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Originally published In Press as doi:10.1074/jbc.M609543200 on December 7, 2006

J. Biol. Chem., Vol. 282, Issue 5, 2821-2831, February 2, 2007
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Characterization of a Novel Intramolecular Chaperone Domain Conserved in Endosialidases and Other Bacteriophage Tail Spike and Fiber Proteins*Formula

David Schwarzer, Katharina Stummeyer, Rita Gerardy-Schahn, and Martina Mühlenhoff1

From the Abteilung Zelluläre Chemie, Zentrum Biochemie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany

Received for publication, October 10, 2006 , and in revised form, November 16, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The largest group of bacteriophages, the order of tailed phages (Caudovirales), has evolved tail spike and fiber proteins for efficient virus-host interactions (1). These specialized adhesins mediate recognition and attachment to the bacterial surface and are the key determinants for host specificity. Since a variety of pathogenic bacteria are protected by thick layers of lipopolysaccharides or capsular polysaccharides, many tail spike proteins possess enzymatic activity. In particular, phages infecting encapsulated bacteria crucially depend on polysaccharide depolymerases (endoglycosidases or lyases) to gain access to the bacterial membrane (2, 3).

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. Interestingly, many spikes and fibers are built up by homotrimers that contain stretches of intertwined subunits like coiled-coil, triple beta-helix, or triple beta-spiral folds, leading to protein complexes that remain stable even in the presence of SDS (4).

One example are endosialidases, the tail spike proteins of Escherichia coli K1-specific phages. E. coli K1, a leading cause of sepsis and meningitis in newborns (5, 6), is encapsulated by {alpha}2,8-linked polysialic acid (7). The K1 capsule protects the bacterium from the immune system (8) but simultaneously provides the attachment site for specialized phages that are equipped with endosialidase tail spikes (9–14). In line with the concept of modular evolution of bacteriophages (15, 16), K1-specific phages evolved by insertion of an endosialidase gene into the tail spike or fiber locus of diverse progenitor phages (17). Thereby, the N-terminal capsid-binding domain of the original spike or fiber protein is kept to ensure proper integration of the newly acquired tail spike into existing tail architectures.

Recently, we solved the crystal structure of the conserved catalytic part of endosialidase endoNF2 (aa 246–911), the tail spike of phage K1F (18). The protein assembles into a catalytic homotrimer with an overall mushroom-like outline. In the cap region, each subunit is folded into a 6-bladed beta-propeller typical for sialidases and a lectin-like beta-barrel domain, which is involved in binding of polysialic acid. The distal part of the protein forms a stalk region, where all three subunits intertwine into a beta-helix fold, which is interrupted by a beta-prism domain. The catalytic part is flanked by the N-terminal capsid binding domain (aa 1–245) and a short C-terminal domain (CTD; aa 912–1064). During maturation, the CTD is released by proteolytic cleavage after a highly conserved serine residue (19). Exchange of this residue by alanine (S911A in endoNF) prevents cleavage but not assembly into active trimers, demonstrating that proteolytic processing is not a prerequisite for complex formation and activity. Although the CTD is not part of the native trimer, folding and trimerization of endosialidases crucially depend on the presence of an intact CTD in the nascent polypeptide. Truncation or even the exchange of a single histidine residue within the domain prevented trimerization and led to accumulation of inactive and predominantly insoluble protein (19, 20). Remarkably, the CTD is not restricted to endosialidases. Homologous domains were found as part of other tail spike and fiber proteins, suggesting a general role in folding and assembling of phage proteins (19).

In the present study, we focused on the structural and functional characterization of the CTD as part of an endosialidase but also of other spike and fiber proteins derived from distinct phages. The CTD acts as an intramolecular chaperone, probably by lowering a high energy barrier on the folding pathway. By analyzing a novel C-terminal chaperone domain, essential for folding and assembly of unrelated proteins, this work contributes not only to our understanding of bacteriophage evolution but might form the basis for engineering highly stable protein complexes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Colominic acid was purchased from Sigma. The vector pBlueScript SK– was obtained from Stratagene and pET expression vectors from Novagen.

Bacteria and Bacteriophages—Bacillus phage GA-1 (DSM 5548), Bacillus pumilus strain G1R (DSM 5549), and E. coli DSM 613 were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), and coliphage T5 was from the American Tissue Culture Collection (ATCC number 11303-B5). The E. coli K5 strain used in this study was isolated from urine at the department of Medical Microbiology of the Medizinische Hochschule Hannover (Hannover, Germany). E. coli BL21(DE3) Gold was purchased from Stratagene.

Cloning of Tail Spike and Fiber Proteins—The coding sequences of the tail genes encoding endoNF, gp12, LTF, and ElmA were amplified from purified phage DNA of coliphage K1F, Bacillus phage GA-1, coliphage T5, and genomic DNA of E. coli K5, respectively, using the following primer pairs: endoNF, MM97 (5'-cgggatccatgtccacgattacacaattc-3') and MM98 (5'-gtccgctcgagcttctgttcaagagcagaaag-3'); {Delta}N-endoNF lacking the first 245 aa, MM147 (5'-cgggatccgctaaaggggatggtgtc-3') and MM98 (5'-gtccgctcgagcttctgttcaagagcagaaag-3'); GA-1 gene 12, DS01 (5'-cgggatccatgcatagaccgccattc-3') and DS02 (5'-ggcctcgagactcaacctttcaagcttc-3'); LTF, DS03 (5'-cgggatccatggctataactaaaataattc-3') and DS04 (5'-ggcctcgagcatacctaatttatcctc-3'); ElmA, DS07 (5'-cgggatccatgacggtctcaaccgaa-3') and DS08 (5'-ggcctcgagattccctgttaattgcaa-3'). Sense and antisense primers are flanked by BamHI and XhoI sites (underlined), respectively. 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 His6 tag. The sequence identity of all PCR products was confirmed by sequencing. The amplified ltf gene showed five sequence deviations from the sequence published by Kaliman et al. (21) but was identical to the recently published sequence of Wang et al. (22).

Site-directed Mutagenesis—Mutagenesis was performed by PCR using the QuikChange site-directed mutagenesis kit (Stratagene) following the manufacturer's guidelines with the following primer pairs (mutated nucleotides are shown in boldface type): MM148 (5'-ccctattgttacttctgccggggagaggaaaacag-3') and MM149 (5'-ctgttttcctctccccggcagaagtaacaataggg-3') for endoNF-N912A; MM150 (5'-gctcgtattcacttcgcggttattgctcagc-3') and MM151 (5'-gctgagcaataaccgcgaagtgaatacgagc-3') for endoNF-G956A; DS45 (5'-gggtgaggagtggggtgttgcgcctgacggaattttctttgc-3') and DS46 (5'-gcaaagaaaattccgtcaggcgcaacaccccactcctcaccc-3') for endoNF-R1035A; DS25 (5'-cgggtgctattaacacagctgacgagagacataaaacgg-3') and DS26 (5'-ccgttttatgtctctcgtcagctgtgttaatagcacccg-3') for gp12-S620A; DS63 (5'-cgggtgctattaacacatctgcagagagacataaaacggac-3') and DS64 (5'-gtccgttttatgtctctctgcagatgtgttaatagcacccg-3') for gp12-D621A; DS65 (5'-aggcgaagaagctagatatgcatttggggttatcgctcaac-3') and DS66 (5'-attgagcgataaccccaaatgcatatctagcttcttcgcct-3') for gp12-H663A; DS67 (5'-gaagaagctagatatcattttgcggttatcgctcaacagattg-3') and DS68 (5'-caatctgttgagcgataaccgcaaaatgatatctagcttcttc-3') for gp12-G665A; DS49 (5'-ggcaacatctacagcatagcacctaccgaatgtcaatgg-3') and DS50 (5'-ccattgacattcggtaggtgctatgctgtagatgttgcc-3') for gp12-R720A; DS27 (5'-ccgtaaatggtacaattaacacagctgatgctagattgaagaacgatgttcg-3') and DS28 (5'-cgaacatcgttcttcaatctagcatcagctgtgttaattgtaccatttacgg-3') for LTF-S1264A; DS41 (5'-cagcgtttacggtgctagccgatgcgcgtttcaagactgc-3') and DS42 (5'-gcagtcttgaaacgcgcatcggctagcaccgtaaacgctg-3') for elmA-S675A. Plasmids containing the corresponding wild-type sequence were used as a template. 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 His6 tag.

Generation of C-terminally Truncated Proteins—Constructs encoding C-terminally truncated proteins were generated by PCR with the following primer pairs: MM124 (5'-ggaattccatatggagataactcag-3') (NdeI) and MM126 (5'-atccgctcgagttaagaagtaacaatagggtt-3') (XhoI) for endoNF{Delta}C (aa 246–911), DS29 (5'-ggttctactctagaagatggtgcagg-3') (XbaI) and DS30 (5'-atccgctcgagttaagatgtgttaatagcacc-3') (XhoI) for gp12{Delta}C (aa 621–740), DS15 5'-gcgatcccgggtcgattc-3' (SmaI) and DS31 (5'-atccgctcgagttaagatgtgttaatagcacc-3') (XhoI) for LTF{Delta}C (aa 1265–1396), DS37 (5'-cggaaactgccccaacg-3') and DS44 (5'-atccgctcgagttaggacagcaccgtaaacgctg-3') (XhoI) for elmA{Delta}C (aa 676–820). 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.

Generation of an Expression Plasmid Encoding the CTD of EndoNF—For separate expression of the endoNF CTD, DNA encoding aa 913–10643 of endoNF was amplified by PCR with the primer pair MM183 5'-GGAATTCCATATGGGGGAGAGGAAAACAGAGC-3' (NdeI site) and MM98 5'-GTCCCTCGAGCTTCTGTTCAAGAGCAGAAAG-3' (XhoI site). The obtained PCR product was subcloned into the NdeI and XhoI restriction sites of pET22b, resulting in a construct encoding the CTD of endoNF with a C-terminal His6 tag.

Generation of Chimera—The chimera {Delta}N-endoNF-gp12 was generated by homologous recombination in E. coli YZ2000 cells (Gene Bridges) (23) using the primers DS69 5'-GCAACCGTTTCACCACTGCATACCTCGGAAGCAACCCTATTGTTACTTCTgacgagagacataaaacggacatagc-3' (uppercase, nt 2684–2733 of the endoNF gene; lowercase, nt 1861–1886 of gene 12) and DS70 5'-gtaatacgactcactatagggcgaattgggtaccgggccccccCTCGAGACTCAACCTTTCAAGCTTCCTTC-3' (lowercase, nt 2198–2220 of gene 12; uppercase, nt 615–673 of pBluescript-SK–; underlined, XhoI site). A plasmid containing the coding sequence of wild-type gp12 was used as a template, and the resulting PCR product was recombined with the linearized plasmid pBluescript-{Delta}N-endoNF containing the coding sequence of endoNF lacking the N-terminal 245 amino acids. The product was subcloned into pET22b-Strep, resulting in a construct encoding aa 246–911 of endoNF with an N-terminal Strep-tag fused to aa 621–740 of gp12 with a C-terminal His6 tag.

Protein Expression and Purification—Proteins were expressed in E. coli BL21-Gold(DE3) in the presence of 100 µg/ml carbenicillin. Bacteria were cultivated in Luria-Bertani broth at 37 °C (endoNF) or in PowerBroth (Athena Enzyme Systems) at 15 °C (gp12, LTF, and ElmA). At an optical density (A600) of 0.6, expression of endoNF was induced by adding 1 mM isopropyl-1-thio-beta-D-galactopyranoside, and bacteria were harvested 2.5 h after induction. Expression of gp12, LTF, and ElmA was induced by adding 0.1 mM isopropyl-1-thio-beta-D-galactopyranoside at an A600 of 1.5, and bacteria were harvested 20 h after induction. Bacterial lysates were obtained by adding 2% (w/v) SDS and 0.1 M beta-mercaptoethanol in 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, followed by incubation at 95 °C for 5 min. Proteins were precipitated by adding 10 volumes of acetone, and pellets were resuspended in sample buffer containing 1.5% (w/v) SDS. For the analysis of soluble and insoluble proteins, bacteria were lysed in BugBuster MasterMix (Novagen), and soluble and insoluble fractions were obtained after centrifugation (22,000 x g, 20 min, 4 °C). For protein purification, 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. To isolate proteolytically matured protein, the minor fraction of noncleaved protein was removed by passing samples over a HisTrap column prior to StrepTactin chromatography. For isolation of CTDs, proteolytically processed and full-length proteins were removed by StrepTactin chromatography before samples were loaded on a HisTrap column. Buffer exchange was performed on a HiPrep 26/10 desalting column (Amersham Biosciences).

Size Exclusion Chromatography—Size exclusion chromatography was performed on a Superdex 200 HR 10/30 column (Amersham Biosciences) equilibrated with 10 mM sodium phosphate buffer, pH 7.4, 300 mM NaCl, and 10 mM Tris-HCl, pH 8.0, for endoNF and gp12, respectively. The column was calibrated using the gel filtration molecular weight markers (MW-GF-200) from Sigma.

SDS-PAGE and Immunoblotting—SDS-PAGE was performed under reducing conditions using 2.5% (v/v) beta-mercaptoethanol and either 1.5% or 1% (w/v) SDS in the sample buffer. For Western blot analysis, proteins were blotted onto nitrocellulose (Whatman). Proteins containing an N-terminal Streptag II were detected by StrepTactin-alkaline phosphatase-conjugate (StrepTactin-AP; IBA) according to the manufacturer's guidelines. His6-tagged proteins were detected with 1 µg/ml penta-His antibody (Qiagen) followed by goat anti-mouse-IgG-AP (Dianova).

Determination of Endosialidase Activity—The enzymatic activity of endoNF variants was determined by means of the thiobarbituric acid assay as described earlier (19).

Circular Dichroism and Secondary Structure Estimation— Purified proteins were analyzed in 10 mM sodium phosphate buffer (pH 7.4) by far-UV CD on a J-715 spectropolarimeter (Jasco) in 1-mm and 0.1-mm path quartz cells. The 1-mm spectra (4-fold accumulation, 250 to 200 nm) were used for scaling the 0.1-mm spectra (16-fold accumulation, 250 to 182 nm) adjusting path length deviations of the 0.1-mm cell. The 0.1-mm spectra were analyzed for estimation of the secondary structure by use of the program CDpro suite (24, 25).

Protein Sequence Analysis—Multiple sequence alignment was performed using the ClustalV method of the MegAlign program version 6.1 (DNAstar Inc.) with the following parameters: gap penalty 15.0 and gap length penalty 10.0. The phylogenetic tree was calculated from the aligned sequences using the maximum likelihood method with molecular clock (Promlk version 3.6a3) in Phylip. The tree was printed with tree view (version 1.6.6).

Thermostability Assay—Purified protein in 1.5% SDS sample buffer was incubated for 5 min at a variety of temperatures in the range of 4–95 °C. Samples were placed on ice until further analysis by SDS-PAGE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Phage Proteins Containing an Endosialidase CTD—Using BLAST search analysis with the C-terminal sequence of endoNF (aa 908–1064), we identified a total of 13 phage proteins containing a similar domain. The identified proteins belong to diverse phages and can be grouped into four protein families (Table 1). Proteins of family I and II are capsular polysaccharide depolymerases specific for the respective host capsule, whereas members of family III and IV are neck appendage and tail fiber proteins, respectively, involved in phage adsorption. According to their different enzymatic or binding specificities, no sequence similarity is observed in the N-terminal part of proteins belonging to different families. The only observed relation between these proteins is found in their CTDs. By multiple sequence alignment, six amino acid clusters with conserved amino acids were identified, which are dispersed in regions of low sequence similarity (see Fig. S1 in the supplemental material). The proteolytic cleavage site Ser{downarrow}Asx identified in endosialidases (19) is located in cluster I and is strictly conserved in all proteins. The CTDs are of variable length (105–163 aa) with overall amino acid sequence similarities ranging from 10.8 to 44.9% (see Table 1). A phylogenetic tree based on the 13 CTD sequences revealed that similarities are not necessarily highest between members of one family (Fig. 1A), since CTDs of family I and III are split up into two different branches.


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  		TABLE 1
Bacteriophage proteins containing an endosialidase CTD

 
Maturation of Proteins with Endosialidase CTD—To investigate whether all proteins containing the endosialidase CTD undergo proteolytic processing, representatives of each family shown in Table 1 were selected and compared with endoNF: the K5 lyase ElmA, the neck appendage protein gp12 of Bacillus phage GA-1, and the LTF (L-shaped tail fiber protein) of coliphage T5 (see Fig. 1B for a schematic representation). Proteins were expressed in E. coli BL21 with an N-terminal Strep-tag and a C-terminal His6 tag. Protein maturation was analyzed by Western blotting, and results are shown in Fig. 2. Since the N-terminal capsid binding domain of endosialidases is not required for trimerization and activity (17, 18), an endoNF variant lacking the first 245 amino acids ({Delta}N-endoNF) was used in this study (see Fig. 1B for schematic representation). For each selected protein, two mutants were generated by (i) deletion of the respective CTD ({Delta}C) and (ii) alanine substitution of the highly conserved serine in the Ser{downarrow}Asx motif.

Notably, all investigated proteins were proteolytically cleaved, resulting in a large N-terminal (Fig. 2, A–D) and a short C-terminal fragment (Fig. 2E). Depending on the cleavage efficiency, variable amounts of full-length proteins were observed (Fig. 2, A–D), which were detectable by the N- and C-terminal epitope tags (see upper and lower panels in Fig. 2, A–D, respectively). Proteolytically processed proteins (marked with an asterisk) were only detectable by the N-terminal Strep-tag due to loss of the His6-tagged CTD during maturation. In all cases, the apparent molecular mass of the mature protein was similar to the mass of the respective truncated variant lacking the CTD ({Delta}C), indicating that all proteins were cleaved at the predicted Ser{downarrow}Asx cleavage site. Moreover, exchange of the highly conserved serine residue to alanine prevented cleavage, resulting in the expression of full-length proteins (Fig. 2, A–D). In summary, these results demonstrate that cleavage at the Ser{downarrow}Asx site is used for release of CTDs in proteins of all four families and that this cleavage is independent of the N-terminal protein context.

Functional Role of the CTD in Nonendosialidase Proteins— To investigate the functional role of the CTD in a protein context other than endosialidases, the neck appendage protein gp12 was chosen as a model protein and compared with endoNF. The two molecules derive from completely unrelated phages, namely Bacillus phage GA-1, infecting the Gram-positive soil bacterium B. pumilus, and phage K1F, infecting Gram-negative, pathogenic E. coli K1, respectively. EndoNF and gp12 were expressed in E. coli BL21 with an N-terminal Strep-tag and a C-terminal His6 tag, and the soluble fraction of the obtained bacterial lysates was analyzed as depicted in Fig. 3. Like endoNF, gp12 formed an oligomeric complex that is resistant to SDS at room temperature. By omitting the boiling step prior to SDS-PAGE, a high molecular weight band appeared for either protein, whereas in parallel, the intensity of the band corresponding to monomeric, proteolytically processed protein (endoNF* and gp12*) decreased. SDS-resistant complexes were only detectable by the N-terminal Strep-tag, demonstrating that they are built up exclusively by mature protein lacking the CTD. In nonboiled samples, a larger proportion of gp12* than {Delta}N-endoNF* was found in the monomeric state (compare second lanes from the left in Fig. 3, A and B), an indication that gp12 oligomers are less stable in the presence of SDS than endoNF trimers.

Similar to the results obtained for endoNF, truncated gp12, which lacks the CTD already in the nascent protein (gp12{Delta}C), was found exclusively as an insoluble, SDS-sensitive species (lane i). This observation demonstrates that the presence of the CTD in the nascent protein is essential for proper folding of endoNF and gp12. However, in both cases, release of the CTD during maturation is not a prerequisite for oligomerization, since SDS-resistant complexes were observed for the noncleavable mutants endoNF-S911A and gp12-S620A.


Figure 1
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  		FIGURE 1.
Phage proteins sharing a homologous CTD. A, phylogenetic tree based on the amino acid sequence alignment of 13 CTDs derived from the phage proteins listed in Table 1. CTDs derived from proteins with similar N-terminal parts are depicted in the same shade of gray and numbered according to the protein families given in Table 1: endosialidases (Ia and Ib), K5 capsule depolymerases (II), neck appendage proteins (IIIa and IIIb), and tail fiber proteins (IV). Accession numbers of the used protein sequences are given in the legend to Fig. S1 in the supplemental material. B, schematic representation of the four different phage proteins analyzed in the present study: the endosialidase of phage K1F, lacking the N-terminal capsid-binding domain ({Delta}N-endoNF), the neck appendage protein gp12 of Bacillus phage GA-1, the L-shaped tail fiber protein of coliphage T5 (LTF), and the K5 lyase ElmA of an E. coli K5 prophage. The nonrelated N-terminal parts are shown in different shades of gray, whereas homologous CTDs are shown in white with the amino acid numbers given above. Molecular masses are indicated for the primary translation products containing an N-terminal Strep-tag and a C-terminal His6 tag, and the values in parentheses indicate the masses for the respective cleavage products generated by proteolytic processing at a highly conserved serine residue as indicated by an arrow.

 
Mutation of Single Conserved Amino Acids Interferes with the Chaperone Function of the CTD—To further characterize the chaperone function of the CTD, we performed a comparative mutagenesis study with endoNF and gp12. Previously, we have shown that alanine substitution of a single histidine residue located in the center of the CTD (His-954 in endoNF) disturbed proper folding of endosialidases, leading to the accumulation of monomeric, inactive protein (19). In the present study, the identification of more proteins with a homologous CTD revealed that the targeted histidine is not conserved in all proteins but is substituted by isoleucine in a subset of neck appendage proteins (see cluster III in Fig. S1). However, this position is followed by a glycine (Gly-956 in endoNF) that is strictly conserved in all proteins (see Fig. S1). To prove that highly conserved residues are essential for the chaperone function of the CTD in endoNF and gp12, the following amino acids were exchanged by alanine: the Asx residue that is part of the cleavage site (endoNF-N912A and gp12-D612A), the glycine residue in cluster III (endoNF-G956A and gp12-G665A), and an arginine residue in cluster V (endoNF-R1035A and gp12-R720A). In addition, the mutants endoNF-H954A and gp12-H663A were analyzed. Notably, all mutated proteins were expressed only partially as soluble proteins, and endoNF variants were hardly detectable in the soluble fraction (Fig. 3A). For none of the mutants, release of the CTD was observed, and exclusively uncleaved, full-length proteins were detected. However, in contrast to the noncleavable mutants endoNF-S911A and gp12-S620A described above, all amino acid exchanges within the CTD prevented the formation of SDS-resistant complexes. In addition, the endoNF mutants N912A, H954A, G956A, and R1035A completely lost enzymatic activity, highlighting the importance of a functional CTD for folding and assembly of the catalytic trimer. Together, the data presented in Fig. 3 demonstrate that the CTD plays a crucial role in folding and assembling not only of endosialidases but also of other, unrelated phage proteins.

CTD Swapping between endoNF and gp12 Is Compatible with the Assembly of Active endoNF Trimers—Assuming similar functions of the C-terminal part in all tail spike and fiber proteins listed in Table 1, our next question was whether CTDs are interchangeable between proteins belonging to different families. To address this point, the chimera {Delta}N-endoNF-gp12 was constructed by replacing the CTD of endoNF by the respective fragment of gp12 (Fig. 4A). The resulting construct was expressed in E. coli BL21 with N-terminal Strep- and C-terminal His6 tag. Released protein fragments were purified by affinity chromatography using a Strep-Tactin column for isolation of {Delta}N-endoNF* and a Ni2+-chelating column for isolation of CTDs. Proteins were analyzed by SDS-PAGE showing N-terminal fragments in Fig. 4B and CTDs in Fig. 4D. Notably, the chimera was proteolytically processed, releasing N- and C-terminal fragments of the expected molecular masses. Irrespective of the origin of the CTD (chimera or wild-type protein), processed {Delta}N-endoNF* fragments formed trimeric complexes as determined by gel filtration (Table 2). However, different complex stabilities were observed in the presence of SDS. Trimers obtained after maturation of wild-type endoNF were stable in the presence of 1.5% SDS (see Fig. 4B, lane 2). By contrast, complexes derived from the chimera were SDS-sensitive under these conditions, and exclusively monomeric species were observed (Fig. 4B, lane 4). Only in the presence of ≤1% SDS, resistant oligomers were detectable as indicated by the appearance of high molecular weight bands in nonboiled samples (Fig. 4B, lane 8). Despite slight differences in SDS resistance, similar molar activities were observed for either {Delta}N-endoNF* variant (Fig. 4C), demonstrating that the gp12 CTD is able to mediate folding and assembly of catalytically active endosialidase trimers. CTDs released from chimeric proteins had, in perfect agreement with the corresponding fragments derived from wild-type gp12, an apparent molecular mass of 15 kDa (Fig. 4D) and, notably, were found to attain the same oligomeric state (see Table 2).


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  		TABLE 2
Quaternary structure of endoNF and gp12

The oligomerization state of the indicated protein fragments was determined for affinity-purified proteins by size exclusion chromatography. The molecular masses of the monomeric species were calculated from respective amino acid sequences including epitope tags. The oligomeric state was calculated from the values obtained by gel filtration divided by the mass calculated for the monomer.

 
Structural Characterization of the CTD of endoNF—In contrast to the {Delta}N-endoNF* trimer for which the crystal structure has been solved (18), no structural information is available for an endosialidase CTD. To learn more about the structural characteristics of this intramolecular chaperone domain, we analyzed the secondary structure of the CTD of endoNF purified from two different sources: (i) as cleavage product generated by proteolytic processing and (ii) as a separately expressed protein. For this purpose, a construct encoding only the CTD of endoNF was generated and expressed in the absence of the N-terminal catalytic part. Both CTD variants were affinity-purified on Ni2+-chelating columns by C-terminally added His6 tags. As shown by size exclusion chromatography, either CTD variant formed hexameric complexes (Table 2). Most probably, these complexes are dimers of trimers, and dimerization is presumably mediated by N-terminal regions of the CTD trimer, which become accessible only after cleavage. Accordingly, the non-cleavable mutant {Delta}N-endoNF-S911A was found exclusively as a trimer, and no dimerization via the trimeric CTDs was observed (Table 2).


Figure 2
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  		FIGURE 2.
Proteolytic maturation of phage proteins containing an endosialidase CTD. A–D, Western blot analysis of {Delta}N-endoNF (A), gp12 (B), LTF (C), and ElmA (D). For each protein, the following variants were expressed in E. coli BL21: wild type, C-terminally truncated protein lacking the CTD ({Delta}C), and a mutant in which the highly conserved serine residue that is part of the cleavage site Ser{downarrow}Asx was exchanged by alanine. All three variants were expressed with an N-terminal Strep-tag, and wild-type and serine mutants were additionally epitope-tagged with a C-terminal His6. Bacterial lysates were separated by SDS-PAGE followed by Western blot analysis using StrepTactin-AP (top) and anti-penta-His antibody (bottom) for N-or C-terminal detection, respectively. Bands corresponding to full-length and proteolytically processed protein (marked with an asterisk) are indicated by an arrow. E, Western blot analysis of released CTDs. Bacterial lysates of E. coli BL21 expressing {Delta}N-endoNF, gp12, LTF, or ElmA were separated by 14% SDS-PAGE, and the C-terminally His6-tagged protein fragments were detected by Western blot analysis with anti-penta-His antibody.

 


Figure 3
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  		FIGURE 3.
Complex formation of {Delta}N-endoNF and gp12 of Bacillus phage GA-1. For {Delta}N-endoNF and gp12, the following variants containing an N-terminal Strep-tag and C-terminal His6 tag were expressed in E. coli BL21: wild-type protein, a variant lacking the CTD, including His6 tag ({Delta}C), and a set of mutants with the indicated amino acid exchanges. The soluble protein fractions were analyzed by Western blotting using StrepTactin-AP (top) and anti-penta-His antibody (bottom) for N- and C-terminal detection, respectively. Prior to electrophoresis, samples were incubated for 5 min in the presence of SDS either at room temperature or at 95 °C as indicated. 1.5 and 1% SDS were used for the analysis of endoNF and gp12, respectively. In the case of the C-terminally truncated variants, also the insoluble protein fraction (indicated with i) was analyzed. Results obtained for {Delta}N-endoNF and gp12 are shown in A and B, respectively. The enzymatic activity of {Delta}N-endoNF variants was determined in the soluble fractions by the thiobarbituric acid assay, and results are given in the upper lane of A.

 
To determine if the CTD is an independent folding unit, we compared the secondary structure of CTDs obtained by separate expression and derived from proteolytically processed endoNF. Purified CTDs were analyzed by CD. As summarized in Fig. 5, almost identical CD spectra were obtained for both variants, demonstrating that the CTD represents an autonomous domain that folds independently of any N-terminal protein context. Interestingly, the CTD was found to be predominantly {alpha}-helical. This structural characteristic clearly separates the domain from the proteolytically processed {Delta}N-endoNF* trimer, which is built up almost exclusively by beta-folds (18).

Impact of N- and C-terminal Domains on the Thermostability of endoNF Trimers—Analysis of truncated endosialidases demonstrated that the N-terminal capsid binding domain is not essential for trimer assembly and activity (17, 18). However, so far, nothing is known of whether N-terminal capping or CTD release stabilize the catalytic trimer. Aiming to understand in more detail the impact of N- and C-terminal domains on thermostability of endosialidases, a comparative analysis of the following endoNF variants was performed: (i) wild-type, (ii) {Delta}N-endoNF lacking the capsid binding domain, and (iii) {Delta}N-endoNF-S911A lacking the capsid binding domain but retaining the CTD (see Fig. 6 for schematic representation of the isolated fragments). Equal aliquots of the purified proteins were incubated for 5 min in the presence of 1.5% SDS at temperatures ranging from 4 to 95 °C, and trimer stability was monitored by SDS-PAGE. Dissociation of wild-type trimers (Fig. 6A) into monomers with an apparent molecular mass of 100 kDa started at 49 °C and was completed at 60 °C. An identical melting behavior was observed for {Delta}N-endoNF* (Fig. 6B), demonstrating that the N-terminal capsid binding domain has no significant influence on the stability of the functionally folded trimer. By contrast, the noncleavable mutant {Delta}N-endoNF-S911A showed a different picture (Fig. 6C). At temperatures above 40 °C, an additional band of low electrophoretic mobility became visible, paralleled by dissociation of the complex as indicated by appearance of monomeric species. Most likely, the additional high molecular weight band represents a partially unfolded and therefore more loosely packed intermediate, a topological variant that forms before disintegration of the trimeric complex. Interestingly, the noncleavable variant {Delta}N-endoNF-S911A seems to be less stable, and disintegration of trimers was observed already at temperatures 6 °C lower than for complexes built up by proteolytically processed {Delta}N-endoNF* fragments. This observation suggests that cleavage of the CTD increases complex stability by increasing the energy barrier for the unfolding process.


Figure 4
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  		FIGURE 4.
Analysis of an endoNF-gp12 chimera. A, schematic representation of the generated chimera containing the catalytic part of endoNF (aa 246–911) and the CTD of gp12 (aa 621–740). B, chimeric {Delta}N-endoNF-gp12 was expressed in E. coli BL21 as a variant containing an N-terminal Strep-tag and a C-terminal His6 tag. Proteins were analyzed by Western blotting using StrepTactin-AP for detection. Soluble protein fractions incubated in the presence of 1.5% SDS are shown in lanes 1–4. Affinity-purified proteolytically processed protein ({Delta}N-endoNF*) derived either from wild-type {Delta}N-endoNF (lanes 5 and 6) or the chimera (lanes 7 and 8) was incubated in the presence of 1.0% SDS prior to electrophoresis. C, relative molar activity of affinity-purified {Delta}N-endoNF* derived from either {Delta}N-endoNF or chimeric {Delta}N-endoNF-gp12. Activities were determined by the thiobarbituric acid assay. Data are means ± S.D. of two independent experiments performed in duplicates, and values obtained for {Delta}N-endoNF-derived fragments were set to 100%. D, CTDs released from {Delta}N-endoNF, the chimera, and gp12 were affinity-purified by Ni2+-chelating chromatography. Proteins were separated by SDS-PAGE and analyzed by Western blotting with anti-penta-His antibody.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we characterized a novel C-terminal intramolecular chaperone domain, essential for assembling and folding of endosialidases and other tail spike and fiber proteins of diverse phages. Altogether, 13 phage proteins belonging to four different protein families were identified that contain this CTD: (i) endosialidases, the tail spike proteins of E. coli K1-specific phages that are required for depolymerization of the polysialic acid capsule of the host (26, 27); (ii) K5 lyases, the capsule-degrading tail spike proteins of phages infecting E. coli K5 (28, 29); (iii) the L-shaped tail fibers of coliphage T5, which accelerate phage adsorption by reversible binding to the O-antigen of the host (30); and (iv) the neck appendage protein gp12 of Bacillus phages GA-1, {varphi}29, PZA, and B103, which forms appendages that are essential for phage attachment (31).


Figure 5
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  		FIGURE 5.
Secondary structure analysis of the CTD of endoNF. The CTD of endoNF was purified after release from wild-type endoNF and after separate expression of aa 913–1064. For both variants, the secondary structure content was analyzed by far-UV CD spectroscopy. The CD signals are given as mean residue weight ellipticity [{Theta}]MRW. Spectra obtained for CTDs derived from proteolytically processed endoNF and from separate expression are shown as solid and dashed lines, respectively. Secondary structure proportions were calculated using CDpro suite (24, 25).

 


Figure 6
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  		FIGURE 6.
Thermostability of endoNF variants. Wild-type endoNF, {Delta}N-endoNF, and the mutant {Delta}N-endoNF-S911A were expressed in E. coli BL21. Proteolytically processed N-terminal parts of endoNF (endoNF*) and {Delta}N-endoNF ({Delta}N-endoNF*) and full-length {Delta} N-endoNF-S911A were affinity-purified on StrepTactin columns. Schematic representations of the purified fragments are shown on the right. For each variant, aliquots of purified protein were incubated in the presence of 1.5% SDS for 5 min at the indicated temperatures. After separation by 6% SDS-PAGE, proteins were visualized by Western blotting with StrepTactin-AP. Results obtained for endoNF*, {Delta}N-endoNF*, and {Delta}N-endoNF-S911A are shown in A–C, respectively. Bands corresponding to monomeric and trimeric protein species are indicated with an arrow.

 
By analyzing a representative of each family (endoNF, ElmA, LTF, and gp12 of Bacillus phage GA-1), we showed that independent of the N-terminal sequence, the CTD is released during protein maturation. Proteolytic cleavage occurs at a highly conserved Ser{downarrow}Asx cleavage site. The essential role of the serine residue was confirmed by alanine substitution, which prevented cleavage but did not affect folding and complex assembly.

Within the CTD, we identified four critical amino acids (Asn-912, His-954, Gly-956, and Arg-1035 in endoNF), which are essential for proper function of the chaperone domain. As shown for endoNF and gp12, single amino acid exchanges prevented the formation of functional oligomers, and only inactive monomeric species were observed. Notably, single alanine substitutions of the strictly conserved glycine and arginine residues led to the expression of full-length proteins, although the cleavage site was not affected. Since oligomerization seems to be a prerequisite for proteolytic processing, cleavage at the Ser{downarrow}Asx site might be mediated by an autocatalytic reaction. By losing the capability to trimerize, the mutants H954A, G956A, and R1035A might have lost the spatial conditions required for interor intramolecular self-cleavage.

As shown for endoNF and gp12, cleavage is not a prerequisite for oligomerization, and both noncleavable mutants, endoNF-S911A and gp12-S620A, formed SDS-resistant complexes. Whether cleavage is required for assembly onto phage particles is not yet known. However, in Bacillus phage {varphi}29, which is closely related to GA-1, only processed gp12 was found as a building block of the neck appendages (32, 33). Using gel filtration, we determined the oligomeric state of mature GA-1 gp12 to be hexameric. This is in agreement with the stoichiometry found in {varphi}29 particles, where 60 gp12 subunits form the 10 neck appendages (34, 35). However, because no crystal structure is available for gp12, it is unknown whether the hexameric neck appendages are arranged as dimers of trimers.


Figure 7
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  		FIGURE 7.
Proposed folding pathway of endoNF. In the schematic representation of endoNF, the CTD is shown in gray, and the folded beta-propeller is shown as a black circle.

 
In contrast to gp12 of Bacillus phage GA-1, only very weak similarity (~11% amino acid identity) was observed between CTDs of the three neck-appendage proteins of Bacillus phages {varphi}29, PZA, and B103, and the CTD of endosialidases. In the phylogenetic tree based on all identified endosialidase-like CTDs, they form an individual branch clearly separate from CTDs of other neck appendage proteins (branch IIIa and -b in Fig. 1). Nevertheless, the cleavage site and all amino acids shown in this study to be essential for CTD function are highly conserved. Moreover, the cleavage site seems to be functional, since for mature gp12 of {varphi}29 a molecular mass of 73,888 Da was determined by mass spectrometry (34), which is in agreement with a molecular mass of 73,963 Da calculated for cleavage after Ser-691 within the Ser{downarrow}Asx cleavage site.

Proteolytic maturation is a common theme among phage proteins involving phage-derived proteases or autocatalytic processes (36–40). In contrast to proenzymes like mammalian digestive serine proteases, the function of proteolytic maturation of phage proteins is not restricted to enzyme activation. In the case of the major head proteins of coliphage T4, cleavage is required for head expansion and DNA packaging (41). Similarly, release of the endosialidase CTD is not prerequisite for an active enzyme (19) but might provide a mechanism to increase protein stability. Based on the results obtained in the present study, we propose a folding pathway as depicted exemplarily for endoNF in Fig. 7. Folding of the nascent polypeptide chain starts with formation of independent domains like the endosialidase beta-propeller and the CTD. Upon release from the ribosome, monomers assemble into trimers. This step crucially depends on a functional CTD, since alanine substitutions of critical amino acid residues within this domain prevented oligomerization and caused accumulation of SDS-sensitive species. CTD-assisted trimer assembly may involve several partially folded intermediates, leading to an SDS-resistant protrimer that still contains CTDs. By generation of the noncleavable variant endoNF-S911A, we mimicked the protrimer state. Before dissociation into monomers, trimeric endoNF-S911A became partially unfolded, and an SDS-resistant intermediate with lower electrophoretic mobility than the native trimer appeared. Compared with processed complexes, full-length timers showed decreased thermostability, indicating that CTD release stabilizes the native trimer. Thus, proteolytic cleavage may lead to a kinetically stable protein, a trimer that is trapped in a specific conformation due to an unusually high unfolding barrier. In this model, the role of the CTD would reside in overcoming the even higher energy barrier during the folding process. Consequently, mutants with a defective or missing CTD are unable to adopt the transition state conformation required for the formation of functional trimers.

A similar mechanism to increase protein stability was described for the {alpha}-lytic protease of Lysobacter enzymogenes, a secreted serine protease involved in degradation of other soil bacteria (42). Folding of {alpha}-lytic protease essentially depends on the Pro domain, which stabilizes the folding transition state and the native state of {alpha}-lytic protease. Release of the Pro domain traps the mature protein in a metastable conformation of high rigidity and low susceptibility to proteases (42). In contrast to the endosialidase CTD, the Pro region is located at the N terminus. In addition, Pro can be expressed in trans as a separate polypeptide chain to assist folding of {alpha}-lytic protease, whereas the endosialidase CTD acts only in cis as part of the precursor protein (19). These differences may reflect the fact that Pro mediates folding of a monomeric protein, whereas the endosialidase CTD is involved in folding and assembling of an oligomeric complex.

Increasing longevity through kinetic stability seems to be a general mechanism observed for SDS-resistant proteins (43). Many of these are viral proteins and form oligomeric complexes with a high content of beta-folds. Interestingly, some also possess an assembly module. In the tail spike protein of Salmonella phage P22, an endorhamnosidase with triple beta-helix domain (44), the C-terminal domain is essential for folding and assembly of the SDS-resistant trimer (45, 46). However, unlike the endosialidase CTD, the dorsal fin domain of P22 remains part of the native trimer. This is also the case for the human adenovirus type 2 adhesin (Ad2), which forms trimeric fibers with a triple-beta-spiral shaft (47). The globular C-terminal domain is not only involved in receptor binding but also required for complex assembly. Another example for C-terminally located folding domains is the "foldon," the 27-aa CTD of fibritin, a segmented coiled-coil homotrimer that forms the fibrous whiskers of coliphage T4 (48). The foldon forms a beta-propeller and is essential for correct fibritin assembly. Interestingly, function of the foldon is independent of the N-terminal protein part, and the foldon can substitute the corresponding assembly domain of Ad2 (49). Moreover, the foldon was successfully used to stabilize HIV gp120, collagen, and T4 long tail fibers (50–52). However, in all cases the C-terminal assembly domain remains part of the trimeric protein complex and is composed of beta-folds. In contrast, CD analysis performed in the present study revealed that the endosialidase CTD is predominantly {alpha}-helical, although the catalytic trimer of endoNF is an all-beta-protein (18). Both proteolytically released and separately expressed CTDs showed identical secondary and quaternary structures, demonstrating that the CTD represents an independent folding unit. Although this domain is trimeric in the noncleaved mutant endoNF-S911A, hexamers were observed for processed CTDs, suggesting that dimerization of trimers occurs after release from the protrimer. Interestingly, gp57, a phage-derived chaperone involved in assembly of the T4 long and short tail fibers (53, 54), is also largely {alpha}-helical and forms trimers that dimerize to hexamers (55). Future studies aiming at solving the three-dimensional structures will reveal whether both proteins adopt similar folds despite lack of sequence similarity.

Similar to the foldon of fibritin, which can substitute the CTD of Ad2, the C-terminal chaperone domain characterized in the present study promotes stable complex formation independent of the N-terminal protein context. In the endoNF-gp12-chimera, the CTD of gp12 mediated the formation of SDS-resistant endoNF trimers, which were proteolytically processed and enzymatically as active as wild-type proteins. However, compared with wild-type trimers, a slightly decreased SDS resistance was observed for chimera-derived complexes, indicating minor structural differences. Sequence variations observed between CTDs of endoNF and gp12 might, therefore, reflect an adaptation to the respective N-terminal part.

In contrast to the P22 tail spike, Ad2, and fibritin, the C-terminal chaperone domain of endosialidases, K5 lyases, LTF, and gp12 is removed after completing the job. Proteolytic processing may have evolved not only to increase the unfolding barrier but also to avoid structural constraints imposed by the presence of the CTD.


   FOOTNOTES
 
* This work was supported by the Deutsche Forschungsgemeinschaft (DFG) in the framework of DFG Research Unit 548 (Ge801/7-1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence should be addressed. Tel.: 49-511-532-9807; Fax: 49-511-532-3956; E-mail: muehlenhoff.martina{at}mh-hannover.de.

2 The abbreviations used are: endoNF, endo-N-acetylneuraminidase or endosialidase (EC 3.2.1.129) of bacteriophage K1F; aa, amino acids; CTD, C-terminal domain; gp12, gene product 12. Back

3 Cleavage of endoNF occurs after the highly conserved Ser-911, releasing a CTD that starts with Asn-912. Asparagine residues, in particular in front of a glycine, are very labile and can undergo spontaneous degradation. Consequently, N-terminal sequencing of the released CTD revealed Gly-913 as the first amino acid (19), and the construct generated for separate expression of the endoNF CTD was designed accordingly. Back


   ACKNOWLEDGMENTS
 
We thank Prof. Dr. Robert Seckler (Institut für Physikalische Biochemie, Universität Potsdam, Potsdam-Golm, Germany) for access to the circular dichroism facility and helpful discussions. Miriam Schiff and Jasmin Hotzy are acknowledged for expert technical assistance.



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 DISCUSSION
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D. Schwarzer, K. Stummeyer, T. Haselhorst, F. Freiberger, B. Rode, M. Grove, T. Scheper, M. von Itzstein, M. Muhlenhoff, and R. Gerardy-Schahn
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