P22 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. 

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).

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.

Steinbacher S, BaxaU, Miller S, Weintraub A, SecklerR, HuberR (1996) Crystal stracUire of phage P22 tailspike protein complexed with Salmonella sp. O-anfigtai receptors. Proc Natl... 

Based on the same principle, there are monomeric / -helical proteins that carry at their extremities a cluster of helical or nonrepetitive structures that could act as a capping element covering their exposed ends (Emsley et al., 1996 Lietzke et al, 1994 Petersen et al, 1997 Steinbacher et al, 1994). For example, the last 40 residues of pectate lyase C form a large loop that partially covers the surface of the /Hielix (Yoder et al, 1993). The fibrous (or otherwise elongated) domain of these natural /f-stranded proteins is not stable in isolation, as for example in the case of the P22 tailspike where bacterially expressed isolated /Hielix domain, at high concentrations, forms fibrous aggregates that bind Congo red (Schuler et al, 1999). 

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...

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

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... 

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). 

Recent reports on facilitation of protein folding by molecular chaperones 46-48) provide another possibility the difference between these two pathways could be a consequence of lack of certain cellular components in the in vitro reaction. It was observed that the products of gr< -bearing plasmid were able to rescue some temperature sensitive P22 tailspike mutants in Salmonella at the restrictive temperature (39 C), though very weakly 49), On the other hand, high yield of the native protein was also reported in the refolding (reconstitution) of the acid urea denatured tailspike polypeptide chains without the addition of cellular factors at low temperatures (10°C) (57). This in vitro result indicates that auxiliary factors inside the cell are not absolutely required for the folding of this protein, at least under these experimental conditions. However, this does not rule out that the cellular factors could play a role under in vivo conditions. 

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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