The elongated adhesin proteins that govern the attachment of many viruses and phages to cells use their extended lateral surfaces to recognize cell surface polysaccharides and lipopolysaccharides. Examples include the Adenovirus penton fiber, T4 short tail fiber, Bordetella pertussis
toxin, the bacteriophage associated Hyaluronate lyase (Hylp2), and a variety of phage tailspikes.1-8
The majority of these proteins are multimeric, folding and assembling into very stable structures that can survive the diverse environments they are exposed to.9
The structures of three bacteriophage tailspikes have been determined to high resolution (): Salmonella
P22 tailspike, Bacillus
Ø29 appendage (tailspike), and E. coli
These three proteins have major β-helix domains, with triple β-helix regions, and complex trimeric C-terminal domains. Among these three proteins, the folding, assembly and off-pathway aggregation pathways of the Salmonella
P22 tailspike have been characterized both in vivo and in vitro. They include monomeric, dimeric and protrimer partially folded intermediates.14-18
Figure 1. Structures of the C-terminal and auto-chaperone domains in Salmonella phage P22 tailspike and homologous proteins. (A) The structure of Salmonela phage P22 tailspike (PDB 1TYU). C-terminal domain remains in mature protein. (B) The structure (more ...)
Many phage structural proteins require chaperones to assist in folding and assembly. The first identified chaperone, GroEL/ES was found associated with phage λ capsid morphogenesis.19
Other early reports of chaperone that aid folding and assembly are associated with phage T4 head assembly,20
T5 tail assembly,21
and P22 coat protein folding.22-24
The initial efforts to identify a chaperone for P22 tailspike folding and assembly were unsuccessful,25
and the ability of the purified fully denatured chains to refold and assemble the native biologically active state in vitro argued against such a function.16-18,26
Recent reports reveal that K1F and Ø29 tailspikes utilize an intra-molecular chaperone domain (IMC). An emerging class of chaperones are the IMC/auto-chaperone segments of proteins that are necessary for the efficient folding and assembly of their own chains. They were initially identified as N-terminal pro-peptides or pro-sequences needed for maturation of exported proteases such as Subtilisin.27
Chen and Inouye refer to this class as type I.28
Another class, type II, is represented by the C- and N-terminal pro-peptides of collagen in which the pro-peptides align and tether the strands before the collagen triple helix is formed. These registration sequences are subsequently cleaved off.29
Recently, non-cleaved type II IMC-like domains have been reported in β-helical trimeric autotransporter proteins. The autotransporter Pertactin protein of Bordetella
, folds the C-terminus domain in the outer membrane, creating a channel for the rest of the chains to move through, and serving as the template for the folding of the N-terminal “passenger” domain.30,31
Recent studies of the assembly of tailspikes of the Bacillus
subtilis phage Ø29 and E. coli
phage K1F have shown the processes to be governed by their C-terminal domains.13,32
The crystal structures of these adhesins are close analogs to the tailspike of the Salmonella
bacteriophage P22 ().
The phage P22 tailspike is predominantly β-sheet ().10,11
The N-terminal domain (residues 1–124) is comprised of anti-parallel β-sheets forming a trimeric mushroom or dome-like structure that is required for binding to the phage capsid. The major structural domain (residues 143–539) is comprised of 13 rungs of parallel β-sheets in a β-helical conformation (). The length of the domain is presumably for the binding and cleavage of the lipopolysaccharide on the host cell surface.11,33
The three β-helical domains are bound through predominantly hydrophilic interfaces.10
Figure 2. Structure of P22 tailspike with cysteine locations, and schematic of the folding and assembly pathway. (A) P22 tailspike single chain crystal structure with the N-terminal head domain deleted. Yellow spheres indicate cysteine residues. (more ...)
After the 13th rung the parallel β-helix motif terminates and the three chains twist around each other and intertwine to form an interdigitated triple β-helix domain (residues 540–546) (). The chains (residues 547–612) then separate to form 3 sides of a triangular β-prism, essentially an oligomeric left handed β-helix. The subunit interfaces constitute a single buried hydrophobic core (). The chains then twist by a loop-short α-helix-loop that put three pairs of cysteines—C613 and C635—in a distinctive even plane, forming a ring-like conformation, termed the cysteine annulus (). The tailspike ends in a triple bladed motif termed the caudal fin.
The structures of the tailspikes of Bacillus
phage Ø29, and E. coli
phage K1F share similarities with the P22 tailspike (). The large structural domain region contains the elongated binding and catalytic domain, β-helical for P22 and Ø29 (), and for K1F, three β-propellers and a β-barrel (). Further along is the shaft region with different β-sheet structures: a triple β-helix in the P22 tailspike (); a triple β-prism for Ø29 (), and have similar elements to the a triangular β-prism/triple β-helix of K1F ().12,13
These three tailspikes have a trimeric bladed configuration in their C-terminal domains (, bottom panels). The tailspikes of Ø29 and K1F have “tentacles” that extended up to the triple β-helix shaft. P22 tailspike lacks these tentacles.
When the Ø29 appendage and K1F tailspike are expressed without the C-termini, the resulting tailspike chains aggregated.13,34
Ø29 and K1F mutants that retain the C-termini domain trimerize successfully. These results show that the C-termini domains are important for folding and assembly.
The C-termini of the Ø29 and K1F chains acting as IMC/autochaperones raise the question of the role of the C-terminal trimeric domain in the folding and assembly of the P22 tailspike. Unlike Ø29 and K1F, the P22 C-terminal domain is not cleaved after native trimer folding. However, the isolated C-terminal domain (residues 537–666) of P22 tailspike can function as an independent oligomerization domain.35
When three maltose binding protein were tethered to a trimeric P22 tailspike C-terminal domain, the chimera chains assembled into a P22 tailspike-like trimer. This suggests a type II IMC function is exhibited by the C-terminal domain of P22 tailspike.35
In earlier searches for cellular chaperones for P22 tailspike folding and assembly, infected cells were treated with iodoacetamide (IAM) with the intent to block ATP synthesis, in the hope of trapping chaperone complexes.25
IAM did indeed result in blocking intracellular tailspike folding and assembly. However, it turned out to be due to direct reaction of the IAM with reactive C-terminal cysteines in tailspike folding intermediates.25
These cysteine thiols are not reactive in the native state, and in fact are strongly hydrogen bonded.36
The in vivo and in vitro refolding pathway of the P22 tailspike proceeds through a number of folding intermediates (). The polypeptide chains emerge from the ribosome or out of the chaotropic agent and proceed to form a single chain partially folded intermediate, in which the β-helical domain is somewhat structured.17
The intermediate further folds to form a species competent for chain/chain association yielding the metastable protrimer. The protrimer then proceeds to the mature thermostable native tailspike trimer.16-18,26,37-40
The partially folded monomeric intermediates are very sensitive to temperature, and shift from productive folding to aggregation a few degrees above physiological temperature.15,41,42
As a result of this thermolability of the early folding intermediate, the chain is the locus of many temperature sensitive folding (tsf
The metastable protrimer is trapped in the cold, and can be distinguished from the native state by its retarded migration in native-PAGE. The protrimer has not acquired the detergent, thermal or protease resistance, found in the native state.37,38
The protrimer can be converted into the native state in the absence of exogenous proteins or factors.
Robinson and King reported that the protrimer intermediate formed in vitro contains transient inter-chain disulfide bonds, which must be reduced during maturation to the native state.45
The addition of reducing reagents to P22 tailspike folding intermediates retarded mobility of monomeric intermediate through native-PAGE, also suggesting the presence of intra-chain disulfide bonds, presumably formed from the reactive cysteines.46
The unusual reactivity of the C-terminal cysteine thiols may be due to interactions with side chains or backbone atoms within some distinctive local conformation, as seen in the reactive site of cysteine proteases. The thiol group of cysteine is deprotonated by nearby basic residues such as histidine before starting cleavage.47,48
Similarly the reduction of disulfides, formed during folding or assembly, may be performed by local side chain, or backbone interaction within folding intermediates. Disulfide exchange between intra- and inter-disulfide bonds among P22 tailspike folding intermediates support this hypothesis.49
The presence of disulfide bonds in intermediates, yet missing from the native state has been carefully studied for bovine pancreatic trypsin inhibitor.50-54
Similarly, the registration peptide of collagen utilizes transient intra-disulfide bonds for nucleation of C-terminal domain to stabilize the chain association stage.55-57
The collagen zipper-like trimer folding proceeds toward the N-terminal domain with using inter-disulfide bonds formation. At the end of trimer folding, both N-terminal and C-terminal domains are processed by procollagen peptidase.
The recent identification of the auto-chaperone function in phage Ø29 and K1F adhesins suggests that the P22 tailspike cysteine annulus may be acting to organize and get in register the tailspike chains for protrimer assembly. In that case one might expect mutants of these cysteine residues to retard folding, without absolutely blocking the overall reaction. Haase-Pettingell et al. showed that the cysteine to serine mutants in vivo retarded the rate of formation of native tailspike.58
The single serine mutations for each C-terminal cysteine decreased folding yields and kinetics, but did not completely inhibit trimer folding. However, double mutations of both cysteines for serine residues C613 and C635 sharply lowered the yield of native trimer.46,58
The homologous tailspike from Det7 of Salmonella enterica
lacks cysteines in this region, Seckler and coworkers have suggested that the transient disulfide bonds are not physiological but an artifact of the relatively slow in vitro refolding process.59
If they do not form disulfide bonds, the in vivo reactivity to IAA (iodoacetic acid) nonetheless indicates an unusual state of cysteine residues during folding and subunit assembly. This conformation dependent reactivity is likely to be a reporter or surrogate for the conformation of the C-terminal portion of the chain needed to carry out its auto-chaperone function.
As noted above, the unusual reactivity of at least two of the eight tailspike cysteines during folding, presumably represents activation by local interactions in some of the partially folded species. If the cysteine annulus motif represents an auto-chaperone function of this domain, it should form early in folding rather than later, as we previously proposed.38,39
The reactivity of those cysteines is likely to serve as a reporter for folding of this region of the chain. Here we examine more carefully the reactivity of the cysteine residues during refolding in vitro, following conformational intermediates separated by native-PAGE, and using mass spectrometry to identify folding and assembly intermediates with reactive cysteines thiols. The results suggest that the conformation maintaining the cysteine thiols in a reactive state forms very early in folding, prior to chain association, and that our earlier model needs to be revised.