PDBsum entry 3c94

Hydrolase

PDB id: 3c94

Contents

Protein chain

458 a.a. *

Ligands

ASP-ILE-PRO-PHE

ILE-PRO-PHE

Metals

_MG ×2

Waters ×42

* Residue conservation analysis

PDB id:

3c94

Name:

Hydrolase

Title:

Exoi/ssb-ct complex

Structure:

Exodeoxyribonuclease i. Chain: a. Synonym: exonuclease i, DNA deoxyribophosphodiesterase, drpase. Engineered: yes. Single-stranded DNA-binding c-terminal tail peptide. Chain: b, c. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 562. Strain: k12. Gene: sbcb, cpea, xona. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Other_details: peptide was chemically synthesized. The sequence is found naturally in e. Coli.

Resolution:

2.70Å R-factor: 0.201 R-free: 0.251

Authors:

D.Lu,J.L.Keck

Key ref:

D.Lu and J.L.Keck (2008). Structural basis of Escherichia coli single-stranded DNA-binding protein stimulation of exonuclease I. Proc Natl Acad Sci U S A, 105, 9169-9174. PubMed id: 18591666 DOI: 10.1073/pnas.0800741105

Date:

15-Feb-08 Release date: 08-Jul-08

Protein chain

P04995  (EX1_ECOLI) -  Exodeoxyribonuclease I from Escherichia coli (strain K12)

Seq: 475 a.a.

Struc: 458 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.1.11.1 - exodeoxyribonuclease I.
• Reaction: Degradation of single-stranded DNA. It acts progressively in a 3'- to 5'-direction, releasing nucleoside 5'-phosphates.

DOI no: 10.1073/pnas.0800741105

Proc Natl Acad Sci U S A 105:9169-9174 (2008)

PubMed id: 18591666

Structural basis of Escherichia coli single-stranded DNA-binding protein stimulation of exonuclease I.

D.Lu, J.L.Keck.

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.

Selected figure(s)

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).

Figures were selected by an automated process.