Nonnative Interactions between Cysteines Direct Productive Assembly of P22 Tailspike Protein

Abstract

Nonnative disulfide bond formation can play a critical role in the assembly of disulfide bonded proteins. During the folding and assembly of the P22 tailspike protein, nonnative disulfide bonds form both in vivo and in vitro. However, the mechanism and identity of cysteine disulfide pairs remains elusive, particularly for P22 tailspike, which contains no disulfide bonds in its native, functional form. Understanding the interactions between cysteine residues is important for developing a mechanistic model for the role of nonnative cysteines in P22 tailspike assembly. Prior in vivo studies have suggested that cysteines 496, 613, and 635 are the most likely site for sulfhydryl reactivity. Here we demonstrate that these three cysteines are critical for efficient assembly of tailspike trimers, and that interactions between cysteine pairs lead to productive assembly of native tailspike.

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

“…However, those oligomeric folding intermediates were dissociated into monomer when DTT was present in the native sample buffer, consistent with earlier data showing intermolecular disulfide bonds in oligomeric folding intermediates (Robinson and King 1997;Danek and Robinson 2004). The monomer band formed in the absence of DTT is more diffuse than the one in the reducing conditions and runs with Figure 1.…”

“…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.…”

“…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.…”

“…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 PAGE consistently showed disulfide-bonded oligomeric species, predominantly as dimers and trimers.…”

Stalled Folding Mutants in the Triple β-Helix Domain of the Phage P22 Tailspike Adhesin

“…29,30 These S-S bonds increase folding efficiency both in vivo and in vitro, and may serve to stabilize the correct association and register en route to the mature reduced trimer. 31,32 This may be analogous to the S-S bonds formed between collagen registration domains before formation of the collagen triple helix. 33 The presence of a CXXC motif beginning at residue 287 supports the notion of internally catalyzed disulfide bonds that are insensitive to normal intracellular redox levels.…”