Chromokinesins NOD and KID Use Distinct ATPase Mechanisms and Microtubule Interactions to Perform a Similar Function

Chromokinesins NOD and KID have similar DNA binding domains and functions during cell division, while their motor domain sequences show significant variations. It has been unclear whether these motors have similar structure, chemistry, and microtubule interactions necessary to follow a similar mechanism of force mediation. We used biochemical rate measurements, cosedimentation, and structural analysis to investigate the ATPase mechanisms of the NOD and KID core domains. These experiments and analysis revealed that NOD and KID have different ATPase mechanisms, microtubule interactions, and catalytic domain structures. The ATPase cycles of NOD and KID have different rate limiting steps. The ATPase rate of NOD was robustly stimulated by microtubules albeit its microtubule affinity was weakened in all nucleotide bound states. KID bound microtubules tightly in all nucleotide states and remained associated with the microtubule for more than 100 cycles of ATP hydrolysis before dissociating. The structure of KID was most similar to conventional kinesin (KIF5). Key differences in the microtubule binding region and allosteric communication pathway between KID and NOD are consistent with our biochemical data. Our results support the model that NOD and KID utilize distinct mechanistic pathways to achieve the same function during cell division.

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Purification and Characterization of the AAA+ Domain of Sinorhizobium meliloti DctD, a σ54-Dependent Transcriptional Activator

Journal of Bacteriology ◽

10.1128/jb.186.11.3499-3507.2004 ◽

2004 ◽

Vol 186 (11) ◽

pp. 3499-3507 ◽

Cited By ~ 22

Author(s):

Hao Xu ◽

Baohua Gu ◽

B. Tracy Nixon ◽

Timothy R. Hoover

Keyword(s):

Dna Binding ◽

Rna Polymerase ◽

Sinorhizobium Meliloti ◽

Atp Hydrolysis ◽

Binding Domain ◽

Stable Complex ◽

Dna Binding Domain ◽

Dna Binding Domains ◽

Binding Domains

ABSTRACT Activators of σ54-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open complex between the promoter and RNA polymerase. These activators are modular, consisting of an N-terminal regulatory domain, a C-terminal DNA-binding domain, and a central activation domain belonging to the AAA+ superfamily of ATPases. The AAA+ domain of Sinorhizobium meliloti C4-dicarboxylic acid transport protein D (DctD) is sufficient to activate transcription. Deletion analysis of the 3′ end of dctD identified the minimal functional C-terminal boundary of the AAA+ domain of DctD as being located between Gly-381 and Ala-384. Histidine-tagged versions of the DctD AAA+ domain were purified and characterized. The DctD AAA+ domain was significantly more soluble than DctD( Δ 1-142), a truncated DctD protein consisting of the AAA+ and DNA-binding domains. In addition, the DctD AAA+ domain was more homogeneous than DctD( Δ 1-142) when analyzed by native gel electrophoresis, migrating predominantly as a single high-molecular-weight species, while DctD( Δ 1-142) displayed multiple species. The DctD AAA+ domain, but not DctD( Δ 1-142), formed a stable complex with σ54 in the presence of the ATP transition state analogue ADP-aluminum fluoride. The DctD AAA+ domain activated transcription in vitro, but many of the transcripts appeared to terminate prematurely, suggesting that the DctD AAA+ domain interfered with transcription elongation. Thus, the DNA-binding domain of DctD appears to have roles in controlling the oligomerization of the AAA+ domain and modulating interactions with σ54 in addition to its role in recognition of upstream activation sequences.

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Dynamics of the HD regulatory subdomain of PARP-1; substrate access and allostery in PARP activation and inhibition

10.1101/2020.09.04.283309 ◽

2020 ◽

Author(s):

Tom E.H. Ogden ◽

Ji-Chun Yang ◽

Marianne Schimpl ◽

Laura E. Easton ◽

Elizabeth Underwood ◽

...

Keyword(s):

Dna Binding ◽

Active Site ◽

Catalytic Domain ◽

Parp Activation ◽

Dna Binding Domains ◽

Binding Domains ◽

Single Strand Breaks ◽

Folded Structure ◽

Early Responder ◽

Parp 1

ABSTRACTPARP-1 is a key early responder to DNA damage in eukaryotic cells. An allosteric mechanism links initial sensing of DNA single-strand breaks by PARP-1’s F1 and F2 domains via a process of further domain assembly to activation of the catalytic domain (CAT); synthesis and attachment of poly(ADP-ribose) (PAR) chains to protein sidechains then signals for assembly of DNA repair components. A key component in transmission of the allosteric signal is the HD subdomain of CAT, which alone bridges between the assembled DNA-binding domains and the active site in the ART subdomain of CAT. Here we present a study of isolated CAT domain from human PARP-1, using NMR-based dynamics experiments to analyse WT apo-protein as well as a set of inhibitor complexes (with veliparib, olaparib, talazoparib and EB-47) and point mutants (L713F, L765A and L765F), together with new crystal structures of the free CAT domain and inhibitor complexes. Variations in both dynamics and structures amongst these species point to a model for full-length PARP-1 activation where first DNA binding and then substrate interaction successively destabilise the folded structure of the HD subdomain to the point where its steric blockade of the active site is released and PAR synthesis can proceed.

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Computational Insights into Allosteric Conformational Modulation of P-Glycoprotein by Substrate and Inhibitor Binding

Molecules ◽

10.3390/molecules25246006 ◽

2020 ◽

Vol 25 (24) ◽

pp. 6006

Author(s):

Juan Xing ◽

Shuheng Huang ◽

Yu Heng ◽

Hu Mei ◽

Xianchao Pan

Keyword(s):

Conformational Changes ◽

Atp Hydrolysis ◽

Molecular Design ◽

Conformational Dynamics ◽

Water Environment ◽

Inhibitor Binding ◽

Binding Domains ◽

Allosteric Communication ◽

P Glycoprotein ◽

Dynamics Simulations

The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) is a physiologically essential membrane protein that protects many tissues against xenobiotic molecules, but limits the access of chemotherapeutics into tumor cells, thus contributing to multidrug resistance. The atomic-level mechanism of how substrates and inhibitors differentially affect the ATP hydrolysis by P-gp remains to be elucidated. In this work, atomistic molecular dynamics simulations in an explicit membrane/water environment were performed to explore the effects of substrate and inhibitor binding on the conformational dynamics of P-gp. Distinct differences in conformational changes that mainly occurred in the nucleotide-binding domains (NBDs) were observed from the substrate- and inhibitor-bound simulations. The binding of rhodamine-123 can increase the probability of the formation of an intermediate conformation, in which the NBDs were closer and better aligned, suggesting that substrate binding may prime the transporter for ATP hydrolysis. By contrast, the inhibitor QZ-Leu stabilized NBDs in a much more separated and misaligned conformation, which may result in the deficiency of ATP hydrolysis. The significant differences in conformational modulation of P-gp by substrate and inhibitor binding provided a molecular explanation of how these small molecules exert opposite effects on the ATPase activity. A further structural analysis suggested that the allosteric communication between transmembrane domains (TMDs) and NBDs was primarily mediated by two intracellular coupling helices. Our computational simulations provide not only valuable insights into the transport mechanism of P-gp substrates, but also for the molecular design of P-gp inhibitors.

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Slow, heterogeneous NFκB single molecule conformational dynamics and implications for function

10.1101/2021.03.02.433616 ◽

2021 ◽

Author(s):

Wei Chen ◽

Wei Liu ◽

Peter Wolynes ◽

Elizabeth A. Komives

Keyword(s):

Dna Binding ◽

Single Molecule ◽

Electrostatic Interactions ◽

Bound States ◽

Conformational Dynamics ◽

Dna Binding Domains ◽

Single Molecule Fret ◽

Binding Domains ◽

Lower Amplitude ◽

Slow Motions

The transcription factor NFκB (RelA-p50) is a multidomain protein that binds DNA and its inhibitor, IκBα with apparently different conformations. We used single-molecule FRET to characterize the interdomain motions of the N-terminal DNA-binding domains in the free protein and also in various bound states. Several surprising results emerged from this study. First, the domains moved with respect to each other on several widely different timescales from hundreds of milliseconds to minutes. The free NFκB displayed stochastic motions leading to a broad distribution of states, ranging from very low-FRET states to high-FRET states. Varying the ionic strength altered the slow motions suggesting that they may be due to different weak electrostatic interactions between the domains creating a rugged energy landscape. Third, the DNA-binding domains continued to be mobile even when the protein was bound to its cognate DNA, but in this case the majority of the states were either high-FRET, a state expected from the available x-ray structures, or low-FRET, a state consistent with one of the DNA-binding domains dissociated. The fluctuations of the DNA-bound states were of lower amplitude and slightly faster frequency. Fourth, the inhibitor, IκBα freezes the domains into a low-FRET state, expected to be incapable of binding DNA. Neutralization of five acidic residues in the IκBα PEST sequence, which was previously shown to impair IκBαs ability to strip NFκB from the DNA, also impaired its ability to freeze the domains into a low-FRET state indicating that the freezing of motions of the DNA-binding domains is essential for efficient molecular stripping.

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NAD+ analog reveals PARP-1 substrate-blocking mechanism and allosteric communication from catalytic center to DNA-binding domains

Nature Communications ◽

10.1038/s41467-018-03234-8 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 65

Author(s):

Marie-France Langelier ◽

Levani Zandarashvili ◽

Pedro M. Aguiar ◽

Ben E. Black ◽

John M. Pascal

Keyword(s):

Dna Binding ◽

Catalytic Center ◽

Dna Binding Domains ◽

Binding Domains ◽

Allosteric Communication ◽

Blocking Mechanism ◽

Parp 1

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Disruption of the Unique ABCG-Family NBD:NBD Interface Impacts Both Drug Transport and ATP Hydrolysis

International Journal of Molecular Sciences ◽

10.3390/ijms21030759 ◽

2020 ◽

Vol 21 (3) ◽

pp. 759

Author(s):

Parth Kapoor ◽

Deborah A. Briggs ◽

Megan H. Cox ◽

Ian D. Kerr

Keyword(s):

Atpase Activity ◽

Drug Transport ◽

Atp Hydrolysis ◽

Contact Region ◽

Chemotherapy Resistance ◽

Dimer Interface ◽

Binding Domains ◽

Related Family ◽

Allosteric Communication ◽

C Terminus

ABCG2 is one of a triumvirate of human multidrug ATP binding cassette (ABC) transporters that are implicated in the defense of cells and tissues against cytotoxic chemicals, but these transporters can also confer chemotherapy resistance states in oncology. Understanding the mechanism of ABCG2 is thus imperative if we are to be able to counter its deleterious activity. The structure of ABCG2 and its related family members (ABCG5/G8) demonstrated that there were two interfaces between the nucleotide binding domains (NBD). In addition to the canonical ATP “sandwich-dimer” interface, there was a second contact region between residues at the C-terminus of the NBD. We investigated this second interface by making mutations to a series of residues that are in close interaction with the opposite NBD. Mutated ABCG2 isoforms were expressed in human embryonic kidney (HEK) 293T cells and analysed for targeting to the membrane, drug transport, and ATPase activity. Mutations to this second interface had a number of effects on ABCG2, including altered drug specificity, altered drug transport, and, in two mutants, a loss of ATPase activity. The results demonstrate that this region is particularly sensitive to mutation and can impact not only direct, local NBD events (i.e., ATP hydrolysis) but also the allosteric communication to the transmembrane domains and drug transport.

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Faculty Opinions recommendation of Excess non-synonymous substitutions suggest that positive selection episodes occurred during the evolution of DNA-binding domains in the Arabidopsis R2R3-MYB gene family.

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

10.3410/f.1015386.196756 ◽

2003 ◽

Author(s):

Elena Alvarez-Buylla

Keyword(s):

Dna Binding ◽

Positive Selection ◽

Gene Family ◽

Synonymous Substitutions ◽

Dna Binding Domains ◽

Binding Domains ◽

Myb Gene ◽

R2r3 Myb

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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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Structure and Function of the CFTR Chloride Channel

Physiological Reviews ◽

10.1152/physrev.1999.79.1.s23 ◽

1999 ◽

Vol 79 (1) ◽

pp. S23-S45 ◽

Cited By ~ 647

Author(s):

DAVID N. SHEPPARD ◽

MICHAEL J. WELSH

Keyword(s):

Cystic Fibrosis ◽

Chloride Channel ◽

Atp Hydrolysis ◽

Current Knowledge ◽

Structure And Function ◽

Binding Domains ◽

Transporter Family ◽

Membrane Spanning ◽

And Function ◽

R Domain

Sheppard, David N., and Michael J. Welsh. Structure and Function of the CFTR Chloride Channel. Physiol. Rev. 79 , Suppl.: S23–S45, 1999. — The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ABC transporter family that forms a novel Cl− channel. It is located predominantly in the apical membrane of epithelia where it mediates transepithelial salt and liquid movement. Dysfunction of CFTR causes the genetic disease cystic fibrosis. The CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. Here we review the structure and function of this unique channel, with a focus on how the various domains contribute to channel function. The MSDs form the channel pore, phosphorylation of the R domain determines channel activity, and ATP hydrolysis by the NBDs controls channel gating. Current knowledge of CFTR structure and function may help us understand better its mechanism of action, its role in electrolyte transport, its dysfunction in cystic fibrosis, and its relationship to other ABC transporters.

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