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  • Crystal and cryoEM structural studies of a cell walldegrading enzyme in the bacteriophage 29 tailYe Xiang, Marc C. Morais, Daniel N. Cohen, Valorie D. Bowman, Dwight L. Anderson, and Michael G. Rossmann

    Department of Biological Sciences, Purdue University, West Lafayette, IN 47907; and Departments of Diagnostic/Biological Sciences and Microbiology,University of Minnesota, Minneapolis, MN 55455

    Contributed by Michael G. Rossmann, April 28, 2008 (sent for review March 20, 2008)

    The small bacteriophage 29 must penetrate the 250- thickexternal peptidoglycan cell wall and cell membrane of the Gram-positive Bacillus subtilis, before ejecting its dsDNA genomethrough its tail into the bacterial cytoplasm. The tail of bacterio-phage 29 is noncontractile and 380 long. A 1.8- resolutioncrystal structure of gene product 13 (gp13) shows that this tailprotein has spatially well separated N- and C-terminal domains,whose structures resemble lysozyme-like enzymes and metallo-endopeptidases, respectively. CryoEM reconstructions of the WTbacteriophage and mutant bacteriophages missing some or mostof gp13 shows that this enzyme is located at the distal end of the29 tail knob. This finding suggests that gp13 functions as atail-associated, peptidoglycan-degrading enzyme able to cleaveboth the polysaccharide backbone and peptide cross-links of thepeptidoglycan cell wall. Comparisons of the gp13 mutants withthe 29 mature and emptied phage structures suggest the se-quence of events that occur during the penetration of the tailthrough the peptidoglycan layer.

    hydrolase infection structure zinc ion

    Bacterial cells, including Bacillus subtilis, are surrounded by apeptidoglycan-containing cell wall. The peptidoglycan con-sists of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues, cross-linked with oligopep-tides at each NAM (1). This highly cross-linked peptidoglycanlayer is not only essential for the integrity of bacteria in the faceof the osmotic pressure created by the high concentration ofmolecules in the cell, but also serves as a physical barrier thatrestricts the passage of macromolecular complexes. In Gram-negative bacteria, the peptidoglycan layer is within the periplas-mic space and is 2575 thick (2), whereas in Gram-positivebacteria the peptiglycan layer is outside the cytoplasmic mem-brane and can be as much as 250 thick (3). Most bacterio-phages have a tail that penetrates the host cell wall and mem-branes to deliver the genome. Bacteriophage tails are largemacromolecular complexes that contain components for recog-nizing and puncturing the host cell and have a channel wideenough to provide passage for ejection of the viral genome (47).The peptidoglycan layer is a major barrier that must be spannedby a phage tail to infect a host cell. Unlike most eukaryoticviruses, infection of a host by tailed bacteriophages usuallyrequires only one virion per bacterium, indicating that tailedbacteriophages have evolved mechanisms that easily surmountthe peptidoglycan barrier. Virus-encoded peptidoglycan degrad-ing enzymes, such as endopeptidases, N-acetylmuramyl-L-alanine amidases, N-acetylglucosaminidase, lysozymes, and lytictransglycosylases, have been found widespread in tailed bacte-riophages (8, 9). These enzymes are known to play a significantrole in facilitating tailed bacteriophage genome entry throughlocalized peptidoglycan degradation or rearrangement (9, 10)and are frequently structural components of the virion. Theirlocation within the virions is varied, but they are located mostlywithin the tail (8, 11, 12).

    Bacteriophage 29 infects the Gram-positive bacterium, B.subtilis, and is one of the smallest known tailed phages (13). The

    mature 29 particle contains a prolate head and a short non-contractible tail. The head and part of the tail are filled with the19-kbp DNA genome and the terminal protein gene product 3(gp3) (7) that is covalently attached to both 5 ends of the DNAgenome (14). Previous studies show that the head consists ofthree proteins: the capsid protein (gp8), the head fibers (gp8.5),and the portal protein (connector) (gp10) (13, 15, 16). The tailhad been found to consist of three proteins: gp11, gp12* (cleavedfrom the gp12 precursor), and gp9, that form the lower collar,appendages, and knob, respectively (Fig. 1) (7, 17). Biochemicaldata (8) have suggested that the peptidoglycan-degrading activ-ity associated with 29 lies in the DNA terminal protein gp3,although the crystal structure of gp3 does not support such afunction (18). However, as gp9 is located at the distal end of thetail, it might be associated with the cell wall-degrading enzymeused by 29.

    The gp13 of bacteriophage 29 consists of 365 amino acidresidues. Although gp13 had not been previously detected in thevirion (19), it was known to be an essential morphogenetic factorthat functions in the phage tail assembly stage and is indispens-able for producing infectious particles (19, 20). In vitro comple-mentation experiments showed that gp13 is required after gp11has assembled onto the phage head. Furthermore, gp13 has beenshown to interact with gp9 in vivo to yield infectious particles(19). Re-examination of gp13 using specific antisera in Westernblots showed that it is a structural component of WT 29,emptied 29, free tails, and the mutant sus13(330) virus (20, 21).This mutant virus has a nonsense stop codon that results inproduction of gp13 that is missing the last 34 aa. On the otherhand, gp13 was not detected by Western blotting in the tail forthe sus13(342) mutant virus (21), which has a nonsense stopcodon that results in the production of a vestigial gp13 consistingof only the 32 N-terminal residues. These results demonstratethat gp9, gp11, gp12, and gp13 are all components of 29tails (21).

    Here, we report cryoEM image reconstructions that suggestgp13 to be located at the end of the tail knob. We also report thatthe crystal structure of gp13 has two domains that resemblelysozyme-like enzymes and metallo-endopeptidases, showingthat it probably functions as a tail-associated peptidoglycan-degrading enzyme.

    Author contributions: Y.X. designed research; Y.X. and M.C.M. performed research; D.N.C.,V.D.B., and D.L.A. contributed new reagents/analytic tools; Y.X. analyzed data; and Y.X.and M.G.R. wrote the paper.

    The authors declare no conflict of interest.

    Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,www.pdb.org (PDB ID codes 3CSQ, 3CSR, 3CSZ, 3CT0, 3CT1, and 3CT5). The cryoEM mapshave been deposited in the Electron Microscopy Data Bank, www.ebi.ac.uk/msd-srv/docs/emdb (accession nos. EMD1506 and EMD5010).

    Present address: Department of Biochemistry and Molecular Biology, University of TexasMedical Branch, Galveston, TX 77555.

    To whom correspondence should be addressed. E-mail: mr@purdue.edu.

    This article contains supporting information online at www.pnas.org/cgi/content/full/0803787105/DCSupplemental.

    2008 by The National Academy of Sciences of the USA

    95529557 PNAS July 15, 2008 vol. 105 no. 28 www.pnas.orgcgidoi10.1073pnas.0803787105

    http://www.pnas.org/cgi/content/full/0803787105/DCSupplementalhttp://www.pnas.org/cgi/content/full/0803787105/DCSupplemental

  • Results and DiscussionThe Structure of gp13. The full-length gp13 (residues 1365)formed very thin needle crystals, too small to detect a diffractionpattern. Hence, the protein was degraded with trypsin and themajor fragment (residues 1347, gp13TD) was repurified andcrystallized. The crystal structure of gp13TD [supporting infor-mation (SI) Text] showed that gp13TD had two spatially wellseparated domains, although the C-terminal domain was poorly

    defined. Therefore, three constructs, gp13NTD (residues 1159,the N-terminal domain), gp13CTD (residues 166365, the C-terminal domain), and gp13C (residues 1334, missing the endof the C-terminal domain), were made to study the domainsindependently. The gp13CTD construct failed to crystallize.Crystals of gp13C were isomorphous with gp13TD, but pro-duced a better electron density map. The resultant refined modelof gp13C (Fig. 2a) gave final Rworking and Rfree factors of 23.8%

    Fig. 1. Diagram of bacteriophage 29 showing its structural components. (Left) A surface-rendered presentation. (Right) A central section.

    Fig. 2. The structure of gp13. (a) Ribbon stereo diagram of gp13C with the N- and C-terminal domains shown in green and purple, respectively. Two flexibleregions that were not clearly visible in the electron density maps are shown in coral. The active sites of the N- and C- terminal domains are indicated by arrows.(b) Stereo diagram showing surface charge distribution of the gp13 N-terminal domain. Sugar substrates are shown in a ball-and-stick representation with N,C, and O atoms colored blue, gray, and green, respectively. Sugar residues at sites A, B, C, and D (black bonds) are from the crystal structure of gp13NTD complexedwith (NAG)4, whereas sugar residues at sites E and F (gray bonds) are based on homology model building. (c) Stereo diagram showing the interaction betweensugar substrates and protein residues. Parts of the protein C backbone are shown in a ball-and-stick presentation with C atoms colored red. Sugar substratesand protein residues are shown in a ball-and-stick presentation and colored the same as in b.

    Xiang et al. PNAS July 15, 2008 vol. 105 no. 28 9553

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    http://www.pnas.org/cgi/data/0803787105/DCSupplemental/Supplemental_PDF#nameddest=STXThttp://www.pnas.org/cgi/data/0803787105/DCSupplemental/Supplemental_PDF#nameddest=STXT

  • and 28.2%, respectively. Crystals of gp13NTD had a differentcrystal form and their structure was determined by molecularreplacement. The rmsd between equivalent C atoms in thegp13C and gp13NTD structures was 0.4 .

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