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Updated: Jun 20, 2026
Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo
Published on: November 7, 2013
Observation of the membrane binding activity and domain structure of gpV, which comprises the tail spike of
Yasuhiro Kageyama1, Masanori Murayama, Takashi Onodera
1Department of Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
Abstract:
The P2 phage virion has tail spike proteins beneath the baseplate and uses them to adsorb to the outer membrane of Escherichia coli during the infection process. Previous immunoelectron microscopic studies suggested that the tail spikes are composed of the gene V product (gpV); however, experimental evidence of its membrane binding activity has yet to be reported. In this study, we purified and characterized recombinant full-length gpV and its C-terminal domain. Limited chymotrypsin proteolysis of gpV produced a C-terminal domain composed of Ser86-Leu211. Our experiments demonstrated that the N- and C-terminal domains have very different melting temperatures: 50 and 74 degrees C, respectively. We also found that gpV binds the E. coli membrane via its C-terminal domain. We conclude that the C-terminal domain of gpV is a stable trimer and serves as the receptor-binding domain for the second step in the phage adsorption process.
Insights
The P2 phage gene V product (gpV) binds Escherichia coli membranes via its C-terminal domain. This stable trimer domain is crucial for phage adsorption during infection.
Area of Science:
- Microbiology
- Molecular Biology
- Structural Biology
Background:
- P2 phage uses tail spike proteins for adsorption to Escherichia coli outer membrane.
- Previous studies implicated gene V product (gpV) in tail spike structure, but its membrane binding activity was unconfirmed.
Purpose of the Study:
- To characterize the recombinant full-length gpV and its C-terminal domain.
- To experimentally confirm the membrane binding activity of gpV and identify the responsible domain.
Main Methods:
- Purification and characterization of recombinant full-length gpV and its C-terminal domain (Ser86-Leu211).
- Limited chymotrypsin proteolysis to generate the C-terminal domain.
- Differential scanning calorimetry to determine melting temperatures of N- and C-terminal domains.
- Assessment of E. coli membrane binding activity of gpV domains.
Main Results:
- The N- and C-terminal domains of gpV exhibit distinct melting temperatures (50°C and 74°C, respectively).
- The C-terminal domain of gpV was identified as the functional unit responsible for binding to E. coli membranes.
- The C-terminal domain forms a stable trimer.
Conclusions:
- The C-terminal domain of P2 phage gpV is a stable trimer.
- This C-terminal domain mediates the second step of phage adsorption by binding to the E. coli outer membrane receptor.
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