Characterization of a Novel Intramolecular Chaperone Domain Conserved in Endosialidases and Other Bacteriophage Tail Spike and Fiber Proteins

Abstract

Folding and assembly of endosialidases, the trimeric tail spike proteins of Escherichia coli K1-specific bacteriophages, crucially depend on their C-terminal domain (CTD). Homologous CTDs were identified in phage proteins belonging to three different protein families: neck appendage proteins of several Bacillus phages, L-shaped tail fibers of coliphage T5, and K5 lyases, the tail spike proteins of phages infecting E. coli K5. By analyzing a representative of each family, we show that in all cases, the CTD is cleaved off after a strictly conserved serine residue and alanine substitution prevented cleavage. Further structural and functional analyses revealed that (i) CTDs are autonomous domains with a high ␣-helical content; (ii) proteolytically released CTDs assemble into hexamers, which are most likely dimers of trimers; (iii) highly conserved amino acids within the CTD are indispensable for CTD-mediated folding and complex formation; (iv) CTDs can be exchanged between proteins of different families; and (v) proteolytic cleavage is essential to stabilize the native protein complex. Data obtained for full-length and proteolytically processed endosialidase variants suggest that release of the CTD increases the unfolding barrier, trapping the mature trimer in a kinetically stable conformation. In summary, we characterize the CTD as a novel C-terminal chaperone domain, which assists folding and assembly of unrelated phage proteins.

Crystal structure of an intramolecular chaperone mediating triple–β-helix folding

“…An inhibition of the cleavage reaction is indicated by a signal at 45 kDa, corresponding to the uncleaved full-length protein; the mutation reported to inhibit cleavage (S911A) is shown as control 5 . A mutation that affects the folding behavior of the triple β-helix leads to insoluble protein as reported previously 6 and is indicated by a signal in the insoluble fraction (P). S911C indicates that a different nucleophilic residue can also embrace the role of the strictly conserved Ser911.…”

“…Folding of endoNF strictly depends on the presence of a C-terminal intramolecular chaperone domain (CIMCD), which is removed after proper assembly of the triple β-helix by autoproteolytic, ATPindependent cleavage 5,6 . EndoNF lacking the CIMCD exclusively forms insoluble protein aggregates during biosynthesis 6 .…”

“…Site-directed mutagenesis has shown that an alanine substitution of Arg1035 disturbs the interaction between the triple β-helix and both β-strands of the tentacle, resulting in an insoluble, incorrectly folded protein 6 . This sensitivity to spatial rearrangements is reflected in the conservation of secondary-structure elements and shows the important role of the tentacles in mediating proper triple-β-helix assembly.…”

“…It was shown that the covalent linkage between the CIMCD and N-terminal pre-protein is necessary for correct folding, indicating that an in trans function of the chaperone is impossible 5 . Furthermore, it could be shown that some of the chaperone domains are exchangeable between the different preproteins 5,6 . While the three-dimensional structures of the CIMCDs are as yet unknown, the crystal structure of N-terminally truncated mature endoNF shows that the homotrimeric enzyme comprises a triple β-helix involved in substrate recognition 7 .…”

“…The crystal structure reveals that these two nonpolar side chains participate in stabilization of the hydrophobic core of the stalk domain, a common pattern in triple β-helices. Mutation of the residues flanking strand β2 (His954, Gly956) led to insoluble protein, without detectable cleavage of the CIMCD 5,6 . Both residues are situated in a pocket between the triple β-helix and the flap surrounding it, forming hydrogen bonds to strand β1.…”