scholarly journals Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides

2019 ◽  
Vol 116 (10) ◽  
pp. 4265-4274 ◽  
Author(s):  
Steven Z. Chou ◽  
Thomas D. Pollard
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Atp Hydrolysis ◽  
Release Site ◽  
Cryo Electron Microscopy ◽  
C Terminus ◽  
Pointed End ◽  
Binding Loop

We used cryo-electron microscopy (cryo-EM) to reconstruct actin filaments with bound AMPPNP (β,γ-imidoadenosine 5′-triphosphate, an ATP analog, resolution 3.1 Å), ADP-Pi(ADP with inorganic phosphate, resolution 3.1 Å), or ADP (resolution 3.6 Å). Subunits in the three filaments have similar backbone conformations, so assembly rather than ATP hydrolysis or phosphate dissociation is responsible for their flattened conformation in filaments. Polymerization increases the rate of ATP hydrolysis by changing the positions of the side chains of Q137 and H161 in the active site. Flattening during assembly also promotes interactions along both the long-pitch and short-pitch helices. In particular, conformational changes in subdomain 3 open up multiple favorable interactions with the DNase-I binding loop in subdomain 2 of the adjacent subunit. Subunits at the barbed end of the filament are likely to be in this favorable conformation, while monomers are not. This difference explains why filaments grow faster at the barbed end than the pointed end. When phosphate dissociates from ADP-Pi-actin through a backdoor channel, the conformation of the C terminus changes so it distorts the DNase binding loop, which allows cofilin binding, and a network of interactions among S14, H73, G74, N111, R177, and G158 rearranges to open the phosphate release site.

scholarly journals Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides

2018 ◽  
Author(s):  
Steven Z. Chou ◽  
Thomas D. Pollard
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Atp Hydrolysis ◽  
Regulatory Proteins ◽  
Actin Assembly ◽  
Cryo Electron Microscopy ◽  
Pointed End ◽  
Binding Loop

AbstractWe used electron cryo-micrographs to reconstruct actin filaments with bound AMPPNP (β,γ-imidoadenosine 5’-triphosphate, an ATP analog), ADP-Pi (ADP with inorganic phosphate) or ADP to resolutions of 3.4 Å, 3.4 Å and 3.6 Å. Subunits in the three filaments have nearly identical backbone conformations, so assembly rather than ATP hydrolysis or phosphate dissociation is responsible for their flattened conformation in filaments. Polymerization increases the rate of ATP hydrolysis by changing the conformations of the three ATP phosphates and the side chains of Gln137 and His161 in the active site. Flattening also promotes interactions along both the long-pitch and short-pitch helices. In particular, conformational changes in subdomain 3 open up favorable interactions with the DNase-I binding loop in subdomain 2 of the adjacent subunit. Subunits at the barbed end of the filament are likely to be in this favorable conformation, while monomers are not. This difference explains why filaments grow faster at the barbed end than the pointed end. Loss of hydrogen bonds after phosphate dissociation may account for the greater flexibility of ADP-actin filaments.Significance StatementActin filaments comprise a major part of the cytoskeleton of eukaryotic cells and serve as tracks for myosin motor proteins. The filaments assemble from actin monomers with a bound ATP. After polymerization, actin rapidly hydrolyzes the bound ATP and slowly dissociates the γ-phosphate. ADP-actin filaments then disassemble to recycle the subunits. Understanding how actin filaments assemble, disassemble and interact with numerous regulatory proteins depends on knowing the structure of the filament. High quality structures of ADP-actin filaments were available, but not of filaments with bound ATP- or with ADP and phosphate. We determined structures of actin filaments with bound AMPPNP (a slowly hydrolyzed ATP analog), ADP and phosphate and ADP by cryo-electron microscopy. These structures show how conformational changes during actin assembly promote ATP hydrolysis and faster growth at one end of the filament than the other.

scholarly journals Cryo-electron microscopy structures of pyrene-labeled ADP-Pi- and ADP-actin filaments

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Steven Z. Chou ◽  
Thomas D. Pollard
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Active Site ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Binding ◽  
Muscle Actin ◽  
Hydrophobic Cavity ◽  
Cryo Electron Microscopy ◽  
Kinetics Of

AbstractSince the fluorescent reagent N-(1-pyrene)iodoacetamide was first used to label skeletal muscle actin in 1981, the pyrene-labeled actin has become the most widely employed tool to measure the kinetics of actin polymerization and the interaction between actin and actin-binding proteins. Here we report high-resolution cryo-electron microscopy structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 Å) or ADP-phosphate (3.0 Å) in the active site. Polymerization buries pyrene in a hydrophobic cavity between subunits along the long-pitch helix with only minor differences in conformation compared with native actin filaments. These structures explain how polymerization increases the fluorescence 20-fold, how myosin and cofilin binding to filaments reduces the fluorescence, and how profilin binding to actin monomers increases the fluorescence.

scholarly journals Structures of cofilin-induced structural changes reveal local and asymmetric perturbations of actin filaments

2020 ◽  
Vol 117 (3) ◽  
pp. 1478-1484 ◽  
Author(s):  
Andrew R. Huehn ◽  
Jeffrey P. Bibeau ◽  
Anthony C. Schramm ◽  
Wenxiang Cao ◽  
Enrique M. De La Cruz ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Actin Filaments ◽  
Structural Changes ◽  
Structural Data ◽  
Binding Mode ◽  
Cryo Electron Microscopy ◽  
Actin Structure ◽  
Cluster A ◽  
Pointed End ◽  
Single Subunit

Members of the cofilin/ADF family of proteins sever actin filaments, increasing the number of filament ends available for polymerization or depolymerization. Cofilin binds actin filaments with positive cooperativity, forming clusters of contiguously bound cofilin along the filament lattice. Filament severing occurs preferentially at boundaries between bare and cofilin-decorated (cofilactin) segments and is biased at 1 side of a cluster. A molecular understanding of cooperative binding and filament severing has been impeded by a lack of structural data describing boundaries. Here, we apply methods for analyzing filament cryo-electron microscopy (cryo-EM) data at the single subunit level to directly investigate the structure of boundaries within partially decorated cofilactin filaments. Subnanometer resolution maps of isolated, bound cofilin molecules and an actin-cofilactin boundary indicate that cofilin-induced actin conformational changes are local and limited to subunits directly contacting bound cofilin. An isolated, bound cofilin compromises longitudinal filament contacts of 1 protofilament, consistent with a single cofilin having filament-severing activity. An individual, bound phosphomimetic (S3D) cofilin with weak severing activity adopts a unique binding mode that does not perturb actin structure. Cofilin clusters disrupt both protofilaments, consistent with a higher severing activity at boundaries compared to single cofilin. Comparison of these structures indicates that this disruption is substantially greater at pointed end sides of cofilactin clusters than at the barbed end. These structures, with the distribution of bound cofilin clusters, suggest that maximum binding cooperativity is achieved when 2 cofilins occupy adjacent sites. These results reveal the structural origins of cooperative cofilin binding and actin filament severing.

scholarly journals The U4 Antibody Epitope on Human Papillomavirus 16 Identified by Cryo-electron Microscopy

2015 ◽  
Vol 89 (23) ◽  
pp. 12108-12117 ◽  
Author(s):  
Jian Guan ◽  
Stephanie M. Bywaters ◽  
Sarah A. Brendle ◽  
Hyunwook Lee ◽  
Robert E. Ashley ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Human Papillomavirus ◽  
Conformational Changes ◽  
Vaccine Development ◽  
Contact Point ◽  
Human Papillomavirus 16 ◽  
Cryo Electron Microscopy ◽  
C Terminus ◽  
L1 Protein

ABSTRACTThe human papillomavirus (HPV) major structural protein L1 composes capsomers that are linked together through interactions mediated by the L1 C terminus to constitute a T=7 icosahedral capsid. H16.U4 is a type-specific monoclonal antibody recognizing a conformation-dependent neutralizing epitope of HPV thought to include the L1 protein C terminus. The structure of human papillomavirus 16 (HPV16) complexed with H16.U4 fragments of antibody (Fab) was solved by cryo-electron microscopy (cryo-EM) image reconstruction. Atomic structures of virus and Fab were fitted into the corresponding cryo-EM densities to identify the antigenic epitope. The antibody footprint mapped predominately to the L1 C-terminal arm with an additional contact point on the side of the capsomer. This footprint describes an epitope that is presented capsid-wide. However, although the H16.U4 epitope suggests the presence of 360 potential binding sites exposed in the capsid valley between each capsomer, H16.U4 Fab bound only to epitopes located around the icosahedral five-fold vertex of the capsid. Thus, the binding characteristics of H16.U4 defined in this study showed a distinctive selectivity for local conformation-dependent interactions with specific L1 invading arms between five-fold related capsomers.IMPORTANCEHuman papillomavirus 16 (HPV16) is the most prevalent oncogenic genotype in HPV-associated anogenital and oral cancers. Here we use cryo-EM reconstruction techniques to solve the structures of the HPV16 capsid complexes using H16.U4 fragment of antibody (Fab). Different from most other antibodies directed against surface loops, H16.U4 monoclonal antibody is unique in targeting the C-terminal arm of the L1 protein. This monoclonal antibody (MAb) is used throughout the HPV research community in HPV serological and vaccine development and to define mechanisms of HPV uptake. The unique binding mode of H16.U4 defined here shows important conformation-dependent interactions within the HPV16 capsid. By targeting an important structural and conformational epitope, H16.U4 may identify subtle conformational changes in different maturation stages of the HPV capsid and provide a key probe to analyze the mechanisms of HPV uptake during the early stages of virus infection. Our analyses precisely define important conformational epitopes on HPV16 capsids that are key targets for successful HPV prophylactic vaccines.

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 Domains of alpha-SNAP required for the stimulation of exocytosis and for N-ethylmalemide-sensitive fusion protein (NSF) binding and activation.

1996 ◽  
Vol 7 (5) ◽  
pp. 693-701 ◽  
Author(s):  
R J Barnard ◽  
A Morgan ◽  
R D Burgoyne
Keyword(s):  
Membrane Proteins ◽  
Fusion Protein ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Atp Hydrolysis ◽  
Coiled Coil ◽  
Protein Protein Interactions ◽  
Permeabilized Cells ◽  
C Terminus ◽  
Adrenal Chromaffin

The binding of alpha-SNAP to the membrane proteins syntaxin, SNAP-25, and synaptobrevin leads to the recruitment of the N-ethylmaleimide-sensitive fusion protein (NSF). ATP hydrolysis by NSF has been suggested to drive conformational changes in one or more of these membrane proteins that are essential for regulated exocytosis. Functional evidence for a role of alpha-SNAP in exocytosis in adrenal chromaffin cells comes from the ability of this protein to stimulate Ca(2+)-dependent exocytosis in digitonin-permeabilized cells. Here we examine the effect of a series of deletion mutants of alpha-SNAP on exocytosis, and on the ability of alpha-SNAP to interact with NSF, to define essential domains involved in protein-protein interactions in exocytosis. Deletion of extreme N- or C-terminal regions of alpha-SNAP produced proteins unable to bind to syntaxin or to stimulate exocytosis, suggesting that these domains participate in essential interactions. Deletion of C-terminal residues abolished the ability of alpha-SNAP to bind NSF. In contrast, deletion of up to 120 N-terminal residues did not prevent the binding of NSF to immobilized alpha-SNAP and such mutants were also able to stimulate the ATPase activity of NSF. These results suggest that the C-terminus, but not the N-terminus, of alpha-SNAP is crucial for interactions with NSF. The involvement of the C-terminus of alpha-SNAP, which contains a predicted coiled-coil domain, in the binding of both syntaxin and NSF would place the latter two proteins in proximity in a ternary complex whereupon the energy derived from ATP hydrolysis by NSF could induce a conformational change in syntaxin required for exocytosis to proceed.

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 Molecular insights into the human ABCB6 transporter

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Guangyuan Song ◽  
Sensen Zhang ◽  
Mengqi Tian ◽  
Laixing Zhang ◽  
Runyu Guo ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Transmembrane Domain ◽  
Atp Hydrolysis ◽  
Toxic Metal ◽  
Bound State ◽  
Metal Resistance ◽  
Blood Group System ◽  
Cryo Electron Microscopy ◽  
Binding Pockets ◽  
Energy Dependent

AbstractABCB6 plays a crucial role in energy-dependent porphyrin transport, drug resistance, toxic metal resistance, porphyrin biosynthesis, protection against stress, and encoding a blood group system Langereis antigen. However, the mechanism underlying porphyrin transport is still unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structures of nanodisc-reconstituted human ABCB6 trapped in an apo-state and an ATP-bound state at resolutions of 3.6 and 3.5 Å, respectively. Our structures reveal a unique loop in the transmembrane domain (TMD) of ABCB6, which divides the TMD into two cavities. It restrains the access of substrates in the inward-facing state and is removed by ATP-driven conformational change. No ligand cavities were observed in the nucleotide-bound state, indicating a state following substrate release but prior to ATP hydrolysis. Structural analyses and functional characterizations suggest an “ATP-switch” model and further reveal the conformational changes of the substrate-binding pockets triggered by the ATP-driven regulation.

scholarly journals Structural basis for polarized elongation of actin filaments

2020 ◽  
Author(s):  
Vilmos Zsolnay ◽  
Harshwardhan H. Katkar ◽  
Steven Z. Chou ◽  
Thomas D. Pollard ◽  
Gregory A. Voth
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Actin Filaments ◽  
Atp Hydrolysis ◽  
Underlying Mechanism ◽  
Structural Basis ◽  
Dnase 1 ◽  
Dynamics Simulations ◽  
Pointed End ◽  
Kinetics Of

AbstractActin filaments elongate and shorten much faster at their barbed end than their pointed end, but the molecular basis of this difference has not been understood. We use all-atom molecular dynamics simulations to investigate the properties of subunits at both ends of the filament. The terminal subunits tend towards conformations that resemble actin monomers in solution, while contacts with neighboring subunits progressively flatten the conformation of internal subunits. At the barbed end the terminal subunit is loosely tethered by its DNase-1 loop to the third subunit, because its monomer-like conformation precludes stabilizing contacts with the penultimate subunit. The motions of the terminal subunit make the partially flattened penultimate subunit accessible for binding monomers. At the pointed end, unique contacts between the penultimate and terminal subunits are consistent with existing cryo-EM maps, limit binding to incoming monomers, and flatten the terminal subunit, which likely promotes ATP hydrolysis and rapid phosphate release. These structures explain the distinct polymerization kinetics of the two ends.Significance StatementEukaryotic cells utilize actin filaments to move, change shape, divide, and transport cargo. Decades of experiments have established that actin filaments elongate and shorten significantly faster from one end than the other, but the underlying mechanism for this asymmetry has not been explained. We used molecular dynamics simulations to investigate the structures of the actin filament ends in the ATP, ADP plus γ-phosphate, and ADP nucleotide states. We characterize the structures of actin subunits at both ends of the filament, explain the mechanisms leading to these differences, and connect the divergent structural properties of the two ends to their distinct polymerization rate constants.

scholarly journals Molecular mechanism for direct actin force-sensing by α-catenin

2020 ◽  
Author(s):  
Lin Mei ◽  
Santiago Espinosa de los Reyes ◽  
Matthew J. Reynolds ◽  
Shixin Liu ◽  
Gregory M. Alushin
Keyword(s):  
Actin Filaments ◽  
Cellular Adhesion ◽  
Actin Binding ◽  
Force Sensing ◽  
Adhesion Protein ◽  
Mechanical Coupling ◽  
Actin Networks ◽  
Cryo Electron Microscopy ◽  
C Terminus ◽  
Associated Proteins

SUMMARYThe actin cytoskeleton mediates mechanical coupling between cells and their tissue microenvironments. The architecture and composition of actin networks are modulated by force, but it is unclear how interactions between actin filaments (F-actin) and associated proteins are mechanically regulated. Here, we employ both optical trapping and biochemical reconstitution with myosin motor proteins to show force greater than one piconewton applied solely to F-actin enhances binding by the essential cell-cell adhesion protein αE-catenin, but not its homolog vinculin. Near atomic-resolution cryo-electron microscopy structures of both proteins bound to F-actin reveal unique rearrangements that facilitate their flexible C-termini refolding to engage distinct interfaces. Truncating α-catenin’s C-terminus eliminates force-activated F-actin binding, and addition of this motif to vinculin confers force-activated binding, demonstrating that α-catenin’s C-terminus is a modular detector of F-actin tension. Our studies establish that piconewton force on F-actin can enhance partner binding, which we propose mechanically regulates cellular adhesion through α-catenin.

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