Ribute significantly to either direct inter-subunit associations or organising the side chain positions to facilitate such interactions. For example, a hydrogen bond linking Glu43 with His46 helps to position the imidazole to also interact with Glu92 (Fig. 5). Glu92, close to the position of the NCS two-fold axis, forms a salt bridge interaction with Arg50 and also a well ordered and buried water molecule. Arg50 then donates a hydrogen bond to the partner subunit Gln93 and the water molecule mediates contact between Arg50 and the partner subunit Glu92. Arg68 forms an intersubunit salt bridge with Glu87, so linking a2 of one subunit with a3 of the partner subunit (Fig. 6). These residues are highlyconserved within SCAN domains and observed to form similar hydrogen bonding patterns where the structures are known. Furthermore, mutation of both the equivalent Arg50 and Arg61 residues to alanines in Zfp206 destabilizes heterodimerization with Zfp110 [26]. Thus, these POR 8 web invariant residues seem to play an important role in both homo- and heterodimerization. Other residues establishing inter-subunit contacts include Lys82, which interacts with Pro77 and Arg61 with Glu115 (Fig. 6). The former makes a hydrogen bond, while the later makes a salt-bridge interaction, which links a2 with the partner subunit a5. The later pair of residues is substituted to a similar lysine glutamine pair in some SCAN domain sequences. Some of the interactions noted in the PEG3-SCAN dimer are absent from the other structures. For example, a hydrogen bond donated from the side chain of Tyr94 on one subunit to the carbonyl of Pro60 (Fig. 7) on the partner cannot occur in otherFigure 4. Superposition of the PEG3-SCAN homodimer (purple) with other SCAN structures. Zfp206 (PDB 4E6S), Znf24 (PDB 3LHR), Znf42 (PDB 2FI2) and Znf174 (PDB 1Y7Q) are shown in cyan, green, yellow and grey, respectively. Superposition was calculated using secondary structure matching [49]. doi:10.1371/journal.pone.0069538.gSCAN Domain of PEGFigure 5. The dimer interface of PEG3-SCAN (I). A hydrogen-bonding network is formed between conserved residues lining the subunitsubunit interface. Water molecules are shown as red spheres, N and O positions are colored blue and red respectively, C positions are purple or green depending on the subunit to which they belong, hydrogen bonds are depicted as dashed lines. The same color scheme is used in Figures 6, 7 and 8. doi:10.1371/journal.pone.0069538.gFigure 6. The dimer interface of PEG3-SCAN (II). A second cluster of hydrogen bonding and salt bridge interactions at the subunit-subunit interface. doi:10.1371/journal.pone.0069538.gSCAN Domain of PEGstructures where phenylalanine replaces the tyrosine. Furthermore, in PEG3-SCAN there is an inter-subunit salt bridge between Glu56 and Lys101 (data not shown). Glutamate replaces the lysine in most other SCAN domains. Such sequence variations may confer a preference of different SCAN domains to form distinctive homo- and heterodimers. Whilst most of the dimer interface excludes water molecules, with the notable exception described above, at the periphery of the SCAN dimer there are five that are involved in mediating subunitsubunit interactions (data not shown). This does not appear to be a major Naringin factor in stabilizing the dimer given the high percentage of the surface area involved in direct association as described above. The dimer interface includes important stabilizing contributions from hydrophobic residues. A h.Ribute significantly to either direct inter-subunit associations or organising the side chain positions to facilitate such interactions. For example, a hydrogen bond linking Glu43 with His46 helps to position the imidazole to also interact with Glu92 (Fig. 5). Glu92, close to the position of the NCS two-fold axis, forms a salt bridge interaction with Arg50 and also a well ordered and buried water molecule. Arg50 then donates a hydrogen bond to the partner subunit Gln93 and the water molecule mediates contact between Arg50 and the partner subunit Glu92. Arg68 forms an intersubunit salt bridge with Glu87, so linking a2 of one subunit with a3 of the partner subunit (Fig. 6). These residues are highlyconserved within SCAN domains and observed to form similar hydrogen bonding patterns where the structures are known. Furthermore, mutation of both the equivalent Arg50 and Arg61 residues to alanines in Zfp206 destabilizes heterodimerization with Zfp110 [26]. Thus, these invariant residues seem to play an important role in both homo- and heterodimerization. Other residues establishing inter-subunit contacts include Lys82, which interacts with Pro77 and Arg61 with Glu115 (Fig. 6). The former makes a hydrogen bond, while the later makes a salt-bridge interaction, which links a2 with the partner subunit a5. The later pair of residues is substituted to a similar lysine glutamine pair in some SCAN domain sequences. Some of the interactions noted in the PEG3-SCAN dimer are absent from the other structures. For example, a hydrogen bond donated from the side chain of Tyr94 on one subunit to the carbonyl of Pro60 (Fig. 7) on the partner cannot occur in otherFigure 4. Superposition of the PEG3-SCAN homodimer (purple) with other SCAN structures. Zfp206 (PDB 4E6S), Znf24 (PDB 3LHR), Znf42 (PDB 2FI2) and Znf174 (PDB 1Y7Q) are shown in cyan, green, yellow and grey, respectively. Superposition was calculated using secondary structure matching [49]. doi:10.1371/journal.pone.0069538.gSCAN Domain of PEGFigure 5. The dimer interface of PEG3-SCAN (I). A hydrogen-bonding network is formed between conserved residues lining the subunitsubunit interface. Water molecules are shown as red spheres, N and O positions are colored blue and red respectively, C positions are purple or green depending on the subunit to which they belong, hydrogen bonds are depicted as dashed lines. The same color scheme is used in Figures 6, 7 and 8. doi:10.1371/journal.pone.0069538.gFigure 6. The dimer interface of PEG3-SCAN (II). A second cluster of hydrogen bonding and salt bridge interactions at the subunit-subunit interface. doi:10.1371/journal.pone.0069538.gSCAN Domain of PEGstructures where phenylalanine replaces the tyrosine. Furthermore, in PEG3-SCAN there is an inter-subunit salt bridge between Glu56 and Lys101 (data not shown). Glutamate replaces the lysine in most other SCAN domains. Such sequence variations may confer a preference of different SCAN domains to form distinctive homo- and heterodimers. Whilst most of the dimer interface excludes water molecules, with the notable exception described above, at the periphery of the SCAN dimer there are five that are involved in mediating subunitsubunit interactions (data not shown). This does not appear to be a major factor in stabilizing the dimer given the high percentage of the surface area involved in direct association as described above. The dimer interface includes important stabilizing contributions from hydrophobic residues. A h.
