PMC:4103454 / 16772-19088 JSONTXT

{"project":"2_test","denotations":[{"id":"24981866-15980472-20021743","span":{"begin":615,"end":619},"obj":"15980472"},{"id":"24981866-17584917-20021744","span":{"begin":1262,"end":1266},"obj":"17584917"}],"text":"Modeling of FAN1 VRR-Nuc Domains Explains Their Oligomeric State and Substrate Specificity\nThe apparent inability of the VRR-Nuc domain of FAN1 to dimerize could in principle result from its interactions with other regions of the full-length protein or be an intrinsic property of the domain. To further investigate this and reconcile the apparent difference in substrate specificity and oligomeric state between bacterially derived VRR-Nuc domains and the FAN1 proteins, we examined sequence alignments of VRR-Nuc domains from each group prepared using the PRALINE multiple alignment server (Simossis and Heringa, 2005; Figures 5A and S4A). The majority of bacterially derived VRR-Nuc domains conform to the αβββαβα topology seen in the crystal structure of stNUC. A subset of these proteins additionally contain a predicted short α-helical section of 8–11 residues between β1-β2, in an equivalent position to the α-helical section of the stalk extension of psNUC. For the FAN1 proteins, however, this is replaced by two strongly predicted α helices approximately 10 and 18 residues long separated by a conserved I/L-G/P dipeptide, giving rise to an αβααββαβ topology that is likely unique among the restriction endonuclease-like superfamily (Knizewski et al., 2007). This difference is also apparent from phylogenetic analysis of VRR-Nuc sequences where FAN1-derived VRR-Nuc domains populate a clade distinct from the bacterial and phage examples (Figures 5B and S4B).\nTo understand how this two-helix insertion might affect the quaternary structure of the FAN1 VRR-Nuc domain, we generated models of the VRR-Nuc domains from hFAN1 and pFAN1, both of which resemble the bacterial VRR-Nuc domains with respect to the β sheet and position of the active site residues (Figures 5C and S4C). Helical wheel representations of the helical insertion suggest that the insertion forms an amphipathic helix-turn-helix motif that packs onto the hydrophobic surface of the central β sheet, effectively blocking the canonical VRR-Nuc domain dimerization interface (Figure 5D). In keeping with the VRR-Nuc domain structures described in this study, the negatively charged active site pocket is surrounded by a ring of positive charge, which also extends along the outer surface of the helical insertion in our models (Figure 5E)."}