Contribution of Tryptophan Residues to the Structural Changes in Perfringolysin O during Interaction with Liposomal Membranes

Nakamura, Megumi; Sekino-Suzuki, Naoko; Mitsui, Ken'ichiro; Ohno‐Iwashita, Yoshiko

doi:10.1093/oxfordjournals.jbchem.a022054

Cited by 46 publications

(33 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This is consistent with our previous findings that a C-terminal tryptic fragment that contains predominantly domain 4 binds to cholesterol and to cholesterol-containing membrane (18). Our findings that the toxin binding to cholesterol in liposomal membrane triggers a conformational change around tryptophan residues in domain 4 also support this view (19,20). Recently, possible roles of the C-terminal region in cell binding were suggested by a report that a monoclonal antibody thought to bind near the C terminus specifically blocks cell binding, although the exact epitope was not identified (21).…”

supporting

confidence: 93%

“…13). We previously suggested that the tryptophan-substituted mutants have some deficiency in membrane insertion activity (13,20) that could cause them to lose cell binding activity. The tryptophan-rich consensus sequence locates in close proximity to one of the two ␤ sheets in domain 4, and distant from the other ␤ sheet to which the C-terminal ␤ strand belongs (Fig.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

C-terminal Amino Acid Residues Are Required for the Folding and Cholesterol Binding Property of Perfringolysin O, a Pore-forming Cytolysin

Shimada

Nakamura

Naito

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

Perfringolysin O (-toxin) is a pore-forming cytolysin whose activity is triggered by binding to cholesterol in the plasma membrane. The cholesterol binding activity is predominantly localized in the ␤-sheet-rich C-terminal half. In order to determine the roles of the C-terminal amino acids in -toxin conformation and activity, mutants were constructed by truncation of the C terminus. While the mutant with a two-amino acid C-terminal truncation retains full activity and has similar structural features to native -toxin, truncation of three amino acids causes a 40% decrease in hemolytic activity due to the reduction in cholesterol binding activity with a slight change in its higher order structure. Furthermore, both mutants were found to be poor at in vitro refolding after denaturation in 6 M guanidine hydrochloride, resulting in a dramatic reduction in cholesterol binding and hemolytic activities. These activity losses were accompanied by a slight decrease in ␤-sheet content. A mutant toxin with a five-amino acid truncation expressed in Escherichia coli is recovered as a further truncated form lacking the C-terminal 21 amino residues. The product retains neither cholesterol binding nor hemolytic activities and shows a highly disordered structure as detected by alterations in the circular dichroism and tryptophan fluorescence spectra. These results show that the C-terminal region of -toxin has two distinct roles; the last 21 amino acids are involved to maintain an ordered overall structure, and in addition, the last two amino acids at the C-terminal end are needed for protein folding in vitro, in order to produce the necessary conformation for optimal cholesterol binding and hemolytic activities.

show abstract

supporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

C-terminal Amino Acid Residues Are Required for the Folding and Cholesterol Binding Property of Perfringolysin O, a Pore-forming Cytolysin

Shimada

Nakamura

Naito

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Domain 4 of perfringolysin O confers the initial interaction of the soluble toxin molecule with cholesterol-containing membranes, prior to conformational changes in the toxin structure and the formation of an oligomeric ␤-barrel-lined pore in the membrane (52). Of these six tryptophan residues Trp-436, Trp-438, and Trp-439 appear to be important for membrane insertion, with Trp-438 being most critical (53,54). Thus the interfacial interaction of one or more tryptophan residues with the membrane appears to be a common property of membrane-interacting loops from quite evolutionarily divergent proteins.…”

Section: Discussionmentioning

confidence: 99%

Molecular Architecture of the Bipartite Fusion Loops of Vesicular Stomatitis Virus Glycoprotein G, a Class III Viral Fusion Protein

Sun

Belouzard

Whittaker

2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

The glycoprotein of vesicular stomatitis virus (VSV G) mediates fusion of the viral envelope with the host cell, with the conformational changes that mediate VSV G fusion activation occurring in a reversible, low pH-dependent manner. Based on its novel structure, VSV G has been classified as class III viral fusion protein, having a predicted bipartite fusion domain comprising residues Trp-72, Tyr-73, Tyr-116, and Ala-117 that interacts with the host cell membrane to initiate the fusion reaction. Here, we carried out a systematic mutagenesis study of the predicted VSV G fusion loops, to investigate the functional role of the fusion domain. Using assays of low pH-induced cell-cell fusion and infection studies of mutant VSV G incorporated into viral particles, we show a fundamental role for the bipartite fusion domain. We show that Trp-72 is a critical residue for VSV G-mediated membrane fusion. Trp-72 could only tolerate mutation to a phenylalanine residue, which allowed only limited fusion. Tyr-73 and Tyr-116 could be mutated to other aromatic residues without major effect but could not tolerate any other substitution. Ala-117 was a less critical residue, with only charged residues unable to allow fusion activation. These data represent a functional analysis of predicted bipartite fusion loops of VSV G, a founder member of the class III family of viral fusion proteins. Vesicular stomatitis virus (VSV)2 is a prototypic virus in the Rhabdoviridae, which includes many important human, animal, and plant pathogens, including Rabies virus (1). VSV is an enveloped virus that is well known to infect cells via a low pHdependent fusion reaction within endosomes (2-4). The virus contains a single envelope protein, termed the glycoprotein (G), which mediates both attachment to host cells and fusion between the virus envelope and the host cell membrane. This fusion event delivers the VSV genome into the host cell for viral replication. In addition to being a critical determinant of viral pathogenesis, VSV G has also served as an important model for protein folding and transport through the secretory pathway of cells (5). VSV G is used extensively in pseudotyped virus systems and as a delivery system for gene therapy applications (6), and, due to the fact that VSV preferentially replicates and destroys immortalized or tumorigenic cells, the virus has been of great interest as an oncolytic agent in anticancer treatment (7).The mature VSV G protein is an ϳ65-kDa type I transmembrane protein containing 511 amino acids that oligomerizes into a homotrimer during transport to the cell surface, where the trimer is then assembled into the viral particle (8). Unlike many other viral glycoproteins (9), VSV G is not subject to proteolytic priming for fusion activation. The fusogenic ability of VSV G has been of significant interest, because, unlike the proposed "spring-loaded" and essentially irreversible metastable state for the pre-fusion state of other fusion proteins such as influenza HA strain X-31 (10), VSV G is apparently fully re...

show abstract

“…Indeed, ILY appears to show more specificity in host-cell type than other members of the CDC family (Nagamune, 1997 ;Nagamune et al, 1996). Despite the differences in the PLO undecapeptide, it shares the three hydrophobic tryptophan residues which have been demonstrated to play a pivotal role in the cytolytic activity of many CDCs (Boulnois et al, 1991 ;Korchev et al, 1998 ;Michel et al, 1990 ;Nakamura et al, 1998 ;Sekino-Suzuki et al, 1996). However, the spacing between the first and second tryptophan is altered by insertion of P %** .…”

Section: Discussionmentioning

confidence: 99%

The variant undecapeptide sequence of the Arcanobacterium pyogenes haemolysin, pyolysin, is required for full cytolytic activity

2002

View full text Add to dashboard Cite

The cholesterol-dependent cytolysins (CDCs) are characterized by an undecapeptide sequence (ECTGLAWEWWR) that is located near the C terminus and within domain 4 of these proteins. Pyolysin (PLO), the CDC of Arcanobacterium pyogenes, has a variant undecapeptide sequence (EATGLAWDPWW). Site-directed mutants were constructed in undecapeptide residues in a recombinant PLO molecule containing a hexahistidine tag (His-PLO). Mutations in each of the three undecapeptide tryptophan residues resulted in low haemolytic activity, confirming the importance of these residues in the protein. Deletion of a proline residue (P 499 ), inserted in PLO, or substitution of this residue with either phenylalanine or glycine resulted in mutant proteins with undetectable or low haemolytic activities, indicating that P 499 is essential for His-PLO haemolytic activity. Substitution of the PLO undecapeptide sequence with a consensus undecapeptide resulted in a His-PLO protein with only 01 % activity, confirming that the variant PLO undecapeptide is required for the full cytolytic activity of this toxin. The presence of the conserved undecapeptide cysteine residue either alone (His-PLO.C 492 ) or in a consensus sequence resulted in His-PLO molecules which were activated in the presence of reducing compounds, confirming the importance of this residue in the thiol-activated nature of many CDC toxins. The ability of His-PLO mutant proteins to bind cholesterol mimicked haemolytic activity, with the exception of His-PLO.C 492 , which, despite having reduced haemolytic activity, showed an increased ability to bind cholesterol compared to His-PLO. Despite reductions in haemolytic activity and cholesterol-binding, all mutant proteins were still able to bind to erythrocyte membranes, suggesting that other regions of PLO may recognize host-cell membranes, through receptors other than cholesterol.

show abstract

Contribution of Tryptophan Residues to the Structural Changes in Perfringolysin O during Interaction with Liposomal Membranes

Cited by 46 publications

References 0 publications

C-terminal Amino Acid Residues Are Required for the Folding and Cholesterol Binding Property of Perfringolysin O, a Pore-forming Cytolysin

C-terminal Amino Acid Residues Are Required for the Folding and Cholesterol Binding Property of Perfringolysin O, a Pore-forming Cytolysin

Molecular Architecture of the Bipartite Fusion Loops of Vesicular Stomatitis Virus Glycoprotein G, a Class III Viral Fusion Protein

The variant undecapeptide sequence of the Arcanobacterium pyogenes haemolysin, pyolysin, is required for full cytolytic activity

Contact Info

Product

Resources

About