the ob-fold (oligonucleotide/oligosaccharide-binding fold) in protein-ssdna interactions
DESCRIPTION
The OB-fold (oligonucleotide/oligosaccharide-binding fold) in Protein-ssDNA Interactions Scott Morrical. Representative Phylogeny of OB-fold Proteins. 8 superfamilies (6 included in this tree) - PowerPoint PPT PresentationTRANSCRIPT
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The OB-fold
(oligonucleotide/oligosaccharide-binding fold)
in Protein-ssDNA Interactions
Scott Morrical
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Representative Phylogeny of
OB-fold Proteins
8 superfamilies (6 included in
this tree)
Largest is nucleic acid
binding proteins (in blue), which
has a deep lineage reflected by its members
spanning the tree
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Literature:
1. Theobald DL, Mitton-Fry RM, & Wuttke DS (2003) Nucleic acid recognition by OB-fold proteins. Annu. Rev. Biophys. Biomol. Struct. 32, 115-133.
2. Bochkarev A, & Bochkareva E (2004) From RPA to BRCA2: Lessons from single-stranded DNA binding by the OB-fold. Curr. Opin. Struct. Biol. 14, 36-42.
3. Bochkareva E, Belegu V, Korolev S, & Bochkarev A (2001) Structure of the major single-stranded DNA binding domain of replication protein A suggests a dynamic mechanism for DNA binding. EMBO J. 20, 612-618.
4. Bochkarev A, Bochkareva E, Frappier L, & Edwards AM (1999) The crystal structure of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding. EMBO J. 16, 4498-4504.
5. Bochkarev A, Pfeutzner RA, Frappier L, & Edwards AM (1997) Structure of the single-stranded DNA-binding domain of replication protein A bound to DNA. Nature 385, 176-181.
6. Yang H, Jeffrey PD, Miller J, Kinnucan E, Sun Y, Thoma NH, Zheng N, Cheng PL, Lee WH, & Pavletich NP (2002) BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure. Science 297, 1837-1848.
7. Horvath MP, Schweiker VL, Bevilacqua JM, Ruggles JA, & Schulz SC (1998) Crystal structure of the Oxytricha nova telomere end-binding protein complexed with single strand DNA. Cell 95, 963-974.
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General OB-fold Features:• Small (70-150 aa) structural motif used for binding single-stranded or highly structured nucleic acids, oligosaccharides; also observed at protein-protein interfaces.
• Found in many, but not all, proteins that bind ssDNA (cf. RecA family).
• Non-conserved sequence, but strongly conserved topology.
• Often described as a Greek key motif:
-- two 3-stranded antiparallel -sheets, where strand 1 is shared by both sheets.
-- -sheets pack orthogonally, forming flattened -barrel with 1-2-3-5-4-1 topology.
-- -helix frequently found between strands 3 & 4, packs against bottom of barrel.
OB-fold domain from streptococcal superantigen SMEZ-2
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General OB-fold Features (cont’d):• Tend to use a common ligand-binding interface centered on -strands 2 & 3.
• Canonical interface is augmented by loops between 1 and 2 (L12), 3 and (L3),
and 4 (L4), and 4 and 5 (L45), which define a cleft that runs across the surface
perpendicular to the axis of the -barrel.
• Most nucleic acid ligands bind within this cleft, typically perpendicular to the antiparallel -strands, with a polarity running 5’ to 3’ from strands 4 and 5 to strand 2 (the so-called “standard polarity”).
• Loops provide ideal recognition for ss nucleic acids, allowing binding through aromatic stacking, hydrogen bonding, hydrophobic packing, and polar interactions.
“Ideal” Canonical
OB-fold from AspRS
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Oxytricha novaTelomere End Binding Protein
OnTEBP
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OB-folds in Proteins that Interact with Highly Structured Nucleic Acids:Structure of Oxytricha nova Telomere End Binding Protein (OnTEBP)
Bound to G4T4G4
2 tandem OB-foldsused for ssDNA
binding
1 OB-foldused for hetero-
dimerization
1 OB-foldused for ssDNA
binding
4 OB-foldstotal
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OnTEBP Bound to G4T4G4
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Radical Induced Fit of OnTEBP-G4T4G4 ComplexN-terminal and - subunit OB-fold motifs clamp down on DNA, provide numerous
aromatic stacking, polar, and hydrophobic interactions to destbilize G-quartetand maintain telomeric DNA in a highly contorted and extensively buried state.
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Human RPA
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Human RPA Heterotrimer Contains 6 OB-fold Motifs
RPA70 DBD-A bound to ss oligowith conserved aromatics in red(missing in RPA70N and RPA14)
Loop flexibility in RPA70 DBD-B+/- ssDNA oligo
+ss
-ss
Structural inserts in RPA70 DBD-C
Zinc ribbon motifinserted in loop L12
3-helixbundle
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Comparison of OB-fold Structuresin RPA14, RPA32, & RPA70 DBD-B
RPA-14 RPA-32 (DBD-D)
RPA-70 (DBD-B)
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ssDNA Binding Mechanism of RPA
Unbound RPA in globular
conformation
Binding of 8 nt by DBD-A and DBD-B
Binding of 13-15 nt by DBD-A, DBD-B,
& DBD-CThe 4 DBDs, + RPA70N and RPA14 occlude ~30 nt in an
extended form
3 C-terminal -helices form trimeric interface
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X-ray Structure of Mouse/RatBrca2 ssDNA-Binding DomainComplexed to Dss1 & ssDNA
Yang et al. (2002) Science 297, 1837-1848
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SequenceConservation of Brca2DNA-BindingDomain
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Structure ofMouse Brca2DNA-BindingDomain:
D = Intact DBD-Dss1 complex
E = Tower deletion DBDmutant bound to Dss1 & oligo dT9
OB-fold: Oligonucleotide/oligosaccharide binding fold,structurally conserved.
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Brca2 Tower/3HB motif is inserted in loop L12 of
OB2 domain (recall RPA DBD-C)
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The Three OB-folds AreStructurally Superimposable& Resemble OB-folds Foundin Human RPA
Interfaces Between OB-foldsAre Arranged to Maintaina Continuous ssDNA-BindingGroove on the Surface
Some Tumor-Derived MutationsIn Brca2 Appear to Affect InterfacesBetween OB-folds. Many Others Map to the Tower Domain & Elsewhere
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Brca2 Contains a 35-ResidueThree-Helix Bundle (3HB) Similarto the Helix-Turn-Helix dsDNABinding Motif
Location: Distal End of Tower Domain(Helices T2, T3,T4)
Contributes to DNA-Binding Activity of Brca2DBD
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DNA Binding Properties of Brca2 DBD & Mutants
Tower is Necessary for ‘Fast Complex’
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Brca2DBDTower-Dss1-dT9 Complex at 3.5 Å5 of 9 ssDNA Residues Resolve, Bound Across OB2 & OB3
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Brca2: Designed to Load Rad51 Onto ssDNA?