ATP-driven Conformational Changes Research Articles

ABC ATPase signature helices in Rad50 link nucleotide state to Mre11 interface for DNA repair

Rad50 ABC-ATPase complex with Mre11 nuclease is essential for dsDNA break repair, telomere maintenance, and ataxia telangiectasia mutated kinase checkpoint signaling. How Rad50 affects Mre11 functions, and how ABC-ATPases communicate nucleotide binding and ligand states across long distances and among protein partners, has remained obscure. Here, structures of Mre11–Rad50 complexes define the Mre11 2-helix Rad50 binding domain (RBD) that forms a 4-helix interface with Rad50 coiled-coils adjoining the ATPase core. Newly identified effector and basic-switch helix motifs extend the ABC-ATPase signature motif to link ATP-driven Rad50 movements to coiled-coils binding Mre11, implying a ~30 Å pull on the linker to the nuclease domain. Both RBD and basic-switch mutations cause clastogen sensitivity. Collective results characterize flexible ATP-dependent Mre11 regulation, defects in cancer-linked RBD mutations, conserved superfamily basic-switches and motifs effecting ATP-driven conformational change, and a unified comprehension of ABC-ATPase activities.

Communication between the AAA+ ring and microtubule-binding domain of dyneinThis paper is one of a selection of papers published in this special issue entitled 8th International Conference on AAA Proteins and has undergone the Journal's usual peer review process.

Dyneins are microtubule motors, the core of which consists of a ring of AAA+ domains. ATP-driven conformational changes of the AAA+ ring are used to drive the movement of a mechanical element (termed the linker domain) that provides the motor's powerstroke and to change the affinity of the motor for microtubules (strong binding during the power stroke and weak binding to allow stepping and recocking of the linker domain). Dynein's microtubule-binding domain (MTBD) is located at the end of a 10 nm long anti-parallel coiled coil (the stalk) and conformational changes that alter the affinity for microtubules must propagate through this coiled coil. A recent crystal structure of dynein's MTBD sheds new light on how this long-range communication along a coiled coil might occur.

X-ray Structure of the Complete ABC Enzyme ABCE1 from Pyrococcus abyssi

The ATP binding cassette enzyme ABCE1 (also known as RNase-L (ribonuclease L) inhibitor, Pixie, and HP68), one of the evolutionary most sequence-conserved enzymes, functions in translation initiation, ribosome biogenesis, and human immunodeficiency virus capsid assembly. However, its structural mechanism and biochemical role in these processes have not been revealed. We determined the crystal structure of Pyrococcus abyssi ABCE1 in complex with Mg(2+) and ADP to 2.8A resolution. ABCE1 consists of four structural domains. Two nucleotide binding domains are arranged in a head-to-tail orientation by a hinge domain, suggesting that these domains undergo the characteristic tweezers-like powerstroke of ABC enzymes. In contrast to all other known ABC enzymes, ABCE1 has a N-terminal iron-sulfur-cluster (FeS) domain. The FeS domain contains two [4Fe-4S] clusters and is structurally highly related to bacterial-type ferredoxins. However, one cluster is coordinated by an unusual CX(4)CX(3/4)C triad. Surprisingly, intimate interactions of the FeS domain with the adenine and ribose binding Y-loop on nucleotide binding domain 1 suggest a linkage between FeS domain function and ATP-induced conformational control of the ABC tandem cassette. The structure substantially expands the functional architecture of ABC enzymes and raises the possibility that ABCE1 is a chemomechanical engine linked to a redox process.

Visualizing the ATPase Cycle in a Protein Disaggregating Machine: Structural Basis for Substrate Binding by ClpB

ClpB is a ring-shaped molecular chaperone that has the remarkable ability to disaggregate stress-damaged proteins. Here we present the electron cryomicroscopy reconstruction of an ATP-activated ClpB trap mutant, along with reconstructions of ClpB in the AMPPNP, ADP, and in the nucleotide-free state. We show that motif 2 of the ClpB M domain is positioned between the D1-large domains of neighboring subunits and could facilitate a concerted, ATP-driven conformational change in the AAA-1 ring. We further demonstrate biochemically that ATP is essential for high-affinity substrate binding to ClpB and cannot be substituted with AMPPNP. Our structures show that in the ATP-activated state, the D1 loops are stabilized at the central pore, providing the structural basis for high-affinity substrate binding. Taken together, our results support a mechanism by which ClpB captures substrates on the upper surface of the AAA-1 ring before threading them through the ClpB hexamer in an ATP hydrolysis-driven step.

The ABC protein turned chloride channel whose failure causes cystic fibrosis

CFTR chloride channels are encoded by the gene mutated in patients with cystic fibrosis. These channels belong to the superfamily of ABC transporter ATPases. ATP-driven conformational changes, which in other ABC proteins fuel uphill substrate transport across cellular membranes, in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. New structural and biochemical information from prokaryotic ABC proteins and functional information from CFTR channels has led to a unifying mechanism explaining those ATP-driven conformational changes.

SecA Dimer Cross-Linked at Its Subunit Interface Is Functional for Protein Translocation

SecA facilitates protein transport across the eubacterial plasma membrane by its association with cargo proteins and the SecYEG translocon, followed by ATP-driven conformational changes that promote protein translocation in a stepwise manner. Whether SecA functions as a monomer or a dimer during this process has been the subject of considerable controversy. Here we utilize cysteine-directed mutagenesis along with the crystal structure of the SecA dimer to create a cross-linked dimer at its subunit interface, which was normally active for in vitro protein translocation.

Nucleotide binding to the homodimeric MJ0796 protein: A computational study of a prokaryotic ABC transporter NBD dimer

Transport by ABC proteins requires a cycle of ATP-driven conformational changes of the nucleotide binding domains (NBDs). We compare three molecular dynamics simulations of dimeric MJ0796: with ATP was present at both NBDs; with ATP at one NBD but ADP at the other; and without any bound ATP. In the simulation with ATP present at both NBDs, the dimeric protein interacts with the nucleotides in a symmetrical manner. However, if ADP is present at one binding site then both NBD–NBD and protein–ATP interactions are enhanced at the opposite site.

X-Ray Structure of RLI, an Essential Twin Cassette ABC ATPase Involved in Ribosome Biogenesis and HIV Capsid Assembly

The ABC ATPase RNase-L inhibitor (RLI) emerges as a key enzyme in ribosome biogenesis, formation of translation preinitiation complexes, and assembly of HIV capsids. To help reveal the structural mechanism of RLI, we determined the Mg2+-ADP bound crystal structure of the twin cassette ATPase of P. furiosus RLI at 1.9 A resolution and analyzed functional motifs in yeast in vivo. RLI shows similarities but also differences to known ABC enzyme structures. Twin nucleotide binding domains (NBD1 and NBD2) are arranged to form two composite active sites in their interface cleft, indicating they undergo the ATP-driven clamp-like motion of the NBDs of ABC transporters. An unusual "hinge" domain along the NBD1:NBD2 interface provides a frame for association and possibly ATP-driven conformational changes of the NBDs. Our results establish a first structural basis for ABC domain heterodimers and suggest that RLI may act as mechanochemical enzyme in ribosome and HIV capsid biogenesis.

Mechanism of Inhibition of Human KSP by Monastrol: Insights from Kinetic Analysis and the Effect of Ionic Strength on KSP Inhibition

Kinesin motor proteins utilize the energy from ATP hydrolysis to transport cellular cargo along microtubules. Kinesins that play essential roles in the mechanics of mitosis are attractive targets for novel antimitotic cancer therapies. Monastrol, a cell-permeable inhibitor that specifically inhibits the kinesin Eg5, the Xenopus laevis homologue of human KSP, can cause mitotic arrest and monopolar spindle formation. In this study, we show that the extent of monastrol inhibition of KSP microtubule-stimulated ATP hydrolysis is highly dependent upon ionic strength. Detailed kinetic analysis of KSP inhibition by monastrol in the presence and absence of microtubules suggests that monastrol binds to the KSP-ADP complex, forming a KSP-ADP-monastrol ternary complex, which cannot bind to microtubules productively and cannot undergo further ATP-driven conformational changes.

Keeping up with protein folding

Protein and misfolding in the cellular environment has emerged as a major research theme over the past fifteen years. The implications are broad, ranging from the production of recombinant proteins in a soluble and bioactive form to the understanding of the etiology of neurodegenerative diseases. The basic principle enunciated by Afinsen – that all the information required for a polypeptide to reach a proper conformation is contained within its amino acid sequence [1] – remains unchallenged. However, it is now well established that many proteins require the assistance of modulators to adopt a native structure as they emerge from ribosomes, regain their original conformation following exposure to stress, or traffic to their ultimate destination [2]. One of the most extensively characterized group of modulators are molecular chaperones, a ubiquitous class of proteins that help other polypeptides reach a proper conformation or cellular location without affecting rates or becoming part of the final structure. Molecular function by transiently binding hydrophobic stretches of amino acids that should normally be buried within the core of their substrates but have become solvent-exposed due to improper de novo or imposition of cellular stress (e.g., temperature increase). As a result of the formation of chaperone-substrate complexes, interactive species are shielded from each other and from the solvent and on-pathway is facilitated. Three functionally distinct sets of cooperate in the conformational quality control of the proteome. At the heart of chaperone networks are folding chaperones which use conformational changes fueled by ATP hydrolysis to promote the net refolding/unfolding of bound substrates. Archetypal are the DnaK-DnaJ-GrpE (Hsp70-Hsp40) and GroEL-GroES (Hsp60-Hsp10) systems of E. coli. Holding chaperones (e.g., small heat shock proteins) passively stabilize partially folded species until become available for their refolding. In doing so, they alleviate titration of by unfolding intermediates. Finally, disaggregating chaperones such as E. coli ClpB and S. cerevisiae Hsp104 employ ATP-driven conformational changes to solubilize protein aggregates and transfer partially folded species to for subsequent refolding. Foldases embody the second group of modulators. These enzymes accelerate rate-limiting steps along the pathway and include peptidyl-prolyl cis/trans isomerases (PPIases), which catalyze the trans to cis isomerization of peptidyl-prolyl bonds, and thiol-disulfide oxidoreductases, which promote the formation and isomerization of disulfide bridges (e.g., the Dsb proteins of the E. coli periplasm). The final participants in the protein quality control machinery are proteases which degrade irreversibly damaged polypeptides that cannot be rescued by the above pathways. Nevertheless, nature loves to frustrate reductionist efforts and it has recently become apparent that functional lines are often blurred. For instance, PPIases and oxidoreductases can combine chaperone and foldase functions, certain protease components remodel polypeptide substrates and can thus be viewed as chaperones, some proteases have the ability to switch from hydrolase to chaperone function, and there is evidence that certain proteins classified as may exhibit protease activity (see for instance [3-10]). To keep readers abreast of the fast moving field of protein folding, Microbial Cell Factories has commissioned a series of reviews by experts in the topic. The first of these already appeared in January [11]. In Unscrambling and egg: protein disaggregation by AAA+ proteins, Weibezahn, Bukau and Mogk discuss the discovery, functional characterization and mechanism of action of Hsp104/ClpB, two orthologous ATPases that have the unique ability to fragment protein aggregates. The remodeling activity of other AAA+ proteins (e.g., ClpA, ClpX and ClpC) which are best known more for their role in proteolysis is also examined. The authors close their thorough review by presenting enticing evidence for the roles of AAA+ proteins in the propagation of neurodegenerative diseases. In the second paper of the series [12], Miot and Betton review the mechanisms of protein quality control in the periplasmic space of E. coli. After a brief discussion of the biogenesis of proteins destined for export from the bacterial cytoplasm, the authors describe the players involved in the periplasmic folding-degradation decision and how cells sense and respond to extracytoplasmic stress. The second half of the review provides a historical perspective on how the maltose binding protein has served as a useful model to understand the mechanisms of conformational quality control in the periplasm. With forthcoming reviews on disulfide bridge formation and in vitro refolding, it is a great time to turn to Microbial Cell Factories to keep up with protein folding.

The Structure of ClpB: A Molecular Chaperone that Rescues Proteins from an Aggregated State

Molecular chaperones assist protein folding by facilitating their “forward” folding and preventing aggregation. However, once aggregates have formed, these chaperones cannot facilitate protein disaggregation. Bacterial ClpB and its eukaryotic homolog Hsp104 are essential proteins of the heat-shock response, which have the remarkable capacity to rescue stress-damaged proteins from an aggregated state. We have determined the structure of Thermus thermophilus ClpB ( TClpB) using a combination of X-ray crystallography and cryo-electron microscopy (cryo-EM). Our single-particle reconstruction shows that TClpB forms a two-tiered hexameric ring. The ClpB/Hsp104-linker consists of an 85 Å long and mobile coiled coil that is located on the outside of the hexamer. Our mutagenesis and biochemical data show that both the relative position and motion of this coiled coil are critical for chaperone function. Taken together, we propose a mechanism by which an ATP-driven conformational change is coupled to a large coiled-coil motion, which is indispensable for protein disaggregation.

Functional and molecular identification of novel members of the ubiquitous membrane fusion proteins alpha- and gamma-SNAP (soluble N-ethylmaleimide-sensitive factor-attachment proteins) families in Dictyostelium discoideum.

The soluble N-ethylmaleimide-sensitive-factor-attachment proteins (SNAP) are eukaryotic soluble proteins required for membrane fusion. Based on their initial identification in bovine brain cytosol, they are divided in alpha/beta and gamma subfamilies. SNAPs act as adapters between N-ethylmaleimide-sensitive factor (NSF), a hexameric ATPase, and membrane SNARE proteins (SNAP receptors). Within the NSF/SNAP/SNARE complex, SNAPs contribute to the catalysis of an ATP-driven conformational change in the SNAREs, resulting in dissociation of the complex. We have constructed a Dictyostelium discoideum strain overexpressing a c-myc-tagged form of D. discoideum NSF (NSF-myc). Its immunoprecipitation from detergent-solubilized membrane extracts reveals two associated polypeptides with apparent molecular masses of 33 and 36 kDa (p33 and p36) that are absent in NSF-myc immunoprecipitates from cytosol. Analysis of trypsin-digested peptides by microsequencing and mass spectrometry and comparison with cDNA sequences identify p33 and p36 as the D. discoideum homologues of alpha- and gamma-SNAP, respectively. The alpha-/gamma-SNAP molar ratio is close to 3 in vegetative amoebae from this organism. The molecular identification of gamma-SNAP in plants (Arabidopsis thaliana) and insects (Drosophila melanogaster) documents, for the first time, the wide distribution of the gamma subtype. Altogether, these results suggest a specific role for gamma-SNAP, distinct from that of alpha-SNAP.

The power stroke of the DnaK/DnaJ/GrpE molecular chaperone system

The molecular chaperone DnaK, the Hsp70 homolog of Escherichia coli, acts in concert with the co-chaperones DnaJ and GrpE. The aim of this study was to identify the particular phase of the peptide binding-release cycle of the DnaK/DnaJ/GrpE system that is directly responsible for the chaperone effects. By real-time kinetic measurements of changes in the intrinsic fluorescence of DnaK and in the fluorescence of dansyl-labeled peptide ligands, the rates of the following steps in the chaperone cycle were determined: (1) binding of target peptide to fast-binding-and-releasing, low-affinity DnaK·ATP; (2) DnaJ-triggered conversion of peptide·DnaK·ATP (T state) to slowly-acting, high-affinity peptide·DnaK·ADP·Pi (R state); (3) switch from R to T state induced by GrpE-facilitated ADP/ATP exchange; (4) release of peptide. Under conditions approximating those in the cell, the apparent rate constants for the T→R and R→T conversion were 0.04 s−1 and 1.0 s−1, respectively. The clearly rate-limiting T→R conversion renders the R state a minor form of DnaK that cannot account for the chaperone effects. Because DnaK in the absence of the co-chaperones is chaperone-ineffective, the T state has also to be excluded. Apparently, the slow, ATP-driven conformational change T→R is the key step in the DnaK/DnaJ/GrpE chaperone cycle underlying the chaperone effects such as the prevention of protein aggregation, disentangling of polypeptide chains and, in the case of eukaryotic Hsp70 homologs, protein translocation through membranes or uncoating of clathrin-coated vesicles.