Structural Analysis of Bacillus
subtilis SPP1 Phage Helicase Loader Protein G39P*
Scott
Bailey
§,
Svetlana E.
Sedelnikova,
Pablo
Mesa¶,
Sylvia
Ayora¶
,
Jon P.
Waltho**,
Alison E.
Ashcroft,
Andrew J.
Baron,
Juan C.
Alonso¶, and
John B.
Rafferty§§
From the Krebs Institute for Biomolecular Research,
Department of Molecular Biology and Biotechnology, University of
Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom,
¶ Departamento de Biotecnología Microbiana, Centro
Nacional de Biotecnologia, CSIC, Campus Universidad Autonoma de Madrid,
Cantoblanco, 28049 Madrid, Departamento de Biología
Molecular, Universidad Autónoma de Madrid, 28049 Madrid, Spain, and Astbury Centre
for Structural Molecular Biology, School of Biology, University of
Leeds, Leeds LS2 9JT, United Kingdom
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.
*
This work was supported in part by European Commission
Grants BIO4-CT98-0106 and QLK2-CT-2000-00634 and an EMBL grant
under the "Human Capital and Mobility" Programme. The Krebs
Institute is a designated Biotechnology and Biological Sciences
Research Council Biomolecular Sciences Centre and a member of the North of England Structural Biology Centre.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1NO1) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§
Present address: Dept. of Molecular Biophysics and Biochemistry,
Yale University, Bass Center, Rm. 415, 266 Whitney Ave., New Haven, CT
06520-8114.
**
Lister Institute Research Fellow.
§§
Royal Society Olga Kennard Fellow. To whom correspondence should
be addressed. Tel.: 44-114-2222809; Fax: 44-114-2728697; E-mail:
j.rafferty@sheffield.ac.uk.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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