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)

Largest is nucleic acid

binding proteins (in blue), which

has a deep lineage reflected by its members

spanning the tree

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.

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

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

Oxytricha novaTelomere End Binding Protein

OnTEBP

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

OnTEBP Bound to G4T4G4

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.

Human RPA

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

Comparison of OB-fold Structuresin RPA14, RPA32, & RPA70 DBD-B

RPA-14 RPA-32 (DBD-D)

RPA-70 (DBD-B)

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

X-ray Structure of Mouse/RatBrca2 ssDNA-Binding DomainComplexed to Dss1 & ssDNA

Yang et al. (2002) Science 297, 1837-1848

SequenceConservation of Brca2DNA-BindingDomain

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.

Brca2 Tower/3HB motif is inserted in loop L12 of

OB2 domain (recall RPA DBD-C)

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

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

DNA Binding Properties of Brca2 DBD & Mutants

Tower is Necessary for ‘Fast Complex’

Brca2DBDTower-Dss1-dT9 Complex at 3.5 Å5 of 9 ssDNA Residues Resolve, Bound Across OB2 & OB3

Brca2: Designed to Load Rad51 Onto ssDNA?