Single-Stranded DNA-Binding Proteins: Multiple Domains for Multiple Functions  Thayne H. Dickey, Sarah E. Altschuler, Deborah S. Wuttke  Structure  Volume 21, Issue 7, Pages 1074-1084 (July 2013) DOI: 10.1016/j.str.2013.05.013 Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 1 Overview of ssDNA-Binding Domains Proteins are colored tan, DNA is colored cyan, oxygen is colored red, nitrogen is colored blue, and sulfur is colored yellow. Regions of interest are highlighted in green. (A) OB-A from RPA70 (Protein Data Bank [PDB] ID code 1JMC) binds ssDNA using aromatic and cation-π-stacking interactions, hydrophobic interactions, and base-mediated H bonds. Variable loop regions are shown in green. (B) KH1 from PCBP1 (PDB ID code 3VKE) binds ssDNA with more phosphate-backbone contacts, but no intermolecular stacking interactions. The conserved GXXG motif is shown in green. (C) RRM1 from hnRNP A1 (PDB ID code 2UP1) binds ssDNA using a variety of interactions similar to those seen in the OB folds and KH domains. Conserved RNP sequence motifs are shown in green. (D) Two of the four subunits of the Why2 complex (PDB ID code 3N1J) are shown in tan and green. The interface has several hydrophobic and stacking interactions, but almost no base-mediated H bonds. Structure 2013 21, 1074-1084DOI: (10.1016/j.str.2013.05.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 2 Base-Mediated H Bonds Are Common at Sequence-Specific Interfaces, but Do Not Always Confer Specificity (A) Pot1pN (tan) binds ssDNA (cyan) with many H bonds that confer high specificity (PDB ID code 1QZH). (B) Schematic and high-resolution structures of nucleotide shuffling by TEBP. TEBP accommodates an additional 3′ nucleotide by shuffling bases to the adjacent 5′ binding site, flipping T8 into solvent, and resetting the binding register. Cognate telomeric sequence is shown in cyan (PDB ID code 1JB7) and noncognate is shown in yellow (PDB ID code 1PH1). (C) hnRNP A1 accommodates the unnatural base 2-aminopurine (2AP) by base flipping. 2AP (DNA and protein are shown in yellow) (PDB ID code 1U1P) maintains stacking interactions and maximizes H bonding by 180° rotation around the glycosidic bond relative to the cognate guanine (cyan) (PDB ID code 2UP1). (D) Pot1pC binds two different ssDNA sequences—GGTTACGGT (tan and cyan) (PDB ID code 4HIK) and GGTAACGGT (green and yellow) (PDB ID code 4HIO)—in globally altered binding modes. DNA base and backbone positions differ as well as loop 2/3 of the protein backbone. Structure 2013 21, 1074-1084DOI: (10.1016/j.str.2013.05.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 3 ssDNA-Binding Domains Frequently Occur in Multiple Copies Each domain is represented by a shape (circles, OB folds; crescents, RRMs), and separate polypeptide chains are colored differently. (A) As is the case with SSB, single-domain proteins can come together to form obligate complexes. (B) Additionally, single proteins themselves, such as FBP, can contain multiple homologous ssDNA-binding domains. Structure 2013 21, 1074-1084DOI: (10.1016/j.str.2013.05.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 4 Multiple Domains Can Be Used for a Variety of Purposes (A) As with SSB, DNA sliding can be achieved by keeping one or more domains bound while another domain repositions itself on the ssDNA ligand. (B) Handoff of an ssDNA ligand could be achieved by sequential dissociation and association of domains. Structure 2013 21, 1074-1084DOI: (10.1016/j.str.2013.05.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 5 Polypeptide Linkers Often Connect Domains within a Protein (A) These linkers may reduce specificity of a protein by accommodating spacers of different lengths between two binding sites, as seen in S. pombe Pot1. (B) These linkers may also allow conformational flexibility necessary to bind adjacent ssDNA-binding sites, as seen in the structures of individual KH domains. Structure 2013 21, 1074-1084DOI: (10.1016/j.str.2013.05.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 6 Many ssDNA-Binding Proteins Have Multiple Functions that Can Be Regulated in a Variety of Ways (A) Protein-protein interactions can affect ssDNA-binding ability. (B) Posttranslational modifications can affect protein-protein interactions and/or cellular localization. (C) Alternative splicing, illustrated here by a reduction in linker length, can affect protein-protein interactions, posttranslational modification, and/or cellular localization. (D) ssDNA sequence, length, or shape may affect function in as-yet unknown ways. Structure 2013 21, 1074-1084DOI: (10.1016/j.str.2013.05.013) Copyright © 2013 Elsevier Ltd Terms and Conditions