Structural analysis of Bacillus subtilis SPP1 phage helicase loader protein G39P.
The Bacillus subtilis SPP1 phage-encoded protein G39P is a loader and inhibitor of the phage G40P replicative helicase involved in the initiation of DNA replication. We have carried out a full x-ray crystallographic and preliminary NMR analysis of G39P and functional studies of the protein, including assays for helicase binding by a number of truncated mutant forms, in an effort to improve our understanding of how it both interacts with the helicase and with the phage replisome organizer, G38P. Our structural analyses reveal that G39P has a completely unexpected bipartite structure comprising a folded N-terminal domain and an essentially unfolded C-terminal domain. Although G39P has been shown to bind its G40P target with a 6:6 stoichiometry, our crystal structure and other biophysical characterization data reveal that the protein probably exists predominantly as a monomer in solution. The G39P protein is proteolytically sensitive, and our binding assays show that the C-terminal domain is essential for helicase interaction and that removal of just the 14 C-terminal residues abolishes interaction with the helicase in vitro. We propose a number of possible scenarios in which the flexibility of the C-terminal domain of G39P and its proteolytic sensitivity may have important roles for the function of G39P in vivo that are consistent with other data on SPP1 phage DNA replication.
Bailey
Scott
S
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom.
Sedelnikova
Svetlana E
SE
Mesa
Pablo
P
Ayora
Sylvia
S
Waltho
Jon P
JP
Ashcroft
Alison E
AE
Baron
Andrew J
AJ
Alonso
Juan C
JC
Rafferty
John B
JB
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D004265:DNA Helicases / Q000378:metabolism; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C119220:G38P protein, Bacteriophage SPP1; C119221:G39P protein, Bacteriophage SPP1; D047908:Intracellular Signaling Peptides and Proteins; D017361:Viral Nonstructural Proteins; D004265:DNA Helicases