Structure, stability, and biological activity of bacteriophage T4 gene product 9 probed with mutagenesis and monoclonal antibodies.
Gene product (gp) 9 connects the long tail fibers and triggers the structural transition of T4 phage baseplate at the beginning of infection process. Gp9 is a parallel homotrimer with 288 amino acid residues per chain that forms three domains. To investigate the role of the gp9 amino terminus, we have engineered a set of mutants with deletions and random substitutions in this part. The structure of the mutants was probed using monoclonal antibodies that bind to either N-terminal, middle, or C-terminal domains. Deletions of up to 12 N-terminal residues as well as random substitutions of the second, third and fourth residues yielded trimers that failed to incorporate in vitro into the T4 9(-)-particles and were not able to convert them into infectious virions. As detected using monoclonal antibodies, these mutants undergo structural changes in both N-terminal and middle domains. Furthermore, deletion of the first twenty residues caused profound structural changes in all three gp9 domains. In addition, N-terminally truncated proteins and randomized mutants formed SDS-resistant "conformers" due to unwinding of the N-terminal region. Co-expression of the full-length gp9 and the mutant lacking first 20 residues clearly shows the assembly of heterotrimers, suggesting that the gp9 trimerization in vivo occurs post-translationally. Collectively, our data indicate that the aminoterminal sequence of gp9 is important to maintain a competent structure capable of incorporating into the baseplate, and may be also required at intermediate stages of gp9 folding and assembly.
Kurochkina
Lidia P
LP
Laboratory of Molecular Bioengineering, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 16/10 Miklukho-Maklaya Street, 117997 Moscow, Russia. lpk@ibch.ru
Vishnevskiy
Alexandr Yu
AY
Zhemaeva
Lyuba V
LV
Sykilinda
Nina N
NN
Strelkov
Sergei V
SV
Mesyanzhinov
Vadim V
VV
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000737:chemistry*; D018360:Crystallography, X-Ray; D019281:Dimerization; D017353:Gene Deletion; D006860:Hydrogen Bonding; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014771:Virion / Q000378:metabolism
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D014764:Viral Proteins; C071681:gene 9 protein, Enterobacteria phage T4