 |
PDBsum entry 3c94
|
|
|
|
References listed in PDB file
|
 |
|
Key reference
|
|
|
Title
|
|
Structural basis of escherichia coli single-Stranded DNA-Binding protein stimulation of exonuclease i.
|
|
|
Authors
|
|
D.Lu,
J.L.Keck.
|
|
|
Ref.
|
|
Proc Natl Acad Sci U S A, 2008,
105,
9169-9174.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Bacterial single-stranded DNA (ssDNA)-binding proteins (SSBs) play essential
protective roles in genome biology by shielding ssDNA from damage and preventing
spurious DNA annealing. Far from being inert, ssDNA/SSB complexes are dynamic
DNA processing centers where many different enzymes gain access to genomic
substrates by exploiting direct interactions with SSB. In all cases examined to
date, the C terminus of SSB (SSB-Ct) forms the docking site for heterologous
proteins. We describe the 2.7-A-resolution crystal structure of a complex formed
between a peptide comprising the SSB-Ct element and exonuclease I (ExoI) from
Escherichia coli. Two SSB-Ct peptides bind to adjacent sites on ExoI.
Mutagenesis studies indicate that one of these sites is important for
association with the SSB-Ct peptide in solution and for SSB stimulation of ExoI
activity, whereas the second has no discernable function. These studies identify
a correlation between the stability of the ExoI/SSB-Ct complex and
SSB-stimulation of ExoI activity. Furthermore, mutations within SSB's C terminus
produce variants that fail to stimulate ExoI activity, whereas the SSB-Ct
peptide alone has no effect. Together, our findings indicate that SSB stimulates
ExoI by recruiting the enzyme to its substrate and provide a structural paradigm
for understanding SSB's organizational role in genome maintenance.
|
|
|
|
|
|
|
|
Figure 1.
Structure of the E. coli ExoI/SSB-Ct complex. (A) Schematic
diagram of E. coli ExoI and SSB colored by structural features
[ExoI, Exonuclease domain (yellow), SH3-like domain (green), and
helical domain (red); SSB, oligonucleotide-binding (OB) domain
followed by ≈60 disordered C-terminal residues (orange)]. The
bar graph depicts evolutionary conservation of the SSB C
terminus (SSB-Ct) sequence among 284 bacterial SSB proteins as
percentage identity. (B) Ribbon diagram of ExoI bound to two
SSB-Ct peptides colored as in A. Mg^2+ ions are in magenta.
Dotted lines represent segments for which electron density was
not observed. (C) Surface representation depicting the binding
sites for two SSB-Ct peptides (A and B) bound to ExoI, colored
as in A. Selected ExoI residues are labeled. (D) Surface
representation as in C colored to model electropositive (blue)
and electronegative (red) potential. (E and F) Detailed views of
the peptide-A and peptide-B sites. The Arg-316 side chain from
apo ExoI (gray) is superimposed.
|
|
Figure 2.
Equilibrium binding highlights the roles of the
peptide-A-binding site and the basic ridge in SSB-Ct binding.
(A–D) Equilibrium binding isotherms of F-SSB-Ct (or peptide
variant) association with ExoI (or Ala variant) as monitored by
fluorescence anisotropy. All data points are the average of
three experiments. Error bars are one standard deviation from
the mean. (A) F-SSB-Ct (black), F-P176S (red), and F-mixed
(orange) ExoI binding isothems. (B) F-SSB-Ct binding by
peptide-A-site ExoI variants (R148A, magenta; Y207A, yellow;
Q311A, green; R316A, red). (C) F-SSB-Ct binding by
peptide-B-site ExoI variants (L331A, yellow; R327A, magenta).
(D) F-SSB-Ct binding by basic ridge (K227A, gray; R338A, blue)
and Mg^2+ binding site (E150A, red; E318A, green; D319A, purple)
ExoI variants. (E) Summary of ExoI variant F-SSB-Ct binding.
Residues are colored to reflect the fold-change observed in
F-SSB-Ct binding affinity relative to wild-type ExoI: <1-fold
binding changes (higher affinity) (teal), 1- to 2-fold (gray),
>2- to 5-fold (salmon), and >5-fold (red).
|
|
|
|
|
|
|
|
|
|
