Solving the crystal structure of FLCN and subsequent bioinformatics studies identified FLCN, FNIP1 and FNIP2 as DENN-family proteins (Nookala et al., 2012, Zhang et al., 2012). The yeast orthologues of FLCN and FNIP1/2 are proposed to be Lst7 and Lst4 respectively. Pacitto et al. (2015) have now solved the crystal structure of Kluyveromyces lactis Lst4, confirming it to be a structural DENN-family protein and functional FNIP orthologue.
Using X-ray crystallography Pacitto et al. solved the structure of the N-terminal longin domain of Lst4 (residues 58-226) to 2.14 Å (PDB ID: 4ZY8). This showed the classic longin domain architecture of a core five-strand β-sheet with a single α-helix on the concave side and two α-helices on the convex side, identifying Lst4 as a DENN-family protein. K. lactis Lst4 is slightly more compact that the more commonly used Saccharomyces cerevisiae protein but there is high conservation of the α-helices and β-sheet strands surface amino acids.
To further validate Lst4 as an orthologue of FNIP1/2 Pacitto et al. demonstrated that Lst4 interacts with Lst7 forming a 1:1 heterodimer similar to FLCN-FNIP1/2. In humans, the DENN domain of FLCN interacts with FNIP1 (Baba et al. 2006). Lst7 does not contain a DENN domain, so Pacitto et al. used truncated C-terminal Lst4 proteins to show that the interaction with Lst7 is dependent on the Lst4 DENN domain. The authors found that this binding was not dependent on the unstructured insertion found in the Lst4 DENN domain, suggesting this region could have an alternative function, potentially in regulating the Lst7-Lst4 complex. Similar insertions of unknown function are found in the longin and DENN domains of human FNIP1/2.
As the Lst7-Lst4 complex contains two longin domains but only one DENN domain, Pacitto et al. propose that the interaction between the Lst7-longin domain and Lst4-DENN domain results in the formation of a functional DENN protein. The Lst4-longin domain is then available for other interactions potentially related to the propagation of DENN-signalling or could, like FNIP1, interact with the AMPK-orthologue Snf1.
The Lst7-Lst4 complex also recapitulates the relocation of FLCN-FNIP1/2 complexes to the lysosomal membranes under starvation conditions. The role of Lst7-Lst4 in TORC1 activity in response to amino acids was recently further elucidated by Péli-Gulli et al. (2015) who reported it as a GAP for the GTPase Gtr2. FLCN-FNIP2 has also been reported to function as GAPs for RagC in mammals (Tsun et al., 2012). However, DENN-family proteins typically function as GEFs for Rab proteins and the FLCN-FNIP1 complex has been reported to act as a GEF for RagA/B GTPases at the lysosomal membrane (Petit et al., 2013). The lack of Lst7-Lst4 GEF activity could be due to the absence of a second DENN domain or as Pacitto et al. suggest the binding of Lst-7 to the DENN-domain of Lst-4 could block the more characteristic DENN-protein GEF activity. Further studies are required to understand the roles of these complexes as GEFs and GAPs in TORC1 signalling and potentially with other GTPases.
The FLCN-FNIP1/2 complexes are unique as dimers of two DENN-family proteins and further studies are needed to clarify their structural arrangements and any impacts this has on function. The confirmation that the yeast orthologues Lst7 and Lst4 share both structure and function with FLCN and FNIP1/2 supports the use of yeast as a eukaryotic model for BHD. Further studies could also help to explain the roles of these proteins and also the impact of FLCN mutations on function. A more detailed understanding of the roles played by FLCN and associated proteins could aid the development of new treatments.
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