Slow Escape from a Helical Misfolded State of the Pore-Forming Toxin Cytolysin A

Dingfelder, Fabian; Macocco, Iuri; Benke, Stephan; Nettels, Daniel; Faccioli, Pietro; Schuler, Benjamin

doi:10.1021/jacsau.1c00175

Cited by 7 publications

(5 citation statements)

References 103 publications

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“…The BF scheme has been extensively validated using plain MD protein folding simulations performed on the Anton supercomputer (23, 25), against the results of deuterium-exchange experiments (56),time-resolved single-molecule FRET (57), and near-UV CD measurements (58). The BF scheme has also beensuccessfully applied to investigate amylodogenic processes (59) and in drug discovery, to identify small molecules that can interfere with the protein folding process (52).…”

Section: Methodsmentioning

confidence: 99%

Long range allostery mediates the regulation of plasminogen activator inhibitor-1 by vitronectin

Kihn

Marchiori

Spagnolli

et al. 2022

Preprint

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The serpin plasminogen activator inhibitor 1 (PAI-1) spontaneously undergoes a massive structural change from a metastable, active conformation, with a solvent accessible reactive center loop (RCL), to a stable, inactive or latent conformation in which the RCL has inserted into the central β sheet. Physiologically, conversion to the latent state is regulated by the binding of vitronectin which retards the rate of this latency transition approximately 2-fold. We investigated the effects of vitronectin on the PAI-1 latency transition using all-atom path sampling simulations in explicit solvent. In simulated latency transitions of free PAI-1, the RCL is quite mobile as is the gate, the region that impedes RCL access to the central β sheet. This mobility allows the formation of a transient salt bridge that facilitates the transition, and this finding rationalizes existing mutagenesis results. Vitronectin binding reduces RCL and gate mobility by allosterically rigidifying structural elements over 40 Å away from the binding site thus blocking the transition to the latent conformation. The effects of vitronectin are propagated by a network of dynamically correlated residues including a number of conserved sites that have previously been identified as important for PAI-1 stability. Simulations also revealed a transient pocket populated only in the vitronectin bound state which corresponds to a cryptic drug binding site identified by crystallography. Overall, these results shed new light on regulation of the PAI-1 latency transition by vitronectin and illustrate the potential of path sampling simulations for understanding functional conformational changes in proteins and for facilitating drug discovery.

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Section: Methodsmentioning

confidence: 99%

Long range allostery mediates the regulation of plasminogen activator inhibitor-1 by vitronectin

Kihn

Marchiori

Spagnolli

et al. 2022

Preprint

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“…Using the X-ray crystal structure data of ClyA, we have previously modeled the entire dodecameric pore complex of ClyA using CHARMM36 and found excellent agreement with the pore dimensions reported in the crystal structure . The CHARMM36 force field has also been used to study the unfolding and refolding mechanism of ClyA in free solvent . Equilibration was monitored by evaluating the root mean squared deviations (RMSD) for the protein.…”

Section: Methodsmentioning

confidence: 90%

“…3 The CHARMM36 force field has also been used to study the unfolding and refolding mechanism of ClyA in free solvent. 27 Equilibration was monitored by evaluating the root mean squared deviations (RMSD) for the protein. Simulation of the wild type and all mutants were performed at three different temperatures: 310, 350, and 400 K. Details of the monomer simulations are given in Supporting Information (see Table S1).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Conformational Flexibility Is a Key Determinant for the Lytic Activity of the Pore-Forming Protein, Cytolysin A

et al. 2022

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Several bacterial infections are mediated by pore-forming toxins (PFTs), a subclass of proteins that oligomerize on mammalian cell membranes forming lytic nanopores. Cytolysin A (ClyA), an α-PFT, undergoes a dramatic conformational change restructuring its two membrane-binding motifs (the β-tongue and the N-terminus helix), during pore formation. A complete molecular picture for this key transition and the driving force behind the secondary structure change upon membrane binding remain elusive. Using all-atom molecular dynamics (MD) simulations of the ClyA monomer and string method based free energy computations with path collective variables, we illustrate that an unfolded β-tongue motif is an on-pathway intermediate during the transition to the helix-turn-helix motif of the protomer. An aggregate of 28 μs of all-atom thermal unfolding MD simulations of wildtype ClyA and its single point mutants reveal that the membrane-binding motifs of the ClyA protein display high structural flexibility in water. However, point mutations in these motifs lead to a distinct reduction in the flexibility, especially in the β-tongue, thereby stabilizing the pretransition secondary structure. Resistance to unfolding was further corroborated by MD simulations of the βtongue mutant motif in the membrane. Combined with the thermal unfolding simulations, we posit that the β-tongue as well as Nterminal mutants that lower the tendency to unfold and disorder the β-tongue are detrimental to pore formation by ClyA and its lytic activity. Erythrocyte turbidity and vesicle leakage assays indeed reveal a loss of activity for the β-tongue mutant, and delayed kinetics for the N-terminus mutants. On the other hand, a point mutation in the extracellular domain that did not abrogate lytic activity displayed similar unfolding characteristics as the wild type. Thus, attenuation of conformational flexibility in membrane-binding motifs correlates with reduced lytic and leakage activity. Combined with secondary structure changes observed in the membrane bound states, our study shows that the tendency to unfold in the β-tongue region is a critical step in the conformational transition and bistability of the ClyA protein and mutants that disrupt this tendency reduced pore formation. Overall, our finding suggests that inherent flexibility in the protein could play a wider and hitherto unrecognized role in membrane-mediated conformational transitions of PFTs and other membrane protein transformations.

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“…The BF scheme has been extensively validated using plain MD protein folding simulations performed on the Anton supercomputer ( 19 , 21 ), against the results of deuterium-exchange experiments ( 54 ), time-resolved single-molecule FRET ( 55 ), and near-UV CD measurements ( 56 ). The BF scheme has also been successfully applied to investigate amylodogenic processes ( 57 ) and in drug discovery, to identify small molecules that can interfere with the protein folding process ( 50 ).…”

Section: Methodsmentioning

confidence: 99%

Long-range allostery mediates the regulation of plasminogen activator inhibitor-1 by cell adhesion factor vitronectin

Kihn¹,

Marchiori²,

Spagnolli³

et al. 2022

Journal of Biological Chemistry

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Slow Escape from a Helical Misfolded State of the Pore-Forming Toxin Cytolysin A

Cited by 7 publications

References 103 publications

Long range allostery mediates the regulation of plasminogen activator inhibitor-1 by vitronectin

Long range allostery mediates the regulation of plasminogen activator inhibitor-1 by vitronectin

Conformational Flexibility Is a Key Determinant for the Lytic Activity of the Pore-Forming Protein, Cytolysin A

Long-range allostery mediates the regulation of plasminogen activator inhibitor-1 by cell adhesion factor vitronectin

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