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, Bochkarev A, & Bochkareva E (2004) From RPA to BRCA2: Lessons from single- stranded DNA binding by the OB-fold. Curr. Opin. Struct. Biol. 14, 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, 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, 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, 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, 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, General OB-fold Features: Small ( 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 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 (contd): Tend to use a common ligand-binding interface centered on -strands 2 & 3. Canonical interface is augmented by loops between 1 and 2 (L 12 ), 3 and (L 3 ), and 4 (L 4 ), and 4 and 5 (L 45 ), 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 nova Telomere 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 G 4 T 4 G 4 2 tandem OB-folds used for ssDNA binding 1 OB-fold used for hetero- dimerization 1 OB-fold used for ssDNA binding 4 OB-folds total OnTEBP Bound to G 4 T 4 G 4 Radical Induced Fit of OnTEBP-G 4 T 4 G 4 Complex N-terminal and - 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. Human RPA Human RPA Heterotrimer Contains 6 OB-fold Motifs 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 +ss -ss Structural inserts in RPA70 DBD-C Zinc ribbon motif inserted in loop L 12 3-helix bundle Comparison of OB-fold Structures in RPA14, RPA32, & RPA70 DBD-B RPA-14RPA-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 nt by DBD-A, DBD-B, & DBD-C The 4 DBDs, + RPA70N and RPA14 occlude ~30 nt in an extended form 3 C-terminal -helices form trimeric interface X-ray Structure of Mouse/Rat Brca2 ssDNA-Binding Domain Complexed to Dss1 & ssDNA Yang et al. (2002) Science 297, Sequence Conservation of Brca2 DNA-Binding Domain Structure of Mouse Brca2 DNA-Binding Domain: D = Intact DBD- Dss1 complex E = Tower deletion DBD mutant bound to Dss1 & oligo dT 9 OB-fold: Oligonucleotide/ oligosaccharide binding fold, structurally conserved. Brca2 Tower/3HB motif is inserted in loop L 12 of OB2 domain (recall RPA DBD-C) 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 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 T 2, T 3,T 4) Contributes to DNA- Binding Activity of Brca2 DBD DNA Binding Properties of Brca2 DBD & Mutants Tower is Necessary for Fast Complex Brca2DBD Tower -Dss1-dT 9 Complex at 3.5 5 of 9 ssDNA Residues Resolve, Bound Across OB2 & OB3 Brca2: Designed to Load Rad51 Onto ssDNA?