Structural basis for polarized elongation of actin filaments

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.

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

Nature Communications ◽

10.1038/s41467-021-27304-6 ◽

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.

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Structural basis of Plasmodium vivax inhibition by antibodies binding to the circumsporozoite protein repeats

eLife ◽

10.7554/elife.72908 ◽

2022 ◽

Vol 11 ◽

Author(s):

Iga Kucharska ◽

Lamia Hossain ◽

Danton Ivanochko ◽

Qiren Yang ◽

John L Rubinstein ◽

...

Keyword(s):

Plasmodium Vivax ◽

Molecular Basis ◽

Parasite Infection ◽

Circumsporozoite Protein ◽

Structural Basis ◽

Structural Studies ◽

Repeat Region ◽

Antibody Recognition ◽

Peptide Conformations ◽

Dynamics Simulations

Malaria is a global health burden, with Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) responsible for the majority of infections worldwide. Circumsporozoite protein (CSP) is the most abundant protein on the surface of Plasmodium sporozoites, and antibodies targeting the central repeat region of CSP can prevent parasite infection. Although much has been uncovered about the molecular basis of antibody recognition of the PfCSP repeats, data remains scarce for PvCSP. Here, we performed molecular dynamics simulations for peptides comprising the PvCSP repeats from strains VK210 and VK247 to reveal how the PvCSP central repeats are highly disordered, with minor propensities to adopt turn conformations. Next, we solved eight crystal structures to unveil the interactions of two inhibitory monoclonal antibodies (mAbs), 2F2 and 2E10.E9, with PvCSP repeats. Both antibodies can accommodate subtle sequence variances in the repeat motifs and recognize largely coiled peptide conformations that also contain isolated turns. Our structural studies uncover various degrees of Fab-Fab homotypic interactions upon recognition of the PvCSP central repeats by these two inhibitory mAbs, similar to potent mAbs against PfCSP. These findings augment our understanding of host-Plasmodium interactions, and contribute molecular details of Pv inhibition by mAbs to unlock structure-based engineering of PvCSP-based vaccines.

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

10.1101/309534 ◽

2018 ◽

Cited By ~ 3

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.

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A single K+-binding site in the crystal structure of the gastric proton pump

10.1101/608851 ◽

2019 ◽

Author(s):

Kenta Yamamoto ◽

Vikas Dubey ◽

Katsumasa Irie ◽

Hanayo Nakanishi ◽

Himanshu Khandelia ◽

...

Keyword(s):

Binding Site ◽

Proton Pump ◽

Atp Hydrolysis ◽

Energy Requirement ◽

Cation Binding ◽

Structural Basis ◽

Cation Binding Site ◽

Gastric Proton Pump ◽

Dynamics Simulations ◽

P Type

AbstractThe gastric proton pump (H+,K+-ATPase), a P-type ATPase responsible for gastric acidification, mediates electro-neutral exchange of H+ and K+ coupled with ATP hydrolysis, but with an as yet undetermined transport stoichiometry. Here we show crystal structures at a resolution of 2.5 Å of the pump in the E2-P transition state, in which the counter-transporting cation is occluded. We found a single K+ bound to the cation-binding site of H+,K+-ATPase, indicating an exchange of 1H+/1K+ per hydrolysis of one ATP molecule. This fulfils the energy requirement for the generation of a six pH unit gradient across the membrane. The structural basis of K+recognition is resolved, supported by molecular dynamics simulations, and this establishes how H+,K+-ATPase overcomes the energetic challenge to generate an H+ gradient of more than a million-fold – the highest cation gradient known in any mammalian tissue – across the membrane.

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Structural basis of Plasmodium vivax inhibition by antibodies binding to the circumsporozoite protein repeats

10.1101/2021.08.31.458469 ◽

2021 ◽

Author(s):

Iga Kucharska ◽

Lamia Hossain ◽

Danton Ivanochko ◽

Qiren Yang ◽

John L Rubinstein ◽

...

Keyword(s):

Plasmodium Vivax ◽

Molecular Basis ◽

Parasite Infection ◽

Circumsporozoite Protein ◽

Structural Basis ◽

Structural Studies ◽

Repeat Region ◽

Antibody Recognition ◽

Peptide Conformations ◽

Dynamics Simulations

Malaria is a global health burden, with Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) responsible for the majority of infections worldwide. Circumsporozoite protein (CSP) is the most abundant protein on the surface of Plasmodium sporozoites, and antibodies targeting the central repeat region of CSP can prevent parasite infection. Although much has been uncovered about the molecular basis of antibody recognition of the PfCSP repeats, data remains scarce for PvCSP. Here, we performed molecular dynamics simulations for peptides comprising the PvCSP repeats from strains VK210 and VK247 to reveal how the PvCSP central repeats are highly disordered, with minor propensities to adopt turn conformations. Next, we solved eight crystal structures to unveil the interactions of two inhibitory monoclonal antibodies (mAbs), 2F2 and 2E10.E9, with PvCSP repeats. Both antibodies can accommodate subtle sequence variances in the repeat motifs and recognize largely coiled peptide conformations that also contain isolated turns. Our structural studies uncover various degrees of Fab-Fab homotypic interactions upon recognition of the PvCSP central repeats by these two inhibitory mAbs, similar to potent mAbs against PfCSP. These findings augment our understanding of host-Plasmodium interactions, and contribute molecular details of Pv inhibition by mAbs to unlock structure-based engineering of PvCSP-based vaccines.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1807028115 ◽

2019 ◽

Vol 116 (10) ◽

pp. 4265-4274 ◽

Cited By ~ 56

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.

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Arginine substitution of a cysteine in transmembrane helix M8 converts Na+,K+-ATPase to an electroneutral pump similar to H+,K+-ATPase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1617951114 ◽

2016 ◽

Vol 114 (2) ◽

pp. 316-321 ◽

Cited By ~ 8

Author(s):

Rikke Holm ◽

Jaanki Khandelwal ◽

Anja P. Einholm ◽

Jens P. Andersen ◽

Pablo Artigas ◽

...

Keyword(s):

Turnover Rate ◽

Atp Hydrolysis ◽

Transmembrane Helix ◽

Cation Binding ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Wild Type ◽

Transport Mode ◽

The Third ◽

Enzyme Turnover

Na+,K+-ATPase and H+,K+-ATPase are electrogenic and nonelectrogenic ion pumps, respectively. The underlying structural basis for this difference has not been established, and it has not been revealed how the H+,K+-ATPase avoids binding of Na+ at the site corresponding to the Na+-specific site of the Na+,K+-ATPase (site III). In this study, we addressed these questions by using site-directed mutagenesis in combination with enzymatic, transport, and electrophysiological functional measurements. Replacement of the cysteine C932 in transmembrane helix M8 of Na+,K+-ATPase with arginine, present in the H+,K+-ATPase at the corresponding position, converted the normal 3Na+:2K+:1ATP stoichiometry of the Na+,K+-ATPase to electroneutral 2Na+:2K+:1ATP stoichiometry similar to the electroneutral transport mode of the H+,K+-ATPase. The electroneutral C932R mutant of the Na+,K+-ATPase retained a wild-type–like enzyme turnover rate for ATP hydrolysis and rate of cellular K+ uptake. Only a relatively minor reduction of apparent Na+ affinity for activation of phosphorylation from ATP was observed for C932R, whereas replacement of C932 with leucine or phenylalanine, the latter of a size comparable to arginine, led to spectacular reductions of apparent Na+ affinity without changing the electrogenicity. From these results, in combination with structural considerations, it appears that the guanidine+ group of the M8 arginine replaces Na+ at the third site, thus preventing Na+ binding there, although allowing Na+ to bind at the two other sites and become transported. Hence, in the H+,K+-ATPase, the ability of the M8 arginine to donate an internal cation binding at the third site is decisive for the electroneutral transport mode of this pump.

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

10.1101/2021.01.05.425498 ◽

2021 ◽

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. 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.

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Structural basis of denuded glycan recognition by SPOR domains in bacterial cell division

Nature Communications ◽

10.1038/s41467-019-13354-4 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 3

Author(s):

Martín Alcorlo ◽

David A. Dik ◽

Stefania De Benedetti ◽

Kiran V. Mahasenan ◽

Mijoon Lee ◽

...

Keyword(s):

Mass Spectrometry ◽

Cell Wall ◽

Cell Division ◽

Molecular Basis ◽

Structural Basis ◽

Bacterial Cell Division ◽

Bacterial Proteins ◽

Daughter Cells ◽

Lytic Transglycosylase ◽

Dynamics Simulations

AbstractSPOR domains are widely present in bacterial proteins that recognize cell-wall peptidoglycan strands stripped of the peptide stems. This type of peptidoglycan is enriched in the septal ring as a product of catalysis by cell-wall amidases that participate in the separation of daughter cells during cell division. Here, we document binding of synthetic denuded glycan ligands to the SPOR domain of the lytic transglycosylase RlpA from Pseudomonas aeruginosa (SPOR-RlpA) by mass spectrometry and structural analyses, and demonstrate that indeed the presence of peptide stems in the peptidoglycan abrogates binding. The crystal structures of the SPOR domain, in the apo state and in complex with different synthetic glycan ligands, provide insights into the molecular basis for recognition and delineate a conserved pattern in other SPOR domains. The biological and structural observations presented here are followed up by molecular-dynamics simulations and by exploration of the effect on binding of distinct peptidoglycan modifications.

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