The OB-fold (oligonucleotide/oligosaccharide-binding fold) in Protein-ssDNA Interactions Scott Morrical

1. The OB-fold (oligonucleotide/oligosaccharide-binding fold) in Protein-ssDNA Interactions Scott Morrical

2. 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

3. Literature: Theobald DL, Mitton-Fry RM, & Wuttke DS (2003) Nucleic acid recognition by OB-fold proteins. Annu. Rev. Biophys. Biomol. Struct. 32, 115-133. 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. 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. 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. 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. 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. 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.

4. 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 b-sheets, where strand 1 is shared by both sheets. • -- b-sheets pack orthogonally, forming flattened b-barrel with 1-2-3-5-4-1 topology. • -- a-helix frequently found between strands 3 & 4, packs against bottom of barrel. OB-fold domain from streptococcal superantigen SMEZ-2

5. General OB-fold Features (cont’d): • Tend to use a common ligand-binding interface centered on b-strands 2 & 3. • Canonical interface is augmented by loops between b1 and b2 (L12), b3 and a (L3a), a and b4 (La4), and b4 and b5 (L45), which define a cleft that runs across the surface perpendicular to the axis of the b-barrel. • Most nucleic acid ligands bind within this cleft, typically perpendicular to the antiparallel b-strands, with a polarity running 5’ to 3’ from strands b4 and b5 to strand b2 (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

6. Oxytricha nova Telomere End Binding Protein OnTEBP

7. 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-folds used for ssDNA binding 1 OB-fold used for ssDNA binding 1 OB-fold used for hetero- dimerization 4 OB-folds total

8. OnTEBP Bound to G4T4G4

9. Radical Induced Fit of OnTEBP-G4T4G4 Complex N-terminal a- and b- subunit OB-fold motifs clamp down on DNA, provide numerous aromatic stacking, polar, and hydrophobic interactions to destabilize G-quartet and maintain telomeric DNA in a highly contorted and extensively buried state.

10. Human RPA

11. Human RPA Heterotrimer Contains 6 OB-fold Motifs Zinc ribbon motif inserted in loop L12 -ss +ss 3-helix bundle RPA70 DBD-A bound to ss oligo with conserved aromatics in red (missing in RPA70N and RPA14) Loop flexibility in RPA70 DBD-B +/- ssDNA oligo Structural inserts in RPA70 DBD-C

12. Comparison of OB-fold Structures in RPA14, RPA32, & RPA70 DBD-B RPA-14 RPA-32 (DBD-D) RPA-70 (DBD-B)

13. ssDNA Binding Mechanism of RPA Unbound RPA in globular conformation Binding of 8 nt by DBD-A and DBD-B 3 C-terminal a-helices form trimeric interface Binding of 13-15 nt by DBD-A, DBD-B, & DBD-C The 4 DBDs, + RPA70N and RPA14 occlude ~30 nt in an extended form

14. X-ray Structure of Mouse/Rat Brca2 ssDNA-Binding Domain Complexed to Dss1 & ssDNA Yang et al. (2002) Science 297, 1837-1848

15. Sequence Conservation of Brca2 DNA-Binding Domain

16. Structure of Mouse Brca2 DNA-Binding Domain: D = Intact DBD- Dss1 complex E = Tower deletion DBD mutant bound to Dss1 & oligo dT9 OB-fold:Oligonucleotide/ oligosaccharide binding fold, structurally conserved.

17. Brca2 Tower/3HB motif is inserted in loop L12 of OB2 domain (recall RPA DBD-C)

18. The Three OB-folds Are Structurally Superimposable & Resemble OB-folds Found in Human RPA Interfaces Between OB-folds Are Arranged to Maintain a Continuous ssDNA-Binding Groove on the Surface Some Tumor-Derived Mutations In Brca2 Appear to Affect Interfaces Between OB-folds. Many Others Map to the Tower Domain & Elsewhere

19. Brca2 Contains a 35-Residue Three-Helix Bundle (3HB) Similar to the Helix-Turn-Helix dsDNA Binding Motif Location: Distal End of Tower Domain (Helices Ta2, Ta3,Ta4) Contributes to DNA- Binding Activity of Brca2 DBD

20. DNA Binding Properties of Brca2 DBD & Mutants Tower is Necessary for ‘Fast Complex’

21. Brca2DBDDTower-Dss1-dT9 Complex at 3.5 Å 5 of 9 ssDNA Residues Resolve, Bound Across OB2 & OB3

22. Brca2: Designed to Load Rad51 Onto ssDNA?