BHD Syndrome was first described in 1977 and although the FLCN gene was discovered in 2002 by Nickerson et al., the function of the protein has remained unknown. The majority of FLCN mutations found in BHD patients result in truncation of the protein and a loss of the C-terminal domain, suggesting an important role for this region. An interesting paper by Nookala et al. (2012) now presents the crystal structure of the C-terminal domain of FLCN, which gives a novel insight into the function of the protein.
Using X-ray crystallography, Nookala et al. determined the structure of the C-terminal domain of FLCN (residues 341-579) to 2 Å (PDB ID: 3V42). The domain is composed of a core β-sheet with helices packed on one side, followed by an all helical region. The authors noted that this fold was remarkably similar to that of DENN1B-S, with both proteins having the same strand order and orientation. In fact, overlaying the structures gave an r.m.s.d. of 2.8 Å over 170 residues, despite there being only an 11% sequence identity.
Differentially expressed in normal cells and neoplasia (DENN) proteins are Rab guanine nucleotide exchange factors (GEFs), which activate Rab GTPases by promoting GDP-GTP exchange. More information on Rabs and DENN domain proteins will be provided over subsequent blog posts. The crystal structure of DENN1B-S was recently solved, bound to its cognate Rab GTPase (Wu et al., 2011).
Since FLCN appears to possess a DENN-like fold, the author next tested whether FLCN has GEF activity towards small GTPases. Using biochemical analysis, the C-terminal domain of FLCN was shown to have GEF activity, in vitro, towards Rab35. This activity was also confirmed with the full length FLCN protein. Rab35 is involved in early endocytic trafficking, recycling events and cytokinesis, and these results now suggest that FLCN may also function in these pathways. Nookala et al. add, however, that FLCN GEF activity towards other small GTPases cannot yet be excluded.
The DENN proteins have three discrete domains, namely the upstream (u-DENN), core (c-DENN) and downstream (d-DENN) regions. The FLCN C-terminal domain maps to the c-DENN and d-DENN regions and, using secondary structure prediction programmes, the FLCN N-terminal domain appears to contain a fold similar to that of the u-DENN domain. Interestingly, FLCN also contains a Zinc-finger domain at the N-terminal of the protein, which is not found in other DENN proteins.
As evidenced from this paper, knowing the structure of a protein can go a long way to helping determine its function. Nookala et al. have proposed a novel function for FLCN as a Rab GEF and this will now open up many new avenues of research. It would be interesting to study endocytic transport in BHD patients, as a dysregulation of this pathway, caused by loss of the FLCN GEF activity, may contribute to the symptoms of BHD. Knowing the structure of FLCN will also assist in the development of drugs and therapies to treat BHD. This new discovery is an exciting step forward and it will no doubt play a big role in shaping the future direction of BHD research.
- Nookala RK, Langemeyer L, Pacitto A, Ochoa-Montano B, Donaldson JC, Blaszczyk BK, Chirgadze DY, Barr FA, Bazan JF, Blundell TL (2012). Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer Open Biol DOI: 10.1098/rsob.120071
- Nickerson ML, Warren MB, Toro JR, Matrosova V, Glenn G, Turner ML, Duray P, Merino M, Choyke P, Pavlovich CP, Sharma N, Walther M, Munroe D, Hill R, Maher E, Greenberg C, Lerman MI, Linehan WM, Zbar B, & Schmidt LS (2002). Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt-Hogg-Dubé syndrome. Cancer cell, 2 (2), 157-64 PMID: 12204536
- Wu X, Bradley MJ, Cai Y, Kümmel D, De La Cruz EM, Barr FA, & Reinisch KM (2011). Insights regarding guanine nucleotide exchange from the structure of a DENN-domain protein complexed with its Rab GTPase substrate. Proceedings of the National Academy of Sciences of the United States of America, 108 (46), 18672-7 PMID: 22065758