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The NMR structure of the gpU tail-terminator protein from bacteriophage lambda: identification of sites contributing
Lizbeth Edmonds1, Amanda Liu, Jamie J Kwan
1Department of Biology, York University, 4700 Keele Street, Canada M3J 1P3.
Abstract:
During the late stages of lambda bacteriophage assembly, the protein gpU terminates tail polymerization and participates at the interface between the mature capsid and tail components. When it engages the lambda tail, gpU undergoes a monomer-hexamer transition to achieve its biologically active form. Towards understanding how gpU participates in multiple protein-protein interactions, we have solved the structure of gpU in its monomeric state using NMR methods. The structure reveals a mixed alpha/beta motif with several dynamic loops at the periphery. Addition of 20 mM MgCl(2) is known to oligomerize gpU in the absence of its protein partners. Multiple image analysis of electron micrographs revealed ring-like structures of magnesium ion saturated gpU with a 30 A pore, consistent with its function as a portal for the passage of viral DNA into the host bacterium. The ability of magnesium ions to promote oligomerization was lost when substitutions were made at a cluster of acidic amino acids in the vicinity of helix alpha2 and the beta1-beta2 loop. Furthermore, substitutions at these sites abolished the biological activity of gpU.
Insights
Lambda bacteriophage protein gpU
Area of Science:
- Structural biology
- Virology
- Molecular biology
Background:
- Lambda bacteriophage protein gpU is crucial for viral assembly, terminating tail polymerization and mediating capsid-tail interactions.
- gpU transitions from a monomer to a hexamer to become biologically active upon engaging the lambda tail.
Purpose of the Study:
- To elucidate the structure of monomeric gpU and understand its role in protein-protein interactions.
- To investigate the mechanism of gpU oligomerization induced by magnesium ions.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy was used to determine the structure of monomeric gpU.
- Electron microscopy and multiple image analysis were employed to study magnesium-induced gpU oligomers.
- Site-directed mutagenesis was used to probe the function of specific amino acid residues.
Main Results:
- The monomeric gpU structure revealed a mixed alpha/beta fold with dynamic peripheral loops.
- Magnesium ion (MgCl2) addition induced the formation of ring-like gpU hexamers with a 30 Å pore, suggesting a portal function.
- Mutations in a cluster of acidic residues near helix alpha2 and the beta1-beta2 loop abolished magnesium-induced oligomerization and biological activity.
Conclusions:
- The structure of monomeric gpU provides insights into its interaction interfaces.
- Magnesium ions facilitate gpU oligomerization, likely through interactions with acidic residues, enabling its function as a portal for DNA translocation.
- Specific acidic residues are critical for both magnesium-induced oligomerization and the biological activity of gpU in bacteriophage assembly.
