In vitro maturation of prehead-like bacteriophage T4 polyheads: structural changes accompanying proteolytic cleavage and lattice expansion.
We have studied cleavage and expansion of the major T4 phage capsid protein gp23 (56 kDa) using polyheads, an aberrant, polymorphic tubular variant of bacteriophage T4D, as a model system. In a first step, we have cleaved the 65-amino-acid-long amino-terminal "delta piece" by limited proteolysis with Staphylococcus aureus V8 protease (type XVII) at exactly the same position, i.e., between residues 65 and 66, at which the phage-coded T4Ppase cleaves, to yield mature gp23* (48.7 kDa) without significantly affecting a second potential cleavage site between amino acids 142 and 143. Negatively stained preparations of thus cleaved polyheads revealed a near-hexagonal lattice with a 11.2-nm lattice constant. One-sided correlation averages of these cleaved/unexpanded polyheads yielded capsomeres that were rounder than those of prehead-like (i.e., uncleaved) control polyheads, with distinct trimers of mass (specifically, trimers of delta pieces in the area where three protomers are shared among three different capsomeres) removed, as revealed by difference maps computed from the correlation averages. Quantitative expansion of the 11.2-nm near-hexagonal lattice into the 13-nm near-hexagonal lattice characteristic of mature phage heads could be induced by pelleting the cleaved/unexpanded polyheads and resuspending them in water. This expansion step was inhibited by the presence of 100 mM phosphate. Cleavage of prehead-like polyheads to the 41-kDa gp23+ either between residues 142 and 143 by V8 protease or between residues 143 and 144 by trypsin rendered the polyheads unable to expand. In contrast, cleaved/expanded gp23* polyheads became resistant to cleavage to gp23+. As revealed by difference maps, the 7.3-kDa mass was additionally missing at the corners where the delta-piece trimers contact the protomers.
Müller
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M
M. E. Müller Institute for Microscopy, University of Basel, Switzerland.
Mesyanzhinov
V V
VV
Aebi
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U
D017122:Bacteriophage T4 / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure*; D002213:Capsid / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D014644:Genetic Variation; D008854:Microscopy, Electron; D008961:Models, Structural; D011110:Polymorphism, Genetic; D012697:Serine Endopeptidases / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology
D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase