Conformational Changes During the ATP Hydrolysis Cycle of the Multidrug Transporter P-Glycoproteinin Response to Substrate Binding

The Pgp (P-glycoprotein) multidrug transporter couples ATP hydrolysis at two cytoplasmic NBDs (nucleotide-binding domains) to the transport of hydrophobic compounds. Orthovanadate (Vi) and fluoroaluminate (AlFx) trap nucleotide in one NBD by forming stable catalytically inactive complexes (Pgp–M2+–ADP–X), which are proposed to resemble the catalytic transition state, whereas the complex formed by beryllium fluoride (BeFx) is proposed to resemble the ground state. We studied the trapped complexes formed via incubation of Pgp with ATP (catalytically forward) or ADP (reverse) and Vi, BeFx or AlFx using Mg2+ or Co2+ as the bivalent cation. Quenching of intrinsic Pgp tryptophan fluorescence by acrylamide, iodide and caesium indicated that conformational changes took place upon formation of the trapped complexes. Trapping with Vi and ATP led to a 6-fold increase in the acrylamide quenching constant, KSV, suggesting that large conformational changes take place in the Pgp transmembrane regions on trapping in the forward direction. Trapping with Vi and ADP gave only a small change in quenching, indicating that the forward- and reverse-trapped complexes are different. TNP (trinitrophenyl)–ATP/TNP–ADP interacted with all of the trapped complexes, however, the fluorescence enhancement differed for the trapped states, suggesting a change in polarity in the nucleotide-binding sites. The nucleotide-binding site of the BeFx-trapped complex was much more polar than that of the Vi and AlFx complexes. Functionally, all the trapped complexes were able to bind drugs and TNP–nucleotides with unchanged affinity compared with native Pgp.

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Conformational Changes in the Multidrug Transporter EmrE Associated with Substrate Binding

Journal of Molecular Biology ◽

10.1016/s0022-2836(03)00895-7 ◽

2003 ◽

Vol 332 (1) ◽

pp. 229-242 ◽

Cited By ~ 60

Author(s):

Christopher G Tate ◽

Iban Ubarretxena-Belandia ◽

Joyce M Baldwin

Keyword(s):

Conformational Changes ◽

Substrate Binding ◽

Multidrug Transporter

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ClpB N-terminal domain plays a regulatory role in protein disaggregation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1512783112 ◽

2015 ◽

Vol 112 (50) ◽

pp. E6872-E6881 ◽

Cited By ~ 42

Author(s):

Rina Rosenzweig ◽

Patrick Farber ◽

Algirdas Velyvis ◽

Enrico Rennella ◽

Michael P. Latham ◽

...

Keyword(s):

Conformational Changes ◽

Atp Hydrolysis ◽

Protein Translocation ◽

Substrate Binding ◽

Regulatory Role ◽

Substrate Interaction ◽

Terminal Domain ◽

Client Proteins ◽

Central Pore ◽

Binding Groove

ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. The ClpB–substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD–substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD–substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD–substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation.

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Faculty Opinions recommendation of Atomic snapshots of an RNA packaging motor reveal conformational changes linking ATP hydrolysis to RNA translocation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1021264.243506 ◽

2004 ◽

Author(s):

Jose Carrascosa

Keyword(s):

Conformational Changes ◽

Atp Hydrolysis ◽

Rna Packaging

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Insights into the roles of charged residues in substrate binding and mode of action of mannuronan C-5 epimerase AlgE4

Glycobiology ◽

10.1093/glycob/cwab025 ◽

2021 ◽

Author(s):

Margrethe Gaardløs ◽

Sergey A Samsonov ◽

Marit Sletmoen ◽

Maya Hjørnevik ◽

Gerd Inger Sætrom ◽

...

Keyword(s):

Optical Tweezers ◽

Conformational Changes ◽

Molecular Mechanisms ◽

Hydrophobic Interactions ◽

Substrate Binding ◽

Interdisciplinary Approach ◽

Product Formation ◽

Titration Calorimetry ◽

Binding Groove ◽

Dynamics Simulations

Abstract Mannuronan C-5 epimerases catalyse the epimerization of monomer residues in the polysaccharide alginate, changing the physical properties of the biopolymer. The enzymes are utilized to tailor alginate to numerous biological functions by alginate-producing organisms. The underlying molecular mechanisms that control the processive movement of the epimerase along the substrate chain is still elusive. To study this, we have used an interdisciplinary approach combining molecular dynamics simulations with experimental methods from mutant studies of AlgE4, where initial epimerase activity and product formation were addressed with NMR spectroscopy, and characteristics of enzyme-substrate interactions were obtained with isothermal titration calorimetry and optical tweezers. Positive charges lining the substrate-binding groove of AlgE4 appear to control the initial binding of poly-mannuronate, and binding also seems to be mediated by both electrostatic and hydrophobic interactions. After the catalytic reaction, negatively charged enzyme residues might facilitate dissociation of alginate from the positive residues, working like electrostatic switches, allowing the substrate to translocate in the binding groove. Molecular simulations show translocation increments of two monosaccharide units before the next productive binding event resulting in MG-block formation, with the epimerase moving with its N-terminus towards the reducing end of the alginate chain. Our results indicate that the charge pair R343-D345 might be directly involved in conformational changes of a loop that can be important for binding and dissociation. The computational and experimental approaches used in this study complement each other, allowing for a better understanding of individual residues’ roles in binding and movement along the alginate chains.

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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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Myosinome: A Database of Myosins from Select Eukaryotic Genomes to Facilitate Analysis of Sequence-Structure-Function Relationships

Bioinformatics and Biology Insights ◽

10.4137/bbi.s9902 ◽

2012 ◽

Vol 6 ◽

pp. BBI.S9902 ◽

Cited By ~ 3

Author(s):

Divya P. Syamaladevi ◽

Margaret S Sunitha ◽

S. Kalaimathy ◽

Chandrashekar C. Reddy ◽

Mohammed Iftekhar ◽

...

Keyword(s):

Conformational Changes ◽

Atp Hydrolysis ◽

Homo Sapiens ◽

Relevant Literature ◽

Myosin Ii ◽

Coiled Coil ◽

Structural Features ◽

Model Organisms ◽

Congenital Diseases ◽

C Elegans

Myosins are one of the largest protein superfamilies with 24 classes. They have conserved structural features and catalytic domains yet show huge variation at different domains resulting in a variety of functions. Myosins are molecules driving various kinds of cellular processes and motility until the level of organisms. These are ATPases that utilize the chemical energy released by ATP hydrolysis to bring about conformational changes leading to a motor function. Myosins are important as they are involved in almost all cellular activities ranging from cell division to transcriptional regulation. They are crucial due to their involvement in many congenital diseases symptomatized by muscular malfunctions, cardiac diseases, deafness, neural and immunological dysfunction, and so on, many of which lead to death at an early age. We present Myosinome, a database of selected myosin classes (myosin II, V, and VI) from five model organisms. This knowledge base provides the sequences, phylogenetic clustering, domain architectures of myosins and molecular models, structural analyses, and relevant literature of their coiled-coil domains. In the current version of Myosinome, information about 71 myosin sequences belonging to three myosin classes (myosin II, V, and VI) in five model organisms ( Homo Sapiens, Mus musculus, D. melanogaster, C. elegans and S. cereviseae) identified using bioinformatics surveys are presented, and several of them are yet to be functionally characterized. As these proteins are involved in congenital diseases, such a database would be useful in short-listing candidates for gene therapy and drug development. The database can be accessed from http://caps.ncbs.res.in/myosinome .

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