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

David Schwarzer,Katharina Stummeyer,Rita Gerardy-Schahn,Martina Mühlenhoff

doi:10.1074/jbc.m609543200

David Schwarzer, Katharina Stummeyer + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m609543200

Copy DOI

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 alpha-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.

Highlights

In mature phage particles, tail spikes and fibers are exposed structures, which require high stability to maintain their functional conformation even under extreme conditions like high salt concentrations, the presence of extracellular proteases, and variations in pH and temperature
We focused on the structural and functional characterization of the C-terminal domain (CTD) as part of an endosialidase and of other spike and fiber proteins derived from distinct phages
Phage Proteins Containing an Endosialidase CTD—Using BLAST search analysis with the C-terminal sequence of endoNF, we identified a total of 13 phage proteins containing a similar domain

Summary

EXPERIMENTAL PROCEDURES

PCR products were ligated into BamHI/ XhoI sites of the expression vector pET22b-Strep, a modified pET22b vector containing the sequence encoding an N-terminal Strep-tag II followed by a thrombin cleavage site (WSHPQFEKGALVPRGS) and a C-terminal His tag. PCR products were subcloned into the BamHI and XhoI sites of pET22b-Strep resulting in a construct with an N-terminal Strep-tag and a C-terminal His tag. PCR products were subcloned into pET22b-Strep using the respective restriction sites given in parentheses, resulting in constructs that encode for proteins with an N-terminal Strep-tag. Bacteria were lysed by sonication in appropriate loading buffer containing protease inhibitors, and soluble fractions were loaded on StrepTactin-Superflow (IBA) and HisTrap HP columns (Amersham Biosciences) according to the manufacturer’s guidelines. Samples were placed on ice until further analysis by SDS-PAGE

RESULTS

Host cell receptor binding

DISCUSSION

Oligomeric state kDa