Structure and Function of the T4 Spackle Protein Gp61.3.

Area of Science:

• Bacteriophage Biology
• Enzymology
• Structural Biology

Background:

• Bacteriophage T4 utilizes two lysozymes: T4 lysozyme (T4L) for progeny release and tail-associated lysozyme (gp5) for host cell envelope penetration.
• The T4 Spackle protein (gp61.3) was hypothesized to inhibit gp5 activity, conferring resistance to superinfection.

Purpose of the Study:

• To elucidate the mechanism by which T4 Spackle protein (gp61.3) interacts with and regulates the activity of the tail-associated lysozyme (gp5).
• To determine the structural basis for gp61.3's inhibitory function.

Main Methods:

• Biochemical assays to assess gp61.3-gp5 interaction and inhibition.
• Protein expression and purification.
• X-ray crystallography to determine the complex structure.

Main Results:

• gp61.3 is secreted and processed in the periplasm, forming a 1:1 complex with the gp5 lysozyme domain (gp5Lys).
• gp61.3 selectively inhibits gp5 activity but not T4L; gp5 overexpression causes cell lysis.
• The crystal structure reveals gp61.3 binds away from the active site, forming a barrier that prevents substrate access and locks gp5 in an inactive conformation.

Conclusions:

• gp61.3 acts as a specific inhibitor of the tail-associated lysozyme gp5, crucial for preventing premature host cell lysis.
• The unique inhibitory mechanism of gp61.3, involving allosteric regulation rather than active site blocking, provides new insights into enzyme inhibition strategies.