A molecular understanding of the catalytic cycle of the nucleotide-binding domain of the ABC transporter HlyB

2005 ◽  
Vol 33 (5) ◽  
pp. 990-995 ◽  
Author(s):  
J. Zaitseva ◽  
S. Jenewein ◽  
C. Oswald ◽  
T. Jumpertz ◽  
I.B. Holland ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Conformational Changes ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Central Element ◽  
Nucleotide Binding ◽  
Single Step ◽  
Catalytic Cycle ◽  
Type I ◽  
Individual Crystal

The ABC transporter (ATP-binding-cassette transporter) HlyB (haemolysin B) is the central element of a type I secretion machinery, dedicated to the secretion of the toxin HlyA in Escherichia coli. In addition to the ABC transporter, two other indispensable elements are necessary for the secretion of the toxin across two membranes in a single step: the transenvelope protein HlyD and the outer membrane protein TolC. Despite the fact that the hydrolysis of ATP by HlyB fuels secretion of HlyA, the essential features of the underlying transport mechanism remain an enigma. Similar to all other ABC transporters, ranging from bacteria to man, HlyB is composed of two NBDs (nucleotide-binding domains) and two transmembrane domains. Here we summarize our detailed biochemical, biophysical and structural studies aimed at an understanding of the molecular principles of how ATP-hydrolysis is coupled to energy transduction, including the conformational changes occurring during the catalytic cycle, leading to substrate transport. We have obtained individual crystal structures for each single ground state of the catalytic cycle. From these and other biochemical and mutational studies, we shall provide a detailed molecular picture of the steps governing intramolecular communication and the utilization of chemical energy, due to ATP hydrolysis, in relation to resulting structural changes within the NBD. These data will be summarized in a general model to explain how these molecular machines achieve translocation of molecules across biological membranes.

scholarly journals Oligomerization of EpsE Coordinates Residues from Multiple Subunits to Facilitate ATPase Activity

2011 ◽  
Vol 286 (12) ◽  
pp. 10378-10386 ◽  
Author(s):  
Marcella Patrick ◽  
Konstantin V. Korotkov ◽  
Wim G. J. Hol ◽  
Maria Sandkvist
Keyword(s):  
Atpase Activity ◽  
Conformational Changes ◽  
Active Site ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Hydrolytic Enzymes ◽  
Point Mutations ◽  
Nucleotide Binding ◽  
Type Ii ◽  
Arginine Residues

EpsE is an ATPase that powers transport of cholera toxin and hydrolytic enzymes through the Type II secretion (T2S) apparatus in the Gram-negative bacterium, Vibrio cholerae. On the basis of structures of homologous Type II/IV secretion ATPases and our biochemical data, we believe that EpsE is active as an oligomer, likely a hexamer, and the binding, hydrolysis, and release of nucleotide cause EpsE to undergo dynamic structural changes, thus converting chemical energy to mechanical work, ultimately resulting in extracellular secretion. The conformational changes that occur as a consequence of nucleotide binding would realign conserved arginines (Arg210, Arg225, Arg320, Arg324, Arg336, and Arg369) from adjoining domains and subunits to complete the active site around the bound nucleotide. Our data suggest that these arginines are essential for ATP hydrolysis, although their roles in shaping the active site of EpsE are varied. Specifically, we have shown that replacements of these arginine residues abrogate the T2S process due to a reduction of ATPase activity yet do not have any measurable effect on nucleotide binding or oligomerization of EpsE. We have further demonstrated that point mutations in the EpsE intersubunit interface also reduce ATPase activity without disrupting oligomerization, strengthening the idea that residues from multiple subunits must precisely interact in order for EpsE to be sufficiently active to support T2S. Our findings suggest that the action of EpsE is similar to that of other Type II/IV secretion ATPase family members, and thus these results may be widely applicable to the family as a whole.

scholarly journals Structural basis of mechano-chemical coupling by the mitotic kinesin KIF14

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Matthieu P.M.H. Benoit ◽  
Ana B. Asenjo ◽  
Mohammadjavad Paydar ◽  
Sabin Dhakal ◽  
Benjamin H. Kwok ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Bound State ◽  
Structural Basis ◽  
Coupling Mechanism ◽  
Developmental Defects ◽  
Microtubule Binding

AbstractKIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers. Like other kinesins, KIF14 couples ATP hydrolysis and microtubule binding to the generation of mechanical work, but the coupling mechanism between these processes is still not fully clear. Here we report 20 high-resolution (2.7–3.9 Å) cryo-electron microscopy KIF14-microtubule structures with complementary functional assays. Analysis procedures were implemented to separate coexisting conformations of microtubule-bound monomeric and dimeric KIF14 constructs. The data provide a comprehensive view of the microtubule and nucleotide induced KIF14 conformational changes. It shows that: 1) microtubule binding, the nucleotide species, and the neck-linker domain govern the transition between three major conformations of the motor domain; 2) an undocked neck-linker prevents the nucleotide-binding pocket to fully close and dampens ATP hydrolysis; 3) 13 neck-linker residues are required to assume a stable docked conformation; 4) the neck-linker position controls the hydrolysis rather than the nucleotide binding step; 5) the two motor domains of KIF14 dimers adopt distinct conformations when bound to the microtubule; and 6) the formation of the two-heads-bound-state introduces structural changes in both motor domains of KIF14 dimers. These observations provide the structural basis for a coordinated chemo-mechanical kinesin translocation model.

G-actin conformational change and polymerization induced by paraquat

1998 ◽  
Vol 76 (4) ◽  
pp. 583-591 ◽  
Author(s):  
Isabella DalleDonne ◽  
Aldo Milzani ◽  
Roberto Colombo
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Structural Changes ◽  
Cell Injury ◽  
Atp Hydrolysis ◽  
Protein Composition ◽  
Dnase I ◽  
Cross Linking ◽  
Cytoskeletal Protein ◽  
Actin Assembly

Paraquat (1,1´-dimethyl-4,4´-bipyridilium dichloride) is a broad-spectrum herbicide that is highly toxic to animals (including man), the major lesion being in the lung. In mammalian cells, paraquat causes deep alterations in the organization of the cytoskeleton, marked decreases in cytoskeletal protein synthesis, and alterations in cytoskeletal protein composition; therefore, the involvement of the cytoskeleton in cell injury by paraquat was suggested. We previously demonstrated that monomeric actin binds paraquat; moreover, prolonged actin exposure to paraquat, in depolymerizing medium, induces the formation of actin aggregates, which are built up by F-actin. In this work we have shown that the addition of paraquat to monomeric actin results in a strong quenching of Trp-79 and Trp-86 fluorescence. Trypsin digestion experiments demonstrated that the sequence 61-69 on actin subdomain 2 undergoes paraquat-dependent conformational changes. These paraquat-induced structural changes render actin unable to completely inhibit DNase I. By using intermolecular cross-linking to characterize oligomeric species formed during paraquat-induced actin assembly, we found that the herbicide causes the formation of actin oligomers characterized by subunit-subunit contacts like those occurring in oligomers induced by polymerizing salts (i.e., between subdomain 1 on one actin subunit and subdomain 4 on the adjacent subunit). Furthermore, the oligomerization of G-actin induced by paraquat is paralleled by ATP hydrolysis.Key words: actin, paraquat, subdomain 2, DNase I, ATP hydrolysis.

scholarly journals Characterization of the Catalytic Cycle of ATP Hydrolysis by Human P-glycoprotein

2001 ◽  
Vol 276 (15) ◽  
pp. 11653-11661 ◽  
Author(s):  
Zuben E. Sauna ◽  
Suresh V. Ambudkar
Keyword(s):  
Multidrug Resistance ◽  
Functional Outcomes ◽  
Atp Hydrolysis ◽  
Substrate Binding ◽  
Nucleotide Binding ◽  
Catalytic Cycle ◽  
Nucleotide Binding Sites ◽  
P Glycoprotein ◽  
Random Manner

P-glycoprotein (Pgp) is a plasma membrane protein whose overexpression confers multidrug resistance to tumor cells by extruding amphipathic natural product cytotoxic drugs using the energy of ATP. An elucidation of the catalytic cycle of Pgp would help design rational strategies to combat multidrug resistance and to further our understanding of the mechanism of ATP-binding cassette transporters. We have recently reported (Sauna, Z. E., and Ambudkar, S. V. (2000)Proc. Natl. Acad. Sci. U. S. A.97, 2515–2520) that there are two independent ATP hydrolysis events in a single catalytic cycle of Pgp. In this study we exploit the vanadate (Vi)-induced transition state conformation of Pgp (Pgp·ADP·Vi) to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site. We find that at the end of the first hydrolysis event there is a drastic decrease in the affinity of nucleotide for Pgp coincident with decreased substrate binding. Release of occluded dinucleotide is adequate for the next hydrolysis event to occur but is not sufficient for the recovery of substrate binding. Whereas the two hydrolysis events have different functional outcomesvis à visthe substrate, they show comparablet12for both incorporation and release of nucleotide, and the affinities for [α-32P]8-azido-ATP during Vi-induced trapping are identical. In addition, the incorporation of [α-32P]8-azido-ADP in two ATP sites during both hydrolysis events is also similar. These data demonstrate that during individual hydrolysis events, the ATP sites are recruited in a random manner, and only one site is utilized at any given time because of the conformational change in the catalytic site that drastically reduces the affinity of the second ATP site for nucleotide binding. In aggregate, these findings provide an explanation for the alternate catalysis of ATP hydrolysis and offer a mechanistic framework to elucidate events at both the substrate- and nucleotide-binding sites in the catalytic cycle of Pgp.

scholarly journals New Biophysical Approaches Reveal the Dynamics and Mechanics of Type I Viral Fusion Machinery and Their Interplay with Membranes

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 413 ◽  
Author(s):  
Mark A. Benhaim ◽  
Kelly K. Lee
Keyword(s):  
Membrane Fusion ◽  
Conformational Changes ◽  
Viral Entry ◽  
Fusion Proteins ◽  
Structural Changes ◽  
Fusion Reaction ◽  
Type I ◽  
Viral Fusion ◽  
Technological Advances ◽  
Viral Fusion Proteins

Protein-mediated membrane fusion is a highly regulated biological process essential for cellular and organismal functions and infection by enveloped viruses. During viral entry the membrane fusion reaction is catalyzed by specialized protein machinery on the viral surface. These viral fusion proteins undergo a series of dramatic structural changes during membrane fusion where they engage, remodel, and ultimately fuse with the host membrane. The structural and dynamic nature of these conformational changes and their impact on the membranes have long-eluded characterization. Recent advances in structural and biophysical methodologies have enabled researchers to directly observe viral fusion proteins as they carry out their functions during membrane fusion. Here we review the structure and function of type I viral fusion proteins and mechanisms of protein-mediated membrane fusion. We highlight how recent technological advances and new biophysical approaches are providing unprecedented new insight into the membrane fusion reaction.

ATP-Induced Conformational Changes of Nucleotide-Binding Domains in an ABC Transporter. Importance of the Water-Mediated Entropic Force

2014 ◽  
Vol 118 (44) ◽  
pp. 12612-12620 ◽  
Author(s):  
Tomohiko Hayashi ◽  
Shuntaro Chiba ◽  
Yusuke Kaneta ◽  
Tadaomi Furuta ◽  
Minoru Sakurai
Keyword(s):  
Abc Transporter ◽  
Conformational Changes ◽  
Nucleotide Binding ◽  
Entropic Force ◽  
Binding Domains

scholarly journals Demonstration of Conformational Changes Associated with Activation of the Maltose Transport Complex

2001 ◽  
Vol 276 (15) ◽  
pp. 12362-12368 ◽  
Author(s):  
Daynene E. Mannering ◽  
Susan Sharma ◽  
Amy L. Davidson
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Binding Protein ◽  
Nucleotide Binding ◽  
Wild Type ◽  
Nucleotide Binding Sites ◽  
Atp Binding Cassette Transporter ◽  
Aqueous Solvent ◽  
In The Wild ◽  
Transport Complex

InEscherichia coli, interaction of a periplasmic maltose-binding protein with a membrane-associated ATP-binding cassette transporter stimulates ATP hydrolysis, resulting in translocation of maltose into the cell. The maltose transporter contains two transmembrane subunits, MalF and MalG, and two copies of a nucleotide-hydrolyzing subunit, MalK. Mutant transport complexes that function in the absence of binding protein are thought to be stabilized in an ATPase-active conformation. To probe the conformation of the nucleotide-binding site and to gain an understanding of the nature of the conformational changes that lead to activation, cysteine 40 within the Walker A motif of the MalK subunit was modified by the fluorophore 2-(4′-maleimidoanilino)naphthalene-6-sulfonic acid. Fluorescence differences indicated that residues involved in nucleotide binding were less accessible to aqueous solvent in the binding protein independent transporter than in the wild-type transporter. Similar differences in fluorescence were seen when a vanadate-trapped transition state conformation was compared with the ground state in the wild-type transporter. Our results and recent crystal structures are consistent with a model in which activation of ATPase activity is associated with conformational changes that bring the two MalK subunits closer together, completing the nucleotide-binding sites and burying ATP in the interface.

scholarly journals Kinesin: switch I & II and the motor mechanism

2002 ◽  
Vol 115 (1) ◽  
pp. 15-23 ◽  
Author(s):  
F. Jon Kull ◽  
Sharyn A. Endow
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Active Site ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Structural Transitions ◽  
Rotational Movement ◽  
Atomic Models ◽  
Binding Cleft

New crystal structures of the kinesin motors differ from previously described motor-ADP atomic models, showing striking changes both in the switch I region near the nucleotide-binding cleft and in the switch II or ‘relay’ helix at the filament-binding face of the motor. The switch I region, present as a short helix flanked by two loops in previous motor-ADP structures, rearranges into a pseudo-β-hairpin or is completely disordered with melted helices to either side of the disordered switch I loop. The relay helix undergoes a rotational movement coupled to a translation that differs from the piston-like movement of the relay helix observed in myosin. The changes observed in the crystal structures are interpreted to represent structural transitions that occur in the kinesin motors during the ATP hydrolysis cycle. The movements of switch I residues disrupt the water-mediated coordination of the bound Mg2+, which could result in loss of Mg2+ and ADP, raising the intriguing possibility that disruption of the switch I region leads to release of nucleotide by the kinesins. None of the new structures is a true motor-ATP state, however, probably because conformational changes at the active site of the kinesins require interactions with microtubules to stabilize the movements.

scholarly journals Molecular mechanisms of substrate-controlled ring dynamics and sub-stepping in a nucleic-acid dependent hexameric motor

2016 ◽  
Author(s):  
Nathan D. Thomsen ◽  
Michael R. Lawson ◽  
Lea B. Witkowsky ◽  
Song Qu ◽  
James M. Berger
Keyword(s):  
Nucleic Acid ◽  
Conformational Changes ◽  
Molecular Motors ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Atp Hydrolysis ◽  
Ring Closure ◽  
Structural Studies ◽  
Saxs Data ◽  
Structural Methods

ABSTRACTRing-shaped hexameric helicases and translocases support essential DNA, RNA, and protein-dependent transactions in all cells and many viruses. How such systems coordinate ATPase activity between multiple subunits to power conformational changes that drive the engagement and movement of client substrates is a fundamental question. Using the E. coli Rho transcription termination factor as a model system, we have employed solution and crystallographic structural methods to delineate the range of conformational changes that accompany distinct substrate and nucleotide cofactor binding events. SAXS data show that Rho preferentially adopts an open-ring state in solution, and that RNA and ATP are both required to cooperatively promote ring closure. Multiple closed-ring structures with different RNA substrates and nucleotide occupancies capture distinct catalytic intermediates accessed during translocation. Our data reveal how RNA-induced ring closure templates a sequential ATP-hydrolysis mechanism, provide a molecular rationale for how the Rho ATPase domains distinguishes between distinct RNA sequences, and establish the first structural snapshots of substepping events in a hexameric helicase/translocase.SIGNIFICANCEHexameric, ring-shaped translocases are molecular motors that convert the chemical energy of ATP hydrolysis into the physical movement of protein and nucleic acid substrates. Structural studies of several distinct hexameric translocases have provided insights into how substrates are loaded and translocated; however, the range of structural changes required for coupling ATP turnover to a full cycle of substrate loading and translocation has not been visualized for any one system. Here, we combine low-and high-resolution structural studies of the Rho helicase, defining for the first time the ensemble of conformational transitions required both for substrate loading in solution and for substrate movement by a processive hexameric translocase.

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