Tailspike protein

In the first edition of this book this chapter was entitled "Antiparallel Beta Structures" but we have had to change this because an entirely unexpected structure, the p helix, was discovered in 1993. The p helix, which is not related to the numerous antiparallel p structures discussed so far, was first seen in the bacterial enzyme pectate lyase, the stmcture of which was determined by the group of Frances Jurnak at the University of California, Riverside. Subsequently several other protein structures have been found to contain p helices, including extracellular bacterial proteinases and the bacteriophage P22 tailspike protein. 

A more complex p helix is present in pectate lyase and the bacteriophage P22 tailspike protein. In these p helices each turn of the helix contains three short p strands, each with three to five residues, connected by loop regions. The p helix therefore comprises three parallel p sheets roughly arranged as the three sides of a prism. However, the cross-section of the p helix is not quite triangular because of the arrangement of the p sheets. Two of the sheets are... 

The number of helical turns in these structures is larger than those found so far in two-sheet p helices. The pectate lyase p helix consists of seven complete turns and is 34 A long and 17-27 A in diameter (Figure 5.30) while the p-helix part of the bacteriophage P22 tailspike protein has 13 complete turns. Both these proteins have other stmctural elements in addition to the P-helix moiety. The complete tailspike protein contains three intertwined, identical subunits each with the three-sheet p helix and is about 200 A long and 60 A wide. Six of these trimers are attached to each phage at the base of the icosahedral capsid. 

Steinbacher, S., et al. Crystal structure of P22 tailspike protein interdigitated subunits in a thermostable trimer. Science 265 383-386, 1994. 

Schuler, B., Furst, F., Osterroth, F., Steinbacher, S., Huber, R., and Seckler, R. (2000). Plasticity and steric strain in a parallel beta-helix Rational mutations in the P22 tailspike protein. Proteins 39, 89-101. 

Steinbacher, S., Seckler, R., Miller, S., Steipe, B., Huber, R., and Reinemer, P. (1994). Crystal structure of P22 tailspike protein Interdigitated subunits in a thermostable trimer. Science 265, 383-386. 

Figure 2-17 Wire model of the tailspike protein of bacteriophage P22 of Salmonella. Three of these fishshaped molecules associate as a trimer to form the spike. From Steinbacher et al.123...

Pectate lyase C from the plant pathogen Erwinia, which causes soft-rot in many different plants, has a parallel P barrel structure (Fig. 13-3)77 which is similar to that of the tailspike protein shown in Fig. 2-17 and represents what may be a very large structural family of proteins.78 The location of the active site is not... 

Carbonell X, Villaverde A, Peptide display on functional tailspike protein of bacteriophage P22, Gene, 176 225-229, 1996. 

Fig. 18. The P22 tailspike protein, (a) The main domain (Steinbacher et al, 1994). The protein is trimeric, and the 3-fold axis is parallel to the long axis of the monomer. The G-terminal sheets and the connecting loops link the subunits together, (b) A trimer of the N-terminal head-binding domain seen down the 3-fold axis (Steinbacher et al., 1997).

The tailspike protein from bacteriophage P22 is well suited for the second approach. The tailspike is a structural protein of P22. It is the last protein to bind to virus capsids during morphogenesis. The tailspike is also an endorhamnosidase which cleaves the 0-antigen protruding from its host cell Salmonella upon... 

In the presence of SDS, the unfolding rate accelerates and can be measured at lower temperature. SDS inhibits the refolding of the 7 species back to the native. There is no detectable aggregation in SDS and the transformation from N to 7 and then to M is quantitative. The unfolding mechanism of tailspike protein... 

Here, and k2 are the unfolding rate constants for the unfolding transition from N to I and from I to respectively. The unfolding kinetics can be quantitatively analyzed from the scanned intensities of the Coomassie blue stained tailspike bands on SDS gels. Figure 1 depicts results from a typical thermal unfolding experiment for wild type tailspike protein, which was performed in Tris buffer 8) and 2% SDS at 65 C. Kinetic analysis yields two rate constants 1.1 x 10 s and 4.0 x 10 s for the conversion from N to I and from I to A/, respectively. 

Figure 1. Thermal unfolding of wild type tailspike protein at 65 C. Thermal unfolding was performed by incubating 0.4 mg/ml tailspike prepared in 50 mM Tris (pH 8), 1.7 mM 2-mercaptoethanol and 2% SDS at 65°C. Samples were taken at the indicated times. The reaction was quenched by mixing the samples with SDS sample buffer (62.5 mM Tris at pH 7, 2.1 mM 2-mercaptoethanol, 10% glycerol, 0.012% Bromophenol blue dye and 2% SDS) in the cold. Then the samples were electrophoresed through SDS-PAGE at about 20°C and the proteins were stained with Coomassie blue.

Figure 2. Dependence of unfolding rate constants on pH. Wild type tailspike protein was prepared in 50 mM Tris, 1.7 mM 2-mercaptoethanol and 2% SDS and adjust to different pH values by 1 N HCl. Thermal unfolding was done at 65°C and followed by SDS-PAGE at about 20°C. Sample pH values shown here have been corrected to 65°C. kj (a) and k2 ( ) shown in log are the thermal unfolding rate constants for the conversions from N to I and from I to M, respectively. The linear lines through the data points are the results of least-square fit to each individual pH phase for both kj and k2 data. The calculated slopes of the fitting lines for kj are -0.46 and 0.35 for the low and high pH phases, respectively and for k2 are -1.9 and 1.1 for the low and high pH phases, respectively.

The intracellular folding mechanism of the P22 tailspike has been extensively characterized by genetic analysis (Figure 3). The maturation process of tailspike protein inside the cell proceeds through several defined intermediate stages. Three newly synthesized polypeptide chains first fold into conformations which are ready for association and then fold/associate to a trimeric intermediate, the protrimer, and finally fold into the native trimer 34,44). The half time for the monomeric chains to fold into a SDS- resistant native trimer in vivo at 30°C is about 5 min (56). 

Figure 3. Intracellular folding pathway of P22 tailspike proteins. The newly synthesized wild type or mutant polypeptide chains at 30°C first fold into partially folded monomeric intermediates. These species fold and associate to form a protrimer intermediate. Further folding results in a thermostable native tailspike. At 40°C, the folding is inhibited and tsf mutants act by blocking an early step in chain folding, prior to association. However, if infected cells are shifted to 30 C, the mutant chains continue through the productive pathway.

Other bacteriophages have been tested for polypeptide display. The minor fibrous protein fibritin of phage T4 has been fused at the C-terminus with a polypeptide of 53 residues [7]. An antigenic peptide has been fused to the C-terminus of the tailspike protein of Salmonella typhimurium P22 bacteriophage [8]. 

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