The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry

AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore towards a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilisation induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ~20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible, and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH.

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Large conformational changes in FtsH create an opening for substrate entry

10.1101/209445 ◽

2017 ◽

Author(s):

Vanessa Carvalho ◽

Roland Kieffer ◽

Nick de Lange ◽

Andreas Engel ◽

Marie-Eve Aubin-Tam

Keyword(s):

Conformational Changes ◽

Protein Quality ◽

Atp Hydrolysis ◽

Transmembrane Helix ◽

Vital Role ◽

Full Length ◽

Aquifex Aeolicus ◽

Cytosolic Domain ◽

Central Pore ◽

Cytoplasmic Structures

AbstractAAA+ proteases are degradation machines, which exploit ATP hydrolysis to unfold protein substrates and translocate them through a central pore towards a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. Although cytoplasmic structures are described, the full-length structure of bacterial FtsH is unknown, and the route by which substrates reach the central pore remains unclear. We use electron microscopy to determine the 3D map of the full-lengthAquifex aeolicusFtsH hexamer. Moreover, detergent solubilisation induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. FtsH structures reveal that the cytosolic domain can tilt with respect to the membrane. A flexible linker of ~20 residues between the second transmembrane helix and the cytosolic domain permits the observed large tilting movements, thereby facilitating the entry of substrate proteins towards the central pore for translocation.

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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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Hierarchical coupling between ATP hydrolysis and Hsp90’s client binding site

10.1101/2020.02.15.950725 ◽

2020 ◽

Author(s):

Steffen Wolf ◽

Benedikt Sohmen ◽

Björn Hellenkamp ◽

Johann Thurn ◽

Gerhard Stock ◽

...

Keyword(s):

Binding Site ◽

Single Molecule ◽

Information Transfer ◽

Atp Hydrolysis ◽

Structural Information ◽

Substrate Binding ◽

Signal Propagation ◽

Nonequilibrium Molecular Dynamics ◽

Time Resolved ◽

Substrate Binding Site

I.ABSTRACTSeveral indicators for a signal propagation from a binding site to a distant functional site have been found in the Hsp90 dimer. Here we determined a time-resolved pathway from ATP hydrolysis to changes in a distant folding substrate binding site. This was possible by combining single-molecule fluorescence-based methods with extensive atomistic nonequilibrium molecular dynamics simulations. We find that hydrolysis of one ATP effects a structural asymmetry in the full Hsp90 dimer that leads to the collapse of a central folding substrate binding site. Arg380 is the major mediator in transferring structural information from the nucleotide to the substrate binding site. This allosteric process occurs via hierarchical dynamics that involve timescales from picoto milliseconds and length scales from Ångstroms to several nanometers. We presume that similar hierarchical mechanisms are fundamental for information transfer through many other proteins.

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Hydrogen exchange reveals Hsp104 architecture, structural dynamics, and energetics in physiological solution

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816184116 ◽

2019 ◽

Vol 116 (15) ◽

pp. 7333-7342 ◽

Cited By ~ 12

Author(s):

Xiang Ye ◽

Jiabei Lin ◽

Leland Mayne ◽

James Shorter ◽

S. Walter Englander

Keyword(s):

Hydrogen Exchange ◽

Atp Hydrolysis ◽

Physiological Solution ◽

Mass Spectrometry Analysis ◽

Structural Element ◽

Spectrometry Analysis ◽

Exchange Mass Spectrometry ◽

Substrate Protein ◽

Central Pore ◽

The One

Hsp104 is a large AAA+ molecular machine that can rescue proteins trapped in amorphous aggregates and stable amyloids by drawing substrate protein into its central pore. Recent cryo-EM studies image Hsp104 at high resolution. We used hydrogen exchange mass spectrometry analysis (HX MS) to resolve and characterize all of the functionally active and inactive elements of Hsp104, many not accessible to cryo-EM. At a global level, HX MS confirms the one noncanonical interprotomer interface in the Hsp104 hexamer as a marker for the spiraled conformation revealed by cryo-EM and measures its fast conformational cycling under ATP hydrolysis. Other findings enable reinterpretation of the apparent variability of the regulatory middle domain. With respect to detailed mechanism, HX MS determines the response of each Hsp104 structural element to the different bound adenosine nucleotides (ADP, ATP, AMPPNP, and ATPγS). They are distinguished most sensitively by the two Walker A nucleotide-binding segments. Binding of the ATP analog, ATPγS, tightly restructures the Walker A segments and drives the global open-to-closed/extended transition. The global transition carries part of the ATP/ATPγS-binding energy to the somewhat distant central pore. The pore constricts and the tyrosine and other pore-related loops become more tightly structured, which seems to reflect the energy-requiring directional pull that translocates the substrate protein. ATP hydrolysis to ADP allows Hsp104 to relax back to its lowest energy open state ready to restart the cycle.

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The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1–NBD2 heterodimer

Biochemical Journal ◽

10.1042/bj20060968 ◽

2006 ◽

Vol 401 (2) ◽

pp. 581-586 ◽

Cited By ~ 29

Author(s):

Fiona L. L. Stratford ◽

Mohabir Ramjeesingh ◽

Joanne C. Cheung ◽

Ling-JUN Huan ◽

Christine E. Bear

Keyword(s):

Atpase Activity ◽

Atp Hydrolysis ◽

Nucleotide Binding ◽

Vital Role ◽

Atp Binding ◽

Primary Role ◽

Binding Domains ◽

Membrane Spanning ◽

Atp Binding And Hydrolysis

CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory ‘R’ domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1–NBD2 heterodimer.

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A host dTMP-bound structure of T4 phage dCMP hydroxymethylase mutant using an X-ray free electron laser

Scientific Reports ◽

10.1038/s41598-019-52825-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Si Hoon Park ◽

Jaehyun Park ◽

Sang Jae Lee ◽

Woo Seok Yang ◽

Sehan Park ◽

...

Keyword(s):

Electron Density ◽

Active Site ◽

Free Electron ◽

Free Electron Laser ◽

Structural Information ◽

Bacteriophage T4 ◽

Vital Role ◽

Spectrometric Analysis ◽

X Ray ◽

High Resolution Structure

Abstract The hydroxymethylation of cytosine bases plays a vital role in the phage DNA protection system inside the host Escherichia coli. This modification is known to be catalyzed by the dCMP hydroxymethylase from bacteriophage T4 (T4dCH); structural information on the complexes with the substrate, dCMP and the co-factor, tetrahydrofolate is currently available. However, the detailed mechanism has not been understood clearly owing to a lack of structure in the complex with a reaction intermediate. We have applied the X-ray free electron laser (XFEL) technique to determine a high-resolution structure of a T4dCH D179N active site mutant. The XFEL structure was determined at room temperature and exhibited several unique features in comparison with previously determined structures. Unexpectedly, we observed a bulky electron density at the active site of the mutant that originated from the physiological host (i.e., E. coli). Mass-spectrometric analysis and a cautious interpretation of an electron density map indicated that it was a dTMP molecule. The bound dTMP mimicked the methylene intermediate from dCMP to 5′-hydroxymethy-dCMP, and a critical water molecule for the final hydroxylation was convincingly identified. Therefore, this study provides information that contributes to the understanding of hydroxymethylation.

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Msp1/ATAD1 in Protein Quality Control and Regulation of Synaptic Activities

Annual Review of Cell and Developmental Biology ◽

10.1146/annurev-cellbio-031220-015840 ◽

2020 ◽

Vol 36 (1) ◽

pp. 141-164

Author(s):

Lan Wang ◽

Peter Walter

Keyword(s):

Quality Control ◽

Mitochondrial Membrane ◽

Protein Import ◽

Protein Quality ◽

Atp Hydrolysis ◽

Mitochondrial Protein ◽

Neurotransmitter Receptors ◽

Functional Specialization ◽

Outer Mitochondrial Membrane ◽

Mitochondrial Protein Import

Mitochondrial function depends on the efficient import of proteins synthesized in the cytosol. When cells experience stress, the efficiency and faithfulness of the mitochondrial protein import machinery are compromised, leading to homeostatic imbalances and damage to the organelle. Yeast Msp1 (mitochondrial sorting of proteins 1) and mammalian ATAD1 (ATPase family AAA domain–containing 1) are orthologous AAA proteins that, fueled by ATP hydrolysis, recognize and extract mislocalized membrane proteins from the outer mitochondrial membrane. Msp1 also extracts proteins that have become stuck in the import channel. The extracted proteins are targeted for proteasome-dependent degradation or, in the case of mistargeted tail-anchored proteins, are given another chance to be routed correctly. In addition, ATAD1 is implicated in the regulation of synaptic plasticity, mediating the release of neurotransmitter receptors from postsynaptic scaffolds to allow their trafficking. Here we discuss how structural and functional specialization imparts the unique properties that allow Msp1/ATAD1 ATPases to fulfill these diverse functions and also highlight outstanding questions in the field.

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The use of blue native PAGE in the evaluation of membrane protein aggregation states for crystallization

Journal of Applied Crystallography ◽

10.1107/s0021889808033797 ◽

2008 ◽

Vol 41 (6) ◽

pp. 1150-1160 ◽

Cited By ~ 18

Author(s):

Jichun Ma ◽

Di Xia

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Protein Quality ◽

Structural Information ◽

Protein Complexes ◽

Polyacrylamide Gel Electrophoresis ◽

Size Exclusion ◽

Native Page ◽

Exclusion Chromatography ◽

Blue Native

Crystallization has long been one of the bottlenecks in obtaining structural information at atomic resolution for membrane proteins. This is largely due to difficulties in obtaining high-quality protein samples. One frequently used indicator of protein quality for successful crystallization is the monodispersity of proteins in solution, which is conventionally obtained by size exclusion chromatography (SEC) or by dynamic light scattering (DLS). Although useful in evaluating the quality of soluble proteins, these methods are not always applicable to membrane proteins either because of the interference from detergent micelles or because of the requirement for large sample quantities. Here, the use of blue native polyacrylamide gel electrophoresis (BN–PAGE) to assess aggregation states of membrane protein samples is reported. A strong correlation is demonstrated between the monodispersity measured by BN–PAGE and the propensity for crystallization of a number of soluble and membrane protein complexes. Moreover, it is shown that there is a direct correspondence between the oligomeric states of proteins as measured by BN–PAGE and those obtained from their crystalline forms. When applied to a membrane protein with unknown structure, BN–PAGE was found to be useful and efficient for selecting well behaved proteins from various constructs and in screening detergents. Comparisons of BN–PAGE with DLS and SEC are provided.

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CryoSAXS as a Method for Measuring Low Resolution Macromolecular Structure

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314095916 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C408-C408

Author(s):

Jesse Hopkins ◽

Andrea Katz ◽

Stephen Meisburger ◽

Matthew Warkentin ◽

Richard Gillilan ◽

...

Keyword(s):

Radiation Damage ◽

Path Length ◽

Room Temperature ◽

Structural Information ◽

Total Sample ◽

Free Variable ◽

Low Resolution ◽

X Ray ◽

Atomic Structures ◽

Fixed Path

Small angle X-ray scattering (SAXS) is an increasingly popular technique for obtaining low resolution structural information from macromolecules and complexes in solution. Biomolecular SAXS signals can rapidly degrade due to radiation damage, so that flow or oscillating cells and large total sample volumes may be required. For particularly sensitive or hard to produce samples, such as of light sensitive proteins, metalloenzymes, and large complexes, and studies where multiple buffer conditions are probed sample consumption may be prohibitive. We describe cryo-cooling of samples to 100 K to prevent X-ray induced radiation damage. We identify SAXS-friendly cryoprotectant conditions that suppress ice formation upon cooling, and compare cryoSAXS profiles obtained in window-free variable-path-length cells with room temperature measurements for a variety of standard molecules. We obtain data sufficient for envelope reconstructions using scattering volumes as small as 20 nL, and find good agreement between cryoSAXS data and known atomic structures. We also discuss work on developing low-volume fixed path-length sample holders for cryoSAXS. Cryo-cooled samples can withstand doses that are 2-3 orders of magnitude higher than typically used for SAXS at room temperature, comparable to those used in cryo-crystallography. While practical challenges remain, cryoSAXS opens the possibility of studies exploiting high brightness X-ray sources and mail-in high-throughput SAXS. This work is funded by the NSF (DBI-1152348).

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RNA Packaging Motor: From Structure to Quantum Mechanical Modelling and Sequential-Stochastic Mechanism

Computational and Mathematical Methods in Medicine ◽

10.1080/17486700802168502 ◽

2008 ◽

Vol 9 (3-4) ◽

pp. 351-369 ◽

Cited By ~ 2

Author(s):

Jelena Telenius ◽

Anders E. Wallin ◽

Michal Straka ◽

Hongbo Zhang ◽

Erika J. Mancini ◽

...

Keyword(s):

Conformational Changes ◽

Atp Hydrolysis ◽

Structural Information ◽

Molecular Motor ◽

Structural Similarity ◽

Binding Pocket ◽

Reaction Coordinate ◽

Rna Packaging ◽

Cooperative Action ◽

Empty Capsid

The bacteriophages of theCystoviridaefamily package their single stranded RNA genomic precursors into empty capsid (procapsids) using a hexameric packaging ATPase motor (P4). This molecular motor shares sequence and structural similarity with RecA-like hexameric helicases. A concerted structural, mutational and kinetic analysis helped to define the mechanical reaction coordinate,i.e.the conformational changes associated with RNA translocation. The results also allowed us to propose a possible scheme of coupling between ATP hydrolysis and translocation which requires the cooperative action of three consecutive subunits. Here, we first test this model by preparing hexamers with defined proportions of wild type and mutant subunits and measuring their activity. Then, we develop a stochastic kinetic model which accounts for the catalytic cooperativity of the P4 hexamer. Finally, we use the available structural information to construct a quantum-chemical model of the chemical reaction coordinate and obtain a detailed description of the electron density changes during ATP hydrolysis. The model explains the results of the mutational analyses and yields new insights into the role of several conserved residues within the ATP binding pocket. These hypotheses will guide future experimental work.

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