scholarly journals Analysis of the Structural Mechanism of ATP Inhibition at the AAA1 Subunit of Cytoplasmic Dynein-1 Using a Chemical “Toolkit”

2021 ◽  
Vol 22 (14) ◽  
pp. 7704
Author(s):  
Sayi’Mone Tati ◽  
Laleh Alisaraie
Keyword(s):  
Binding Affinity ◽  
Atp Hydrolysis ◽  
Critical Role ◽  
Motor Protein ◽  
Structural Data ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Motor Domain ◽  
Conformational Search ◽  
Structural Mechanism

Dynein is a ~1.2 MDa cytoskeletal motor protein that carries organelles via retrograde transport in eukaryotic cells. The motor protein belongs to the ATPase family of proteins associated with diverse cellular activities and plays a critical role in transporting cargoes to the minus end of the microtubules. The motor domain of dynein possesses a hexameric head, where ATP hydrolysis occurs. The presented work analyzes the structure–activity relationship (SAR) of dynapyrazole A and B, as well as ciliobrevin A and D, in their various protonated states and their 46 analogues for their binding in the AAA1 subunit, the leading ATP hydrolytic site of the motor domain. This study exploits in silico methods to look at the analogues’ effects on the functionally essential subsites of the motor domain of dynein 1, since no similar experimental structural data are available. Ciliobrevin and its analogues bind to the ATP motifs of the AAA1, namely, the walker-A (W-A) or P-loop, the walker-B (W-B), and the sensor I and II. Ciliobrevin A shows a better binding affinity than its D analogue. Although the double bond in ciliobrevin A and D was expected to decrease the ligand potency, they show a better affinity to the AAA1 binding site than dynapyrazole A and B, lacking the bond. In addition, protonation of the nitrogen atom in ciliobrevin A and D, as well as dynapyrazole A and B, at the N9 site of ciliobrevin and the N7 of the latter increased their binding affinity. Exploring ciliobrevin A geometrical configuration suggests the E isomer has a superior binding profile over the Z due to binding at the critical ATP motifs. Utilizing the refined structure of the motor domain obtained through protein conformational search in this study exhibits that Arg1852 of the yeast cytoplasmic dynein could involve in the “glutamate switch” mechanism in cytoplasmic dynein 1 in lieu of the conserved Asn in AAA+ protein family.

scholarly journals Elucidation of Structural Mechanism of ATP Inhibition at the AAA1 Subunit of Cytoplasmic Dynein 1 Using a Chemical "Toolkit"

2021 ◽  
Author(s):  
Sayi'Mone Martinet Tati ◽  
Laleh Alisaraie
Keyword(s):  
Binding Site ◽  
Binding Affinity ◽  
Atp Hydrolysis ◽  
Critical Role ◽  
Motor Protein ◽  
Structural Data ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Motor Domain ◽  
Conformational Search

Dynein is a cytoskeletal motor protein that carries organelles via retrograde transport in eukaryotic cells. The motor protein belongs to the ATPase family of proteins associated with diverse cellular activities and plays a critical role in transporting cargoes to the minus end of the microtubules. The motor domain of dynein possesses a hexameric head, where ATP hydrolysis occurs. The AAA1 binding site is the leading ATP hydrolytic site, followed by the AAA3 subsite. Small-molecule ATP competitive inhibitors of dynein are thought to target the AAA1 site. The presented work elucidates the structure-activity relationship of dynapyrazole A and B, ciliobrevin A and D in their various protonated states and their 46 analogs for their binding properties in the nucleotide-binding site of the AAA1 subunit and their effects on the functionally essential subsites of the motor domain of cytoplasmic dynein 1, as there is currently no similar experimental structural data available. Ciliobrevin and its analogs bind to the ATP motifs of the AAA1, namely the Walker-A or P-loop, the Walker-B, and the sensor I and II. Ciliobrevin A shows a better binding affinity to the AAA1 binding site of dynein 1 than its D analog. Although the double bond in ciliobrevin A and D was expected to decrease the ligand potency, they show a better affinity to the AAA1 binding site than dynapyrazole A and B, lacking the bond. Protonation of the nitrogen in ciliobrevin A, D, dynapyrazole A, and B at the N9 site of ciliobrevin, and the N7 of the latter increased their binding affinity. Exploring ciliobrevin A geometrical configuration suggests the E isomer has a superior binding profile over the Z due to binding at the critical ATP motifs. Utilizing the refined structure of the motor domain obtained through protein conformational search in this study exhibits that Arg1852 of the yeast cytoplasmic dynein could involve in the "glutamate switch" mechanism in cytoplasmic dynein 1 in lieu of the conserved Asn in AAA+ protein family, as the guanidine moiety of the Arg engages in an H-bond with the carboxylate moiety of Glu1849.

Structure of the γ–ε complex of cyanobacterial F1-ATPase reveals a suppression mechanism of the γ subunit on ATP hydrolysis in phototrophs

2018 ◽  
Vol 475 (18) ◽  
pp. 2925-2939 ◽  
Author(s):  
Satoshi Murakami ◽  
Kumiko Kondo ◽  
Shinya Katayama ◽  
Satoshi Hara ◽  
Ei-ichiro Sunamura ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Atp Hydrolysis ◽  
Critical Role ◽  
Structural Data ◽  
Hairpin Structure ◽  
Critical Function ◽  
F1 Atpase ◽  
Γ Subunit ◽  
Suppression Mechanism ◽  
Biochemical Analyses

F1-ATPase forms the membrane-associated segment of F0F1-ATP synthase — the fundamental enzyme complex in cellular bioenergetics for ATP hydrolysis and synthesis. Here, we report a crystal structure of the central F1 subcomplex, consisting of the rotary shaft γ subunit and the inhibitory ε subunit, from the photosynthetic cyanobacterium Thermosynechococcus elongatus BP-1, at 1.98 Å resolution. In contrast with their homologous bacterial and mitochondrial counterparts, the γ subunits of photosynthetic organisms harbour a unique insertion of 35–40 amino acids. Our structural data reveal that this region forms a β-hairpin structure along the central stalk. We identified numerous critical hydrogen bonds and electrostatic interactions between residues in the hairpin and the rest of the γ subunit. To elaborate the critical function of this β-hairpin in inhibiting ATP hydrolysis, the corresponding domain was deleted in the cyanobacterial F1 subcomplex. Biochemical analyses of the corresponding α3β3γ complex confirm that the clinch of the hairpin structure plays a critical role and accounts for a significant interaction in the α3β3 complex to induce ADP inhibition during ATP hydrolysis. In addition, we found that truncating the β-hairpin insertion structure resulted in a marked impairment of the interaction with the ε subunit, which binds to the opposite side of the γ subunit from the β-hairpin structure. Combined with structural analyses, our work provides experimental evidence supporting the molecular principle of how the insertion region of the γ subunit suppresses F1 rotation during ATP hydrolysis.

scholarly journals Role of a Class Dhc1b Dynein in Retrograde Transport of Ift Motors and Ift Raft Particles along Cilia, but Not Dendrites, in Chemosensory Neurons of Living Caenorhabditis elegans

1999 ◽  
Vol 147 (3) ◽  
pp. 519-530 ◽  
Author(s):  
Dawn Signor ◽  
Karen P. Wedaman ◽  
Jose T. Orozco ◽  
Noelle D. Dwyer ◽  
Cornelia I. Bargmann ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Neuronal Cell ◽  
Motor Protein ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Neuronal Cell Body ◽  
Transport Pathway ◽  
Chemosensory Neurons ◽  
Sensory Cilia

The heterotrimeric motor protein, kinesin-II, and its presumptive cargo, can be observed moving anterogradely at 0.7 μm/s by intraflagellar transport (IFT) within sensory cilia of chemosensory neurons of living Caenorhabditis elegans, using a fluorescence microscope–based transport assay (Orozco, J.T., K.P. Wedaman, D. Signor, H. Brown, L. Rose, and J.M. Scholey. 1999. Nature. 398:674). Here, we report that kinesin-II, and two of its presumptive cargo molecules, OSM-1 and OSM-6, all move at ∼1.1 μm/s in the retrograde direction along cilia and dendrites, which is consistent with the hypothesis that these proteins are retrieved from the distal endings of the cilia by a retrograde transport pathway that moves them along cilia and then dendrites, back to the neuronal cell body. To test the hypothesis that the minus end–directed microtubule motor protein, cytoplasmic dynein, drives this retrograde transport pathway, we visualized movement of kinesin-II and its cargo along dendrites and cilia in a che-3 cytoplasmic dynein mutant background, and observed an inhibition of retrograde transport in cilia but not in dendrites. In contrast, anterograde IFT proceeds normally in che-3 mutants. Thus, we propose that the class DHC1b cytoplasmic dynein, CHE-3, is specifically responsible for the retrograde transport of the anterograde motor, kinesin-II, and its cargo within sensory cilia, but not within dendrites.

scholarly journals Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning

2011 ◽  
Vol 22 (15) ◽  
pp. 2690-2701 ◽  
Author(s):  
Melissa D. Stuchell-Brereton ◽  
Amanda Siglin ◽  
Jun Li ◽  
Jeffrey K. Moore ◽  
Shubbir Ahmed ◽  
...  
Keyword(s):  
Light Chain ◽  
Direct Evidence ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Dynein Light Chain ◽  
Spindle Positioning ◽  
Dynein Motor ◽  
Intermediate Chains ◽  
Activator Complex

Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function.

scholarly journals Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase

2018 ◽  
Vol 115 (43) ◽  
pp. E10041-E10048 ◽  
Author(s):  
J. Brooks Crickard ◽  
Kyle Kaniecki ◽  
Youngho Kwon ◽  
Patrick Sung ◽  
Eric C. Greene
Keyword(s):  
Chromosome Segregation ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Potent Inhibitor ◽  
Chromosomal Rearrangements ◽  
Motor Protein ◽  
Product Formation ◽  
Gross Chromosomal Rearrangements ◽  
Strand Annealing ◽  
Hydrolysis Activity

Cross-over recombination products are a hallmark of meiosis because they are necessary for accurate chromosome segregation and they also allow for increased genetic diversity during sexual reproduction. However, cross-overs can also cause gross chromosomal rearrangements and are therefore normally down-regulated during mitotic growth. The mechanisms that enhance cross-over product formation upon entry into meiosis remain poorly understood. In Saccharomyces cerevisiae, the Superfamily 1 (Sf1) helicase Srs2, which is an ATP hydrolysis-dependent motor protein that actively dismantles recombination intermediates, promotes synthesis-dependent strand annealing, the result of which is a reduction in cross-over recombination products. Here, we show that the meiosis-specific recombinase Dmc1 is a potent inhibitor of Srs2. Biochemical and single-molecule assays demonstrate that Dmc1 acts by inhibiting Srs2 ATP hydrolysis activity, which prevents the motor protein from undergoing ATP hydrolysis-dependent translocation on Dmc1-bound recombination intermediates. We propose a model in which Dmc1 helps contribute to cross-over formation during meiosis by antagonizing the antirecombinase activity of Srs2.

scholarly journals Подвижность ионных носителей заряда в пьезоэлектрических кристаллах Li-=SUB=-2-=/SUB=-B-=SUB=-4-=/SUB=-O-=SUB=-7-=/SUB=-

2020 ◽  
Vol 62 (3) ◽  
pp. 386
Author(s):  
Н.И. Сорокин ◽  
Ю.В. Писаревский ◽  
В.В. Гребенев ◽  
В.А. Ломонов
Keyword(s):  
Crystal Lattice ◽  
Carrier Mobility ◽  
Room Temperature ◽  
Lithium Ion ◽  
Structural Data ◽  
Crystallographic Axis ◽  
Frequency Range ◽  
Impedance Measurements ◽  
Structural Mechanism ◽  
Ag Electrodes

The impedance measurements of Li2B4O7 single crystal with Ag electrodes in the frequency range 1-3*107 Hz at room temperature have been made. The Li2B4O7 crystal (sp. gr. I41cd, Z = 8) was oriented along crystallographic axis c. Contributions from the bulk crystal and crystal / electrode boundaries in the impedance hodograph of the Ag | Li2B4O7 | Ag system were selected. The structural mechanism of lithium-ion transport in Li2B4O7 has been discussed. Based on electrophysical and structural data, the conductivity σdc = 2.3 × 10–9 S / cm, carrier mobility (vacancies VLi) μmob = 6 × 10−10 cm2 / sV and their concentration nmob = 2.4 × 1019 cm – 3 (0.14% of the amount of lithium in the crystal lattice) have been determined.

scholarly journals Molecular Determinants of μ-Conotoxin KIIIA Interaction with the Voltage-Gated Sodium Channel Nav1.7

2019 ◽  
Author(s):  
Ian H. Kimball ◽  
Phuong T. Nguyen ◽  
Baldomero M. Olivera ◽  
Jon T. Sack ◽  
Vladimir Yarov-Yarovoy
Keyword(s):  
Computational Modeling ◽  
Rational Design ◽  
Structural Model ◽  
Critical Role ◽  
Structural Data ◽  
Molecular Probes ◽  
Integrative Approach ◽  
In Silico Docking ◽  
Voltage Gated ◽  
Dynamics Simulations

AbstractThe voltage-gated sodium (Nav) channel subtype Nav1.7 plays a critical role in pain signaling, making it an important drug target. Here we studied the molecular interactions between μ-conotoxin KIIIA (KIIIA) and the human Nav1.7 channel (hNav1.7). We developed a structural model of hNav1.7 using Rosetta computational modeling and performed in silico docking of KIIIA using RosettaDock to predict residues forming specific pairwise contacts between KIIIA and hNav1.7. We experimentally validated these contacts using mutant cycle analysis. Comparison between our KIIIA-hNav1.7 model and the recently published cryo-EM structure of KIIIA-hNav1.2 revealed key similarities and differences between channel subtypes with potential implications for the molecular mechanism of toxin block. Our integrative approach, combining structural data with computational modeling, experimental validation, and molecular dynamics simulations will be useful for engineering molecular probes to study Nav channel function, and for rational design of novel biologics targeting specific Nav channels.

scholarly journals Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sean P. Carney ◽  
Wen Ma ◽  
Kevin D. Whitley ◽  
Haifeng Jia ◽  
Timothy M. Lohman ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Variable Number ◽  
Structural Basis ◽  
Variable Step Size ◽  
Dna Unwinding ◽  
Step Size ◽  
Structural Mechanism ◽  
Dynamics Simulations

AbstractUvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

scholarly journals Functional role of residues involved in substrate binding of human 4-hydroxyphenylpyruvate dioxygenase

2021 ◽  
Author(s):  
Chih-Wei Huang ◽  
Chi-Ching Hwang ◽  
Yung-Lung Chang ◽  
Jen-Tzu Liu ◽  
Sheng-Peng Wu ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Functional Role ◽  
Substrate Binding ◽  
Critical Role ◽  
Intermediate Formation ◽  
Hydroxyl Group ◽  
Double Mutation ◽  
Closed Conformation ◽  
Bond Network

4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E’s Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.

Rab3D directs organelle transport by recruiting cytoplasmic dynein motor protein Tctex-1 to secretory membranes of osteoclast

Bone ◽  
2009 ◽  
Vol 44 ◽  
pp. S160 ◽  
Author(s):  
N.J. Pavlos ◽  
J. Xu ◽  
H. Feng ◽  
P. Ng ◽  
T. Cheng ◽  
...  
Keyword(s):  
Motor Protein ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Dynein Motor
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