution cryo-EM study of native Pma1 hexamers embedded in endogenous lipids. Remarkably, we found that the Pma1 hexamer encircles a liquid-crystalline membrane domain composed of 57 ordered lipid molecules. The Pma1-encircled lipid patch structure likely serves as the building block of the MCP. At pH 7.4, the carboxyl-terminal regulatory α-helix binds to the phosphorylation domains of two neighboring Pma1 subunits, locking the hexamer in the autoinhibited state. The regulatory helix becomes disordered at lower pH, leading to activation of the Pma1 hexamer. The activation process is accompanied by a 6.7 Å downward shift and a 40° rotation of transmembrane helices 1 and 2 that line the proton translocation path. The conformational changes have enabled us to propose a detailed mechanism for ATP-hydrolysis-driven proton pumping across the plasma membrane. Our structures will facilitate the development of antifungal drugs that target this essential protein.

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Mitoribosomal small subunit biogenesis in trypanosomes involves an extensive assembly machinery

Science ◽

10.1126/science.aaw5570 ◽

2019 ◽

Vol 365 (6458) ◽

pp. 1144-1149 ◽

Cited By ~ 20

Author(s):

Martin Saurer ◽

David J. F. Ramrath ◽

Moritz Niemann ◽

Salvatore Calderaro ◽

Céline Prange ◽

...

Keyword(s):

Ribosomal Rna ◽

Conformational Changes ◽

Large Scale ◽

Small Subunit ◽

Ribosomal Assembly ◽

Mitochondrial Ribosomes ◽

Ribonucleoprotein Complexes ◽

Cryo Electron Microscopy ◽

Number Of Factors ◽

Cellular Machinery

Mitochondrial ribosomes (mitoribosomes) are large ribonucleoprotein complexes that synthesize proteins encoded by the mitochondrial genome. An extensive cellular machinery responsible for ribosome assembly has been described only for eukaryotic cytosolic ribosomes. Here we report that the assembly of the small mitoribosomal subunit in Trypanosoma brucei involves a large number of factors and proceeds through the formation of assembly intermediates, which we analyzed by using cryo–electron microscopy. One of them is a 4-megadalton complex, referred to as the small subunit assemblosome, in which we identified 34 factors that interact with immature ribosomal RNA (rRNA) and recognize its functionally important regions. The assembly proceeds through large-scale conformational changes in rRNA coupled with successive incorporation of mitoribosomal proteins, providing an example for the complexity of the ribosomal assembly process in mitochondria.

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AMPylation targets the rate-limiting step of BiP’s ATPase cycle for its functional inactivation

eLife ◽

10.7554/elife.29428 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 33

Author(s):

Steffen Preissler ◽

Lukas Rohland ◽

Yahui Yan ◽

Ruming Chen ◽

Randy J Read ◽

...

Keyword(s):

Atp Hydrolysis ◽

Inverse Correlation ◽

Rate Limiting Step ◽

Hsp70 Chaperone ◽

Tightly Coupled ◽

Client Proteins ◽

Functional Inactivation ◽

Atp Binding And Hydrolysis ◽

Rate Limiting ◽

J Protein

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP contributes to protein folding homeostasis by engaging unfolded client proteins in a process that is tightly coupled to ATP binding and hydrolysis. The inverse correlation between BiP AMPylation and the burden of unfolded ER proteins suggests a post-translational mechanism for adjusting BiP’s activity to changing levels of ER stress, but the underlying molecular details are unexplored. We present biochemical and crystallographic studies indicating that irrespective of the identity of the bound nucleotide AMPylation biases BiP towards a conformation normally attained by the ATP-bound chaperone. AMPylation does not affect the interaction between BiP and J-protein co-factors but appears to allosterically impair J protein-stimulated ATP-hydrolysis, resulting in the inability of modified BiP to attain high affinity for its substrates. These findings suggest a molecular mechanism by which AMPylation serves as a switch to inactivate BiP, limiting its interactions with substrates whilst conserving ATP.

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Molecular insights into the human ABCB6 transporter

Cell Discovery ◽

10.1038/s41421-021-00284-z ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Guangyuan Song ◽

Sensen Zhang ◽

Mengqi Tian ◽

Laixing Zhang ◽

Runyu Guo ◽

...

Keyword(s):

Conformational Changes ◽

Transmembrane Domain ◽

Atp Hydrolysis ◽

Toxic Metal ◽

Bound State ◽

Metal Resistance ◽

Blood Group System ◽

Cryo Electron Microscopy ◽

Binding Pockets ◽

Energy Dependent

AbstractABCB6 plays a crucial role in energy-dependent porphyrin transport, drug resistance, toxic metal resistance, porphyrin biosynthesis, protection against stress, and encoding a blood group system Langereis antigen. However, the mechanism underlying porphyrin transport is still unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structures of nanodisc-reconstituted human ABCB6 trapped in an apo-state and an ATP-bound state at resolutions of 3.6 and 3.5 Å, respectively. Our structures reveal a unique loop in the transmembrane domain (TMD) of ABCB6, which divides the TMD into two cavities. It restrains the access of substrates in the inward-facing state and is removed by ATP-driven conformational change. No ligand cavities were observed in the nucleotide-bound state, indicating a state following substrate release but prior to ATP hydrolysis. Structural analyses and functional characterizations suggest an “ATP-switch” model and further reveal the conformational changes of the substrate-binding pockets triggered by the ATP-driven regulation.

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Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides

10.1101/309534 ◽

2018 ◽

Cited By ~ 3

Author(s):

Steven Z. Chou ◽

Thomas D. Pollard

Keyword(s):

Conformational Changes ◽

Active Site ◽

Actin Filaments ◽

Actin Polymerization ◽

Atp Hydrolysis ◽

Regulatory Proteins ◽

Actin Assembly ◽

Cryo Electron Microscopy ◽

Pointed End ◽

Binding Loop

AbstractWe used electron cryo-micrographs to reconstruct actin filaments with bound AMPPNP (β,γ-imidoadenosine 5’-triphosphate, an ATP analog), ADP-Pi (ADP with inorganic phosphate) or ADP to resolutions of 3.4 Å, 3.4 Å and 3.6 Å. Subunits in the three filaments have nearly identical backbone conformations, so assembly rather than ATP hydrolysis or phosphate dissociation is responsible for their flattened conformation in filaments. Polymerization increases the rate of ATP hydrolysis by changing the conformations of the three ATP phosphates and the side chains of Gln137 and His161 in the active site. Flattening also promotes interactions along both the long-pitch and short-pitch helices. In particular, conformational changes in subdomain 3 open up favorable interactions with the DNase-I binding loop in subdomain 2 of the adjacent subunit. Subunits at the barbed end of the filament are likely to be in this favorable conformation, while monomers are not. This difference explains why filaments grow faster at the barbed end than the pointed end. Loss of hydrogen bonds after phosphate dissociation may account for the greater flexibility of ADP-actin filaments.Significance StatementActin filaments comprise a major part of the cytoskeleton of eukaryotic cells and serve as tracks for myosin motor proteins. The filaments assemble from actin monomers with a bound ATP. After polymerization, actin rapidly hydrolyzes the bound ATP and slowly dissociates the γ-phosphate. ADP-actin filaments then disassemble to recycle the subunits. Understanding how actin filaments assemble, disassemble and interact with numerous regulatory proteins depends on knowing the structure of the filament. High quality structures of ADP-actin filaments were available, but not of filaments with bound ATP- or with ADP and phosphate. We determined structures of actin filaments with bound AMPPNP (a slowly hydrolyzed ATP analog), ADP and phosphate and ADP by cryo-electron microscopy. These structures show how conformational changes during actin assembly promote ATP hydrolysis and faster growth at one end of the filament than the other.

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Coupling of ATPase activity, microtubule binding and mechanics in the dynein motor domain

10.1101/309179 ◽

2018 ◽

Cited By ~ 1

Author(s):

Stefan Niekamp ◽

Nicolas Coudray ◽

Nan Zhang ◽

Ronald D. Vale ◽

Gira Bhabha

Keyword(s):

Atpase Activity ◽

Conformational Change ◽

Conformational Changes ◽

Large Scale ◽

Atp Hydrolysis ◽

Molecular Motor ◽

Coiled Coil ◽

Microtubule Binding ◽

Dynein Motor ◽

In The Wild

The movement of a molecular motor protein along a cytoskeletal track requires communication between enzymatic, polymer-binding, and mechanical elements. Such communication is particularly complex and not well understood in the dynein motor, an ATPase that is comprised of a ring of six AAA domains, a large mechanical element (linker) spanning over the ring, and a microtubule-binding domain (MTBD) that is separated from the AAA ring by a ~135 Å coiled-coil stalk. We identified mutations in the stalk that disrupt directional motion, have microtubule-independent hyperactive ATPase activity, and nucleotide-independent low affinity for microtubules. Cryo-electron microscopy structures of a mutant that uncouples ATPase activity from directional movement reveal that nucleotide-dependent conformational changes occur normally in one half of the AAA ring, but are disrupted in the other half. The large-scale linker conformational change observed in the wild-type protein is also inhibited, revealing that this conformational change is not required for ATP hydrolysis. These results demonstrate an essential role of the stalk in regulating motor activity and coupling conformational changes across the two halves of the AAA ring.

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Structural basis for broad coronavirus neutralization

10.1101/2020.12.29.424482 ◽

2020 ◽

Author(s):

Maximilian M. Sauer ◽

M. Alexandra Tortorici ◽

Young-Jun Park ◽

Alexandra C. Walls ◽

Leah Homad ◽

...

Keyword(s):

Conformational Changes ◽

Cross Reactivity ◽

Structural Basis ◽

Species Barrier ◽

Highly Pathogenic ◽

X Ray Crystallography ◽

Structural Framework ◽

Cryo Electron Microscopy ◽

Generation Structure ◽

Cryptic Epitope

Three highly pathogenic β-coronaviruses crossed the animal-to-human species barrier in the past two decades: SARS-CoV, MERS-CoV and SARS-CoV-2. SARS-CoV-2 has infected more than 64 million people worldwide, claimed over 1.4 million lives and is responsible for the ongoing COVID-19 pandemic. We isolated a monoclonal antibody, termed B6, cross-reacting with eight β-coronavirus spike glycoproteins, including all five human-infecting β-coronaviruses, and broadly inhibiting entry of pseudotyped viruses from two coronavirus lineages. Cryo-electron microscopy and X-ray crystallography characterization reveal that B6 binds to a conserved cryptic epitope located in the fusion machinery and indicate that antibody binding sterically interferes with spike conformational changes leading to membrane fusion. Our data provide a structural framework explaining B6 cross-reactivity with β-coronaviruses from three lineages along with proof-of-concept for antibody-mediated broad coronavirus neutralization elicited through vaccination. This study unveils an unexpected target for next-generation structure-guided design of a pan-coronavirus vaccine.

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Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1807028115 ◽

2019 ◽

Vol 116 (10) ◽

pp. 4265-4274 ◽

Cited By ~ 56

Author(s):

Steven Z. Chou ◽

Thomas D. Pollard

Keyword(s):

Conformational Changes ◽

Active Site ◽

Actin Filaments ◽

Actin Polymerization ◽

Atp Hydrolysis ◽

Release Site ◽

Cryo Electron Microscopy ◽

C Terminus ◽

Pointed End ◽

Binding Loop

We used cryo-electron microscopy (cryo-EM) to reconstruct actin filaments with bound AMPPNP (β,γ-imidoadenosine 5′-triphosphate, an ATP analog, resolution 3.1 Å), ADP-Pi(ADP with inorganic phosphate, resolution 3.1 Å), or ADP (resolution 3.6 Å). Subunits in the three filaments have similar backbone conformations, so assembly rather than ATP hydrolysis or phosphate dissociation is responsible for their flattened conformation in filaments. Polymerization increases the rate of ATP hydrolysis by changing the positions of the side chains of Q137 and H161 in the active site. Flattening during assembly also promotes interactions along both the long-pitch and short-pitch helices. In particular, conformational changes in subdomain 3 open up multiple favorable interactions with the DNase-I binding loop in subdomain 2 of the adjacent subunit. Subunits at the barbed end of the filament are likely to be in this favorable conformation, while monomers are not. This difference explains why filaments grow faster at the barbed end than the pointed end. When phosphate dissociates from ADP-Pi-actin through a backdoor channel, the conformation of the C terminus changes so it distorts the DNase binding loop, which allows cofilin binding, and a network of interactions among S14, H73, G74, N111, R177, and G158 rearranges to open the phosphate release site.

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