Glu289 residue in the pore-forming motif of Vibrio cholerae cytolysin is important for efficient β-barrel pore formation

Abstract

Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging β-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and subsequently transform into transmembrane β-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane β-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals WT-like oligomeric β-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature than that of the WT toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the prepore-like intermediate state that is hindered from converting into the functional β-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC. Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging β-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and subsequently transform into transmembrane β-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane β-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals WT-like oligomeric β-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature than that of the WT toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the prepore-like intermediate state that is hindered from converting into the functional β-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC. Pore-forming toxins (PFTs) are a unique class of protein toxins with potent cell-killing activity. PFTs exert their toxic effects by forming pores in the lipid bilayer of the plasma membrane. Pore formation causes severe damage to the plasma membrane that can eventually lead to the death of the target cells (1Dal Peraro M. van der Goot F.G. Pore-forming toxins: ancient, but never really out of fashion.Nat. Rev. Microbiol. 2016; 14: 77-92Crossref PubMed Scopus (475) Google Scholar). PFTs are produced by a wide variety of organisms, and they play important roles in diverse biological processes. In particular, many pathogenic bacteria employ PFTs as their virulence factors (2Verma P. Gandhi S. Lata K. Chattopadhyay K. Pore-forming toxins in infection and immunity.Biochem. Soc. Trans. 2021; 49: 455-465Crossref PubMed Scopus (19) Google Scholar). Based on the structural signature of the pore-forming motifs, PFTs are generally classified into two distinct categories: (i) α-PFTs that employ α-helical bundles to form membrane-inserted pores and (ii) β-PFTs that utilize β-barrels to generate transmembrane pores (3Iacovache I. Bischofberger M. van der Goot F.G. Structure and assembly of pore-forming proteins.Curr. Opin. Struct. Biol. 2010; 20: 241-246Crossref PubMed Scopus (140) Google Scholar). Vibrio cholerae cytolysin (VCC) is a distinct member in the β-PFT family (4Sengupta N. Mondal A.K. Mishra S. Chattopadhyay K. Dutta S. Single-particle cryo-EM reveals conformational variability of the oligomeric VCC beta-barrel pore in a lipid bilayer.J. Cell Biol. 2021; 220e202102035Crossref PubMed Google Scholar, 5De S. Olson R. Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 7385-7390Crossref PubMed Scopus Google Scholar). VCC is produced by of the pathogenic of a that causes severe Microbiol. Rev. PubMed Google Scholar). VCC is to be a potent virulence of It that activity, and A. from Vibrio cholerae PubMed Scopus Google Scholar, K. N. of of Vibrio PubMed Google Scholar, and of a produced by Vibrio cholerae toxic produced by PubMed Google Scholar). VCC is by the bacteria as an Upon from the the is by the and the form of VCC is K. K. A. M. for of the of Vibrio cholerae of the structural and of the PubMed Google Scholar, Vibrio cholerae is by PubMed Google Scholar). VCC is of a cytolysin that a distinct pore-forming motif from an designated as the motif and VCC harbors a by a the of the cytolysin R. Crystal structure of the Vibrio cholerae cytolysin (VCC) and assembly into a transmembrane Biol. PubMed Scopus Google Scholar). VCC as monomers in A.K. N. Chattopadhyay K. of elevated membrane and an membrane-bound oligomeric intermediate of the beta-barrel pore-forming toxin Vibrio cholerae PubMed Scopus Google Scholar). VCC monomers to the membrane and form transmembrane β-barrel pores S. Olson R. Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 7385-7390Crossref PubMed Scopus Google Scholar, A.K. Chattopadhyay K. of Vibrio cholerae cytolysin in the membrane-bound state membrane and functional pore formation by the Biol. PubMed Scopus (19) Google Scholar, A. S. M. of Vibrio cholerae cytolysin a pore and a for and in the target Biol. PubMed Scopus Google Scholar, A. I. M. S. of Vibrio cholerae cytolysin as an pore-forming Microbiol. PubMed Scopus Google Scholar). binding of VCC the target membrane R. Chattopadhyay K. Vibrio cholerae of the membrane mechanism of a beta-barrel pore-forming PubMed Scopus Google Scholar). VCC specific to to the target membrane. the of the cytolysin of VCC play a crucial in binding to the membrane A.K. Chattopadhyay K. the membrane mechanism of a membrane-damaging beta-barrel pore-forming toxin Vibrio cholerae Microbiol. PubMed Scopus Google Scholar). an pore-formation is dependent on the of VCC with the membrane R. Mondal A.K. R. S. Chattopadhyay K. the of in the pore-formation mechanism of Vibrio cholerae a membrane-damaging beta-barrel pore-forming PubMed Scopus Google Scholar). In to the a specific by the VCC binding to the in the target membrane A.K. K. Chattopadhyay K. of the on the of cholerae for the membrane mechanism of the Biol. PubMed Scopus Google Scholar). VCC the mechanism of membrane pore formation by the pore-forming β-PFTs A.K. Chattopadhyay K. on of beta-barrel pore-forming PubMed Scopus Google Scholar). toxin monomers to the target membrane and form β-barrel pores A.K. Chattopadhyay K. of Vibrio cholerae cytolysin in the membrane-bound state membrane and functional pore formation by the Biol. PubMed Scopus (19) Google Scholar). β-barrel is by the pore-forming by of the toxin S. Olson R. Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 7385-7390Crossref PubMed Scopus Google Scholar). is with a structural of the pore-forming In the pore-forming motif remains as the cytolysin R. Crystal structure of the Vibrio cholerae cytolysin (VCC) and assembly into a transmembrane Biol. PubMed Scopus Google Scholar). the oligomeric pore formation, motif of of the toxin from the cytolysin and inserts into the membrane lipid bilayer in a to form the of the transmembrane β-barrel S. Olson R. Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 7385-7390Crossref PubMed Scopus Google Scholar). mechanism of the pore-forming in formation of oligomeric intermediates that are designated as prepore assembly is an intermediate in the pore-forming of the toxin are to be in a state and are into the membrane lipid bilayer A.K. Chattopadhyay K. on of beta-barrel pore-forming PubMed Scopus Google Scholar, I. S. N. Peraro M. van der Goot F.G. structure of reveals a protein and the pore-formation 2016; PubMed Scopus Google Scholar, M. I. of transmembrane beta-barrel formation of pore-forming PubMed Scopus Google Scholar). pores are membrane of the pore-forming transmembrane with a mechanism of β-barrel pore formation, formation of the prepore for VCC K. Chattopadhyay K. formation by Vibrio cholerae from a the pore-forming PubMed Scopus (19) Google Scholar). Pore-forming motif is the crucial structural of pore-forming of the β-PFTs they common of the transmembrane β-barrel that the of the membrane lipid bilayer is with In pore is by the and A.K. P. Lata K. M. S. Chattopadhyay K. in the pore-forming of the membrane-damaging protein Biol. PubMed Scopus Google Scholar). a structural the pore-forming of the β-PFTs to form pores the of the membrane lipid of the pore-forming motif appears to play a in structural during the of of a of the the pore-forming motif play more crucial roles than In of our we the of the specific of the motif of VCC A.K. P. N. Dutta S. S. Chattopadhyay K. in the of the pore-forming motif regulates oligomeric beta-barrel pore formation by Vibrio cholerae Microbiol. 2021; PubMed Scopus Google Scholar). In the the cytolysin In the oligomeric pore they are from the β-barrel of the residue is to be for the pore-forming of VCC. appears to an structural the of that plays a crucial in the structural of the pore-forming motif during oligomeric pore formation A.K. P. N. Dutta S. S. Chattopadhyay K. in the of the pore-forming motif regulates oligomeric beta-barrel pore formation by Vibrio cholerae Microbiol. 2021; PubMed Scopus Google Scholar). with the of the VCC harbors a of out of into the of the β-barrel are as (4Sengupta N. Mondal A.K. Mishra S. Chattopadhyay K. Dutta S. Single-particle cryo-EM reveals conformational variability of the oligomeric VCC beta-barrel pore in a lipid bilayer.J. Cell Biol. 2021; 220e202102035Crossref PubMed Google S. Olson R. Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 7385-7390Crossref PubMed Scopus Google Microbiol. Rev. PubMed Google and In the we the of for the β-barrel pore-formation mechanism of VCC. In the we the of the for the pore-forming of VCC. we specific that the pore in the oligomeric pore state We mutant of in of of the target with VCC and for their pore-forming the of WT VCC as of the as and of activity, as and to the effects on the and by in the to the of the to the a of with this of E289A and in VCC in a of the and to that of the E289A-VCC and to in their with to that of the WT of the target the to of and the to and mutant to that with the mutation of in a in the of VCC and of the E289A and a of protein and and In this of activity, the of from the with the VCC for a of of this that the E289A and drastically as to the of E289A a in the activity, a in the activity, to of the WT and WT-like the and In mutant severely the protein results that the of a specific of the and the pore-forming motif of is for the membrane-damaging pore-forming of the toxin. form of of VCC as a distinct of structural E289A mutant an with that of In the mutant a that the mutation of in a in the structure to of the of and a of a distinct for the as to that of In the E289A mutant a that to that of the WT protein that the mutation of but E289A the structural integrity of VCC in the of pore-forming ability of the E289A we first binding ability the membrane lipid bilayer of In the of E289A-VCC and with the membrane lipid bilayer of to that with of the binding from the as as of the binding on the the of protein and during that E289A-VCC with the and to the membrane lipid of binding that the E289A mutant with the as with In this VCC with the to to the and for protein and of this that E289A-VCC in the to We E289A mutant form oligomeric assembly an with the membrane. form in the membrane lipid can be on the in the of Based on in the VCC with and for Our that the membrane-bound of E289A-VCC to that by results that the mutation of E289A in VCC binding and ability in the membrane lipid bilayer of to the of E289A mutation on the pore-formation mechanism of we binding and ability of the mutant in the of we first a to the binding of the VCC with In this with the VCC a temperature of temperature VCC binding to but oligomeric pore formation by the membrane-bound toxin and thus of the VCC can be by with the that E289A-VCC to the of to that with we formation by the E289A mutant binding with membrane membrane with the VCC and membrane-bound by and by and with of E289A mutant the in the of results that E289A-VCC of binding with the of and to form oligomeric to with the WT toxin. of the of the membrane-bound oligomeric pores by the β-PFTs S. U. R. of the Vibrio cholerae cytolysin (VCC) with and a Struct. Biol. PubMed Scopus Google Scholar). we to the assembly state of E289A-VCC in the membrane lipid bilayer of and in the of with the A.K. P. N. Dutta S. S. Chattopadhyay K. in the of the pore-forming motif regulates oligomeric beta-barrel pore formation by Vibrio cholerae Microbiol. 2021; PubMed Scopus Google we by with the and membrane and E289A-VCC in the and in the membrane of our that the E289A pore-forming activity, the ability to with the target and form oligomeric assembly with to with the of pore-forming we membrane and ability of In the of mutant WT-like with the membrane and to with the membrane lipid bilayer of binding of mutant with with binding a in ability to form with binding ability to of E289A mutant and It in the membrane in the of binding and formation by the we to assembly state with as as E289A for the membrane-bound of the that the membrane-bound of mutant in and in membrane our results membrane and ability of to structural and thus pore-forming of this Our that the mutation of E289A the pore-forming motif of VCC drastically pore-forming of the without affecting structural integrity and membrane-binding potential of the E289A-VCC oligomeric assembly in the target membranes, to that by structural into the oligomeric by E289A-VCC in the target membranes, cryo-EM to E289A mutant in a for the E289A to with the lipid cryo-EM with of E289A the oligomeric β-PFT pore assembly of class to the of the protein class the of oligomeric assembly with the lipid membrane and the of oligomeric E289A-VCC with lipid membrane distinct the of transmembrane with the lipid to of protein to a for the in membrane analysis of the class the of oligomeric that membrane with that to be membrane It is to that a of for (4Sengupta N. Mondal A.K. Mishra S. Chattopadhyay K. Dutta S. Single-particle cryo-EM reveals conformational variability of the oligomeric VCC beta-barrel pore in a lipid bilayer.J. Cell Biol. 2021; 220e202102035Crossref PubMed Google Scholar). In with the E289A of the in the in transmembrane β-barrel be of the with β-barrel from in the of (4Sengupta N. Mondal A.K. Mishra S. Chattopadhyay K. Dutta S. Single-particle cryo-EM reveals conformational variability of the oligomeric VCC beta-barrel pore in a lipid bilayer.J. Cell Biol. 2021; 220e202102035Crossref PubMed Google to for E289A-VCC in the mutation of E289A in the pore-forming motif pore formation to the of pore analysis of the of E289A-VCC two (i) oligomeric in transmembrane β-barrel formation and (ii) in and β-barrel be and structural state pore in a lipid of the the two of the bilayer architecture of the pore-forming state of the E289A mutant with the from the structure of the VCC pore S. Olson R. Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 7385-7390Crossref PubMed Scopus Google and N. and for PubMed Scopus Google of a for the of the the of of for as and the of the the of the with our for the of in the mutant to the to form pore in this structural state of E289A-VCC structural in the to the of the transmembrane β-barrel a of and Although the of the structure of pore and pore state of of the to the pore state of E289A-VCC the of to the of the transmembrane β-barrel and the of to the pore-forming we lipid with the of this assembly that the of the E289A-VCC on the membrane as for (4Sengupta N. Mondal A.K. Mishra S. Chattopadhyay K. Dutta S. Single-particle cryo-EM reveals conformational variability of the oligomeric VCC beta-barrel pore in a lipid bilayer.J. Cell Biol. 2021; 220e202102035Crossref PubMed Google Scholar). E289A-VCC WT-like structural integrity and membrane of transmembrane β-barrel pore as in our cryo-EM E289A mutant severely pore-forming activity, as to that of the of pore-forming of we the the mutation of E289A a on the of functional pore formation by the membrane-bound toxin this we the kinetics of of with of as and of this that the of and E289A-VCC delayed kinetics of activity, as to of WT toxin. a delayed kinetics of of the mutant rescued with the of the temperature of the and of the kinetics temperature the of to of the the temperature of the that the efficacy of pore-forming of E289A mutant more severely dependent on temperature than that of that the energy of the pore-formation mechanism residue with of the β-PFT into the transmembrane oligomeric β-barrel pore a of structural Upon toxin monomers assemble into the prepore oligomeric the into the membrane lipid bilayer to β-barrel of the of the motif is of the in the β-PFT In the the motif remains the cytolysin In the of oligomeric pore formation in the membrane, of the toxin from the cytolysin and with the of the forming the β-barrel A.K. Chattopadhyay K. on of beta-barrel pore-forming PubMed Scopus Google Scholar, A.K. Chattopadhyay K. Vibrio cholerae mechanism of an beta-barrel pore-forming Biol. PubMed Scopus Google Scholar). It is to that a of the motif is in the of the β-PFT structure and of the that of the pore-forming motif β-PFT pore-formation and the toxins in the prepore-like K. Chattopadhyay K. formation by Vibrio cholerae from a the pore-forming PubMed Scopus (19) Google Scholar, pore assembly of by temperature and by formation in Microbiol. PubMed Scopus Google Scholar, S. I. M. S. A. Pore formation by Vibrio cholerae cytolysin the as beta-barrel toxins from PubMed Scopus (19) Google Scholar). of the in specific the pore-forming motif of the distinct β-PFTs are to play crucial is important to the specific of the for the mechanism of of the β-PFTs in In the we that the of E289A and in the motif the pore-forming efficacy of VCC and that the mutation of in a in the structural integrity of the protein assembly of in the target membranes, as in the structural in this mutant protein in the structural integrity of the mutant pore-forming activity. of pore-forming of E289A our that E289A-VCC to the target and oligomeric assembly with as with and our cryo-EM that E289A-VCC of forming WT-like transmembrane β-barrel pore assembly in the lipid bilayer and oligomeric assembly state for the membrane-bound in transmembrane β-barrel and assembly state with transmembrane β-barrel in the cryo-EM of (4Sengupta N. Mondal A.K. Mishra S. Chattopadhyay K. Dutta S. Single-particle cryo-EM reveals conformational variability of the oligomeric VCC beta-barrel pore in a lipid bilayer.J. Cell Biol. 2021; 220e202102035Crossref PubMed Google Scholar). of this oligomeric assembly with β-barrel the that prepore-like intermediate state in the of membrane pore formation by VCC. formation of prepore oligomeric assembly with β-barrel in the cryo-EM on β-PFTs I. S. N. Peraro M. van der Goot F.G. structure of reveals a protein and the pore-formation 2016; PubMed Scopus Google Scholar, M. I. of transmembrane beta-barrel formation of pore-forming PubMed Scopus Google Scholar, K. S. S. A. of the mutant reveals a intermediate state of pore Biol. Scopus Google Scholar). Based on the of the cryo-EM of this assembly state with β-barrel to be in the of E289A-VCC than that in the cryo-EM of (4Sengupta N. Mondal A.K. Mishra S. Chattopadhyay K. Dutta S. Single-particle cryo-EM reveals conformational variability of the oligomeric VCC beta-barrel pore in a lipid bilayer.J. Cell Biol. 2021; 220e202102035Crossref PubMed Google Scholar). is that the mutation of E289A in the motif to membrane-bound of VCC in the prepore-like assembly state and with into the functional transmembrane We that the pore-forming of E289A-VCC be rescued with the elevated temperature of the with temperature the terms of to of the of pore-forming of the mutant more than that with WT VCC results energy with the pore-formation mechanism of E289A-VCC in into drastically kinetics of of the Based on appears that the mutation of E289A the pore-forming motif of VCC to membrane-bound toxin in the prepore-like intermediate assembly state and compromises the efficacy to generate functional transmembrane β-barrel by the energy with the of and pore-forming of VCC to that the of a the the pore-forming motif of VCC is for pore-formation mechanism. of the β-barrel pore-formation mechanism by structural and membrane of the pore-forming It remains how are in a A.K. Chattopadhyay K. of Vibrio cholerae cytolysin in the membrane-bound state membrane and functional pore formation by the Biol. PubMed Scopus (19) Google Scholar, M. R. M. S. mechanism of the oligomeric the membrane is by a of the lipid membrane and is during PubMed Scopus Google Scholar). It is that the pore-forming motif play roles in the efficacy of the pore-formation with is from our that the mutation of E289A in the pore-forming motif of VCC to of the membrane-bound toxin molecules into the functional transmembrane pores. In the state of are to their a of than from that of are as (i) of the of the motif a of (ii) of the a of and of the cytolysin a of the of the can potential with in the state of VCC. of drastically in the transmembrane oligomeric pore and are in to a toxin in the β-barrel of the two and a of that are by the pore-forming motif of the Based on appears that a of the residue during the of oligomeric β-barrel pore formation by VCC E289A mutation be to a of of the for the functional β-barrel formation be to the of an residue the of mutation of E289A structural of the motif and the efficacy of pore is by our energy and delayed kinetics of E289A pore-forming activity, and is by our that the of and pore-forming of VCC. and be to more of the the residue and in of the membrane-bound VCC molecules into the functional pore In for the first how a specific residue the pore-forming motif of VCC regulates pore-formation mechanism by the energy with the and the to in regulatory roles are by the pore-forming motif of the with by the of of and from in the by the of WT VCC the in the K. Chattopadhyay K. mutation in Vibrio cholerae cytolysin compromises the membrane pore-formation mechanism of the PubMed Scopus Google Scholar). the the of the form into the cells with R. for to from the of the the by the VCC from by with of for VCC by of the protein by by the the from the protein of the in this the and by mutant and the for of the VCC on a temperature a temperature the to for the by with the of the VCC by the the of the the of the and the of the temperature as an for the structure of the of the VCC the the A.K. P. N. Dutta S. S. Chattopadhyay K. in the of the pore-forming motif regulates oligomeric beta-barrel pore formation by Vibrio cholerae Microbiol. 2021; PubMed Scopus Google Scholar, K. Chattopadhyay K. mutation in Vibrio cholerae cytolysin compromises the membrane pore-formation mechanism of the PubMed Scopus Google Scholar). and in of in a that the the of the cells in an of on the of the the of activity, with VCC for of by the in the of the by the of by the cells as the and of the cells with to the activity. of the VCC with by a A.K. P. N. Dutta S. S. Chattopadhyay K. in the of the pore-forming motif regulates oligomeric beta-barrel pore formation by Vibrio cholerae Microbiol. 2021; PubMed Scopus Google Scholar). cells with the in the cells with and the for on a with and with the and as the the K. Chattopadhyay K. mutation in Vibrio cholerae cytolysin compromises the membrane pore-formation mechanism of the PubMed Scopus Google Scholar). the in and to a and the for to generate the lipid on the lipid with for by a of pore to the binding of the VCC with the membrane lipid bilayer of the on a the A.K. P. N. Dutta S. S. Chattopadhyay K. in the of the pore-forming motif regulates oligomeric beta-barrel pore formation by Vibrio cholerae Microbiol. 2021; PubMed Scopus Google Scholar). with the the by the with a of for and with of for with a of the for a of binding of the VCC the protein for with a of by with the the of the by the and of for a of the during the protein the binding of the VCC the during the the of the protein as the of the protein on the VCC with a of in a of in for to for the the with and in of the and the by in the of the the formation by the VCC in the with a of in in for to for with in and into two for and the for by of the the K. Chattopadhyay K. mutation in Vibrio cholerae cytolysin compromises the membrane pore-formation mechanism of the PubMed Scopus Google Scholar). with and subsequently in an for and for from the and with the the of the membrane in a and for the the for and the in and protein in the membrane by a and to be of the membrane with VCC in a of in for to for with in and into two for and the for to by as and with the and on an to the assembly of the VCC with the membrane lipid bilayer of the and membrane VCC with of in a of in with of membrane in a of in for to for and in of the and with plasma a plasma for of the to the plasma and for of the and the with a a a of and for the with the and in of and with a of to and the for to in for to of E289A-VCC as for the to cryo-EM and for for and into by a in with A. S. S. Dutta S. and for single-particle 2021; Scopus Google a of and a of to a of the of and a of for for a of into of a to cryo-EM structure Struct. Biol. PubMed Scopus Google and to of of for 14: PubMed Scopus Google Scholar). in A. N. for single-particle PubMed Scopus Google and the that to for with A. N. and from Struct. Biol. PubMed Scopus Google Scholar). a of to in and class class with the to as for with by of to into with with distinct lipid for to a the in with the of and the and the into A. for cryo-EM structure 14: PubMed Scopus Google Scholar). of by in to and with and to of the of the transmembrane β-barrel of E289A-VCC with a from the of into the of E289A-VCC in for and PubMed Scopus Google Scholar). as the with the E289A in the P. K. and of Biol. 2010; PubMed Scopus Google Scholar). with as an for in N. a for structure Biol. 2010; PubMed Scopus Google Scholar). of the of the cryo-EM in the of the E289A-VCC oligomeric state with and β-barrel the to the pore state of structural in for and PubMed Scopus Google and in and Sci. PubMed Scopus Google Scholar). structural with the protein structural of the state and the oligomeric pore form of VCC from the protein to the and for the oligomeric pore assembly of to the with the E289A-VCC mutant with transmembrane and E289A-VCC mutant with transmembrane for the the cryo-EM of E289A-VCC mutant with transmembrane β-barrel in with the are the and that they of with the of this We the and of for and is by from of and We the and for the cryo-EM the of and from the of A. K. M. and K. A. K. N. M. R. K. I. and S. A. K. N. M. R. K. S. and K. A. K. N. R. I. S. and K. A. K. N. M. R. K. I. and S. A. K. N. S. and K. A. K. N. M. R. K. I. and S. A. K. N. S. and K. A. K. S. and K. and I. S. and K. K. K. with

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