P22 tailspike trimer assembly is governed by interchain redox associations

Danek, Brenda L.; Robinson, Anne S.

doi:10.1016/j.bbapap.2004.04.001

Cited by 5 publications

(8 citation statements)

References 43 publications

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“…These results are similar to previously reported by Danek and Robinson for tailspike monomeric complex treated with oxidized glutathione 65 . Given that the cysteine reactivity must depend on some distinct chain conformation, this sensitive precursor may represent species with the C-terminal chaperone domain folded into a competent conformation.…”

Section: Resultssupporting

confidence: 91%

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The C-terminal cysteine annulus participates in auto-chaperone function forSalmonellaphage P22 tailspike folding and assembly

Takata¹,

Haase-Pettingell

King³

2012

Bacteriophage

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Elongated trimeric adhesins are a distinct class of proteins employed by phages and viruses to recognize and bind to their host cells, and by bacteria to bind to their target cells and tissues. The tailspikes of E. coli phage K1F and Bacillus phage Ø29 exhibit auto-chaperone activity in their trimeric C-terminal domains. The P22 tailspike is structurally homologous to those adhesins. Though there are no disulfide bonds or reactive cysteines in the native P22 tailspikes, a set of C-terminal cysteines are very reactive in partially folded intermediates, implying an unusual local conformation in the domain. This is likely to be involved in the auto-chaperone function. We examined the unusual reactivity of C-terminal tailspike cysteines during folding and assembly as a potential reporter of auto-chaperone function. Reaction with IAA blocked productive refolding in vitro, but not off-pathway aggregation. Two-dimensional PAGE revealed that the predominant intermediate exhibiting reactive cysteine side chains was a partially folded monomer. Treatment with reducing reagent promoted native trimer formation from these species, consistent with transient disulfide bonds in the auto-chaperone domain. Limited enzymatic digestion and mass spectrometry of folding and assembly intermediates indicated that the C-terminal domain was compact in the protrimer species. These results indicate that the C-terminal domain of the P22 tailspike folds itself and associates prior to formation of the protrimer intermediate, and not after, as previously proposed. The C-terminal cysteines and triple β-helix domains apparently provide the staging for the correct auto-chaperone domain formation, needed for alignment of P22 tailspike native trimer.

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Section: Resultssupporting

confidence: 91%

“…A variety of experiments indicate a role for transient disulfide bonds in protrimer folding and assembly 45 , 65 . However, it has been difficult to ascertain if these are essential for efficient folding and assembly, or a byproduct of the slow folding reaction, and the proximity of the reactive 613 and 635 cysteines.…”

Section: Discussionmentioning

confidence: 99%

The C-terminal cysteine annulus participates in auto-chaperone function forSalmonellaphage P22 tailspike folding and assembly

Takata¹,

Haase-Pettingell

King³

2012

Bacteriophage

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“…4A). Alternatively, formation of a transient disulfide bond may be required during the initial protein folding but is dispensable thereafter, as has been demonstrated for several proteins [20,26,27]. We evaluated this possibility by refolding DksA2 in the presence and absence of DTT.…”

Section: Discussionmentioning

confidence: 98%

DksA2, a zinc‐independent structural analog of the transcription factor DksA

et al. 2013

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Transcription factor DksA contains a four-Cys Zn2 +-finger motif thought to be responsible for structural integrity and the relative disposition of its domains. Pseudomonas aeruginosa encodes an additional DksA paralog (DksA2) that is expressed selectively under Zn2+ limitation. Although DksA2 does not bind Zn2+, it complements the Escherichia coli dksA deletion and has similar effects on transcription in vitro. In this study, structural and biochemical analyses reveal that DksA2 has a similar fold, domain structure and RNA polymerase binding properties to those of the E. coli DksA despite the lack of the stabilizing metal ion.

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“…Each chain in the native trimer has eight reduced cysteines: six are located in the β‐helix domain and two are in the C‐terminal domain. Disulfide bonds are formed during tailspike assembly, and they are believed to play critical roles in the registration of the three tailspike chains (Robinson and King 1997; Haase‐Pettingell et al 2001; Danek and Robinson 2003, 2004). Our current model is that intermolecular disulfide bonds form between the C613 on one chain and the C635 on another chain in the C‐terminal domain to help align the three subunits.…”

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confidence: 99%

Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDS

Kim

Robinson

2006

Protein Science

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Each chain of the native trimeric P22 tailspike protein has eight cysteines that are reduced and buried in its hydrophobic core. However, disulfide bonds have been observed in the folding pathway and they are believed to play a critical role in the registration of the three chains. Interestingly, in the presence of sodium dodecyl sulfate (SDS) only monomeric chains, rather than disulfide-linked oligomers, have been observed from a mixture of folding intermediates. Here we show that when the oligomeric folding intermediates were separated from the monomer by native gel electrophoresis, the reduction of intermolecular disulfide bonds did not occur in the subsequent second-dimension SDS-gel electrophoresis. This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers.Keywords: P22 tailspike protein (TSP); transient disulfide bond; assembly; disulfide shuffling; SDS P22 tailspike protein is a member of the elongated b-helix family of viral adhesins that participates in polysaccharide binding during viral infection that includes pectate lyase C and pertactin (Mitraki et al. 2002;Simkovsky and King 2006). Tailspike is a homotrimer and each monomer has 666 amino acid residues. Tailspike has four major domains: the N-terminal head-binding domain; the b-helix domain, which is formed by alternating b strands and turns; a bsheet prism domain, where three chains are interdigitated; and the C-terminal domain (Steinbacher et al. 1994(Steinbacher et al. , 1997. Each chain in the native trimer has eight reduced cysteines: six are located in the b-helix domain and two are in the Cterminal domain. Disulfide bonds are formed during tailspike assembly, and they are believed to play critical roles in the registration of the three tailspike chains (Robinson and King 1997;Haase-Pettingell et al. 2001;Danek andRobinson 2003, 2004). Our current model is that intermolecular disulfide bonds form between the C613 on one chain and the C635 on another chain in the C-terminal domain to help align the three subunits. Site-directed mutagenesis of either C613 or C635 significantly decreased the assembly kinetics of tailspike as well as final yields both in vivo and in vitro (Haase-Pettingell et al. 2001;Danek and Robinson 2003). Since the intermolecular disulfide bonds are absent in the native trimer, reduction of those disulfide bonds is a required step in tailspike folding. How does the reduction occur? The nascent chains fold on the ribosome while translation is in progress, and one could imagine many sources of reductants (Brunschier et al. 1993;Gordon et al. 1994;Sather and King 1994;Clark and King 2001). However, in vitro, purified tailspike manages to fold into native trimer in the absence of redox buffers to yields of 80%-90% (Danek and Robinson 2004). A further troubling issue has been the presence of only two detectable bands on SDS-PAGE-the monomer and the trimer-although nondenaturing P...

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P22 tailspike trimer assembly is governed by interchain redox associations

Cited by 5 publications

References 43 publications

The C-terminal cysteine annulus participates in auto-chaperone function forSalmonellaphage P22 tailspike folding and assembly

The C-terminal cysteine annulus participates in auto-chaperone function forSalmonellaphage P22 tailspike folding and assembly

DksA2, a zinc‐independent structural analog of the transcription factor DksA

Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDS

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