PMC:2728203 / 19005-22166 JSONTXT

{"project":"2_test","denotations":[{"id":"18190927-12531921-62517992","span":{"begin":3159,"end":3161},"obj":"12531921"}],"text":"Putative protein binding site centered on Phe73\nInspection of the solution and crystal structures of the Nab2 N-terminal domain showed the presence of a hydrophobic patch centered on Phe72 and Phe73 (Fig. 6a), suggesting that this region of the surface might represent a putative protein:protein interaction interface. Phe73 in particular was completely exposed on the surface of the protein. We therefore engineered a series of amino acid changes in these positions and assayed these mutants for binding to Mlp1 and Gfd1. Mutation of Phe72 had a negligible effect on the binding of the Nab2 N-terminal domain to either the Mlp1 C-terminal fragment or the Gfd1 C-terminus (Fig. 6b–d). In contrast, mutation of Phe73 to Asp or Ala dramatically reduced the affinity of Nab2 for the Mlp1 C-terminal fragment, whereas mutation to Trp did not decrease its affinity (Fig. 6b). Significantly, these mutations did not alter the binding of the Nab2 N-terminal domain to the Gfd1 C-terminal fragment, consistent with the mutations not altering the overall conformation of the domain (Fig. 6c). Binding experiments using yeast lysates confirmed that mutations in the hydrophobic patch on Nab2 interfered with the interaction between Nab2-N and the intact full-length Mlp1 protein. Equal amounts of GST-Nab2-N variants (WT, F72D, and F73D) were incubated in yeast lysate prepared from cells expressing protein-A-tagged Mlp1, and then, proteins associated with GST-Nab2-N were recovered using glutathione beads. Mlp1 that copurified with GST-Nab2-N was detected by immunoblotting for the protein A tag. As shown in Fig. 6d, full-length Mlp1 copurified with both wild-type Nab2-N and F72D Nab2-N. However, copurification of Mlp1 was not detected with either the GST control or the Nab2-N F73D mutant. Overall, the mutagenesis results are consistent with Phe73 forming a crucial component of the interaction interface between Nab2 and Mlp1 and hydrophobic interactions making an important contribution to the interface.\nIn summary, our results show that the N-terminal domain of Nab2 binds to the C-terminal domain of Mlp1 and that the Mlp1 binding site on Nab2 does not appear to overlap with the Gfd1 binding site. The Nab2 N-terminal domain has a fold based on a five-helix bundle that is analogous to the PWI fold found in SRm160 and other nucleic acid binding proteins, although it does not retain the nucleic acid binding function seen in other PWI domains. The hydrophobic side chain of Phe73 in the Nab2 N-terminal domain is exposed on the surface of the molecule and appears to be a crucial component of the Mlp1 binding site, although, clearly, further work will be required to define the precise nature of the interface between these two molecules. Our results are consistent with a model that envisages that interaction with Mlp proteins occurs soon before mRNA is exported and that Nab2 is important either for targeting the mRNA to the Mlp proteins or for releasing the mature mRNA from the Mlp proteins to enable its transit through the NPC. Such a model would be consistent with the increased nuclear retention of mRNA seen on overexpression of Mlp1 in vivo.21"}