STRUCTURAL TRANSITIONS DURING BACTERIOPHAGE-HK97 HEAD ASSEMBLY

Bacteriophage HK97 builds its head shell from a 42 kDa major head prot ein, but neither this 42 kDa protein nor its processed, 31 kDa form is found in the mature head. Instead, each of the major head-protein sub units is covalently cross-linked into oligomers of five, six or more b y a protein cross-linking reaction that occurs both in vivo and in vit ro. Mutants that block prohead maturation lead to the accumulation of one of two types of proheads, termed Prohead I and Prohead II. Prohead I is assembled from about 415 copies of the 42 kDa (384 amino acids) protein subunit and accumulates in infections by mutant amU4. Followin g assembly, the N-terminal 102 amino acids of each subunit are removed , leaving a prohead shell constructed of 31 kDa subunits, called Prohe ad II, which accumulates in infections by mutant amC2. During DNA pack aging, when the prohead shell expands, all of the head protein subunit s become covalently cross-linked to other subunits. Purified Prohead I I (or, less completely, Prohead I) becomes cross-linked ill vitro in r esponse to any of a number of conditions that induce shell expansion, including conditions commonly used for protein analysis. In vitro cros s-linking occurs efficiently in the absence of added cofactors of enzy mes, and we propose that cross-linking is catalyzed by shell subunits themselves. Shell expansion is easily monitored by observing a decreas e in electrophoretic mobility of Prohead II in agarose gels. Using the mobility shift in agarose gel to monitor expansion and SDS/gel electr ophoresis to monitor cross-linking in vitro, we find that expansion pr ecedes and is required for cross-linking, and we propose that expansio n triggers the cross-linking reaction. Comparison of peptides isolated from Prohead II and in vitro cross-linked Prohead II shows a single a ltered major cross-link peptide in which a lysine, originating from ly sine169 of the protein sequence, is linked to asparagine356, presumabl y derived from the neighboring subunit. Examination of the cross-link- containing peptide by mass spectrometry shows that the cross-link bond is an amide between the side-chains of the lysine and the asparagine residues.