| Escherichia coli bacteriophage HK97 assembles its mature capsid from 415 copies of the major capsid protein gp5. After assembly with maturation protease, the precursor capsid called prohead undergoes proteolysis and several stages of maturation including expansion and crosslinking to form a mature capsid. During capsid assembly and maturation, gp5 proteins assemble with each other and undergo refolding and conformational changes. Thus, some of the gp5 residues must play essential roles in the local network of intra- and inter- protein interactions, prohead assembly, and the different stages of capsid maturation. Here I establish a genetic suppressor approach to investigate the functional features of gp5 residues in vivo. By correlating the results from biochemical, structural, and genetic studies, specific protein interactions during HK97 capsid assembly and maturation have been determined. First I report that gp5 residue V163 facilitates HK97 head protein crosslinking, possibly by providing a local hydrophobic environment to promote crosslinking. I demonstrate that mutant gp5 V163D exhibits a specific defect in the head protein crosslinking reaction. Genetic results show that Val, Leu, Thr, Ile and Cys are tolerated at gp5 residue 163, suggesting a certain limited size and its hydrophobicity are important. Second, I propose that gp5 residues 231 and 178 play central and essential roles in HK97 capsid assembly by mediating assembly of proheads from capsomers. Single substitutions at residue 231 or 178, e.g. D231L, block the assembly of capsomers into proheads. Interestingly, the deficiency of the mutant substitution D231L can be suppressed by substitutions K178V,;K178I, or K178N. Instead of the wild type pair of residues D231 and K178, alternative pairs, D231L/K178V, D231L/K178I, and D231L/K178N, yield viable phage HK97. Structural analysis confirms that this interaction between residues 231 and 178 is involved at the early HK97 prohead assembly from capsomers. Both cases underline that genetic suppressor studies offer useful in vivo data to determine the functional importance of the HK97 gp5 residues. |
