Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer

Mutations in the renal tumour suppressor protein, folliculin, lead to proliferative skin lesions, lung complications and renal cell carcinoma. Folliculin has been reported to interact with AMP-activated kinase, a key component of the mammalian target of rapamycin pathway. Most cancer-causing mutations lead to a carboxy-terminal truncation of folliculin, pointing to a functional importance of this domain in tumour suppression. We present here the crystal structure of folliculin carboxy-terminal domain and demonstrate that it is distantly related to differentially expressed in normal cells and neoplasia (DENN) domain proteins, a family of Rab guanine nucleotide exchange factors (GEFs). Using biochemical analysis, we show that folliculin has GEF activity, indicating that folliculin is probably a distantly related member of this class of Rab GEFs.


Summary
Mutations in the renal tumour suppressor protein, folliculin, lead to proliferative skin lesions, lung complications and renal cell carcinoma. Folliculin has been reported to interact with AMP-activated kinase, a key component of the mammalian target of rapamycin pathway. Most cancer-causing mutations lead to a carboxy-terminal truncation of folliculin, pointing to a functional importance of this domain in tumour suppression. We present here the crystal structure of folliculin carboxy-terminal domain and demonstrate that it is distantly related to differentially expressed in normal cells and neoplasia (DENN) domain proteins, a family of Rab guanine nucleotide exchange factors (GEFs). Using biochemical analysis, we show that folliculin has GEF activity, indicating that folliculin is probably a distantly related member of this class of Rab GEFs.

Introduction
Birt -Hogg-Dubé syndrome (BHD) is an inherited genetic disorder that predisposes individuals to renal cell carcinoma (RCC), benign skin tumours and lung cysts that lead to recurrent spontaneous pneumothorax [1,2]. Although BHD syndrome was first described in 1977 [3], it was not until 2002 that the gene encoding folliculin was identified and its mutation associated with the disease [1]. However, the cellular function of the protein remains unknown.
Folliculin and its interacting partners, FNIP1 and FNIP2, were shown to form a complex with AMP-activated protein kinase (AMPK) [5,6]. The involvement of folliculin, via AMPK, in mammalian target of rapamycin complex 1 (mTORC1) signalling remains unclear, as conflicting evidence has been reported [4,[7][8][9][10]. Folliculin was also reported, in two separate studies, to be involved in the transcriptional regulation of proteins in the transforming growth factor b (TGF-b) pathway. In the first study, Cash et al. [8] showed apoptotic defects in FLCN-deficient cell lines as a direct result of downregulation of a transcription factor, Bim, which is involved in the TGF-b pathway. In the second study, Hong et al. [10] showed that several genes from the TGF-b pathway are differentially expressed in cells with and without folliculin. Additionally, Preston et al. [11]  increases transcriptional activity of hypoxia-inducing factor 1-a (Hif1-a), a phenomenon often seen in RCC. RCC is a complex type of genitourinary cancer with different tumour histologies [2,12], instances of which have increased in the past few decades, accounting for 2-3% of all adult cancers and more than 80% of kidney cancers [13,14]. So far, mutations in seven genes (folliculin, von Hippel-Lindau protein, the proto-oncogene MET, the TSC1 and TSC2 proteins of the tuberous sclerosis complex, fumarate hydratase and succinate dehydrogenase) have been associated with metabolic-disorder-related RCCs [2]. There is a correlation between the histological subtype of RCC and the causal gene mutation; however, interestingly, all histological subtypes have been reported in BHD patients [15].
In BHD patients, the most common germline mutation that can lead to RCCs occurs in the mutation hotspot, exon 11, of the FLCN gene, and produces a truncated folliculin protein that lacks the C-terminal half [16]. It is not known whether tumours from patients carrying these truncating mutations, or from any other identified mutations, express endogenous mutated folliculin. Intriguingly, while the C-terminal domain of folliculin is highly conserved in vertebrates, it is seemingly absent in the putative yeast orthologue, Lethal with Sec13 protein 7 (LST7; [17] and electronic supplementary material, figure S1). The LST7 protein was shown to have an involvement in regulating amino acid transport, through trafficking of the GAP1 general amino acid permease between the Golgi and plasma membrane [17].
In this study, we use structural and biochemical analyses to show that folliculin is probably a distant relative of the differentially expressed in normal cells and neoplasia (DENN) family of guanine nucleotide exchange factors (GEFs) and demonstrate that it possesses in vitro nucleotide exchange activity towards Rab35 GTPase.

Results and discussion
In order to understand the essentiality of the C-terminal region (amino acids 341 -566) and reveal the function of full-length folliculin, we have determined at 2 Å resolution the three-dimensional structure of the C-terminal domain of folliculin, henceforth called folliculin-CT. We have calculated phases by the multi-wavelength anomalous dispersion (MAD) method using anomalous scatterers from selenomethionine (see §4). The folliculin-CT crystallized in C222 1 space group with two molecules in the asymmetric unit related by twofold non-crystallographic symmetry. The fold of folliculin-CT is dominated by an ab architecture with a core b-sheet and helices packed on the one side, followed by an all-helical region ( figure 1 and table 1). The NTPase ab-domain comes high in DALI [18] structural searches of the Protein Data Bank, but this approach does not consider the connectivity between secondary structures. Indeed, the strand topology differs, and the signature Walker A and B motifs [19] conserved for function across NTPase families are absent in the folliculin-CT domain.
Recently, Wu et al. [20] have reported the crystal structure of a DENN domain containing protein with its cognate Rab GTPase, Rab35. The DENN domain family consists of a group of ancient but poorly understood proteins that share common structural features and have been shown to be GEFs for Rab GTPases [21]; they facilitate GDP-GTP exchange, thereby activating the Rab GTPase in vesicular transport [22,23]. Rab GTPases form the essential network of vesicle membrane transport both in exo-and endocytic pathways [22]. Interestingly, folliculin-CT shares remarkable structural similarity with the DENN domain of the DENN1B-S protein; both proteins have the same order and orientation of strands (figure 2). Although the sequence identity is only 11 per cent, a structural alignment using the program BATON (based on COMPARER [24]) shows a r.m.s.d. of 2.8 Å over its 170 core-aligned residues, corroborating the strong similarity and probable homology. The amino acid conservation is evident in the JOY [25] alignment of 10 diverse homologues of both DENN1B and folliculin-CT (see the electronic supplementary material, figure S2).
The structural similarity to the DENN domain protein DENND1B suggests that folliculin might possess GEF activity towards a member of the Ras-superfamily of small GTPases, most likely a Rab GTPase regulator of vesicular trafficking. Indeed, biochemical analysis of a representative set of Rabs reveals that folliculin-CT, in vitro, has GEF activity towards Rab35, and this is confirmed using a subset of Rabs with the full-length folliculin, which shows similar activity to the folliculin-CT (figure 3). These findings suggest that folliculin may act as a Rab GEF in vivo. Rab35 has been implicated in early endocytic trafficking, recycling events and cytokinesis [26][27][28]. Perturbed Rab35-dependent transport may lead to aberrant regulation of specific signalling pathways, separate from those regulated by Rab5 (canonical growth factor pathways, etc.; [29]). However, although the structural similarity to the DENND1B Rab GEF implicates a Rab and the in vitro data presented here suggest that Rab35 might be involved, the divergence of folliculins from the DENN family does not exclude involvement of other members of the 44 member human Rab family [21] or other Ras-like small GTPases.
Wu et al. [20] identified several conserved clusters of residues (within 5 Å radius of Rab35) within the DENN1 subfamily as being essential for Rab binding/GEF activity. However, there is little conservation of the sequences in these regions among the most diverged DENN proteins. In a similar way, the equivalent regions in folliculin are highly conserved within the close orthologues, but are not conserved in the most divergent folliculins. We conclude that, if functions are conserved, there must be complementary changes in the interacting DENNs and their partner GTPases. Therefore, until the physiologically relevant partner of folliculin is identified and a model of the interaction developed, it is not possible to test this hypothesis by mutagenesis of specific interacting residues.
An initial sequence-based classification of the DENN superfamily suggested that the DENN homology region is composed of three distinct modules: upstream or u-DENN, the betterconserved central or core or c-DENN, and downstream or d-DENN regions [21,24]. These modules have been lent a structural form by the DENN1B fold [30], with u-DENN mapping to an N-terminal longin domain, whereas the core DENN and d-DENN modules respectively match the a/b fold and helical tail extension. In order to gain further insights into the overall architecture of the full-length folliculin (and thereby understand its function), we used sensitive methods developed for structure-guided alignment of distant sequences [31], fold recognition and structure prediction [32,33], to build domainlevel alignments of folliculin-like sequences, and to investigate the possibility of structural and functional similarities in the rsob.royalsocietypublishing.org Open Biol 2: 120071 N-terminal and central regions of folliculin and the DENN superfamily. Indeed, folliculin is transitively linked to the DENN superfamily by HMM-HMM alignment [32] to both the core set of DENN sequences [34] as well as a more recently described, outlier branch of DENN proteins related to yeast AVL9, which functions in exocytic transport [35]. The truncated folliculin polypeptide chain found in BHD syndrome is missing the c-DENN and d-DENN modules, and retains only the u-DENN region-which is the only part of the DENN homology region that is kept in the more compact yeast folliculin orthologue, LST7. The predicted u-DENN region in folliculin is linked by an approximately  rsob.royalsocietypublishing.org Open Biol 2: 120071 40þ amino acid disordered segment to the Rab-interacting c-DENN:d-DENN modules; the equivalent linker was removed (for crystallographic purposes) in the structure described for DENN1B [20]. In folliculin, this connector region has a stretch of acidic residues. It also harbours a bipartite tryptophan (WD -WQ) motif, which has been shown to be a kinesin light chain 1 interacting motif [36]; interestingly, the DENN1B protein (isoform 5) has a similar motif in the region spanning residues 629-729 [36]. Given the probable distant homology between folliculin and the larger DENN superfamily, and the close structural resemblance between folliculin and DENN1B protein (the Rab binding c-DENN:d-DENN modules and linker bipartite tryptophan motif ), the N-terminal regions of both proteins were scrutinized for closer architectural similarities. The N-terminal 85 amino acids of folliculin comprise a conserved HxCx 2 H-f28 -56 residuesg-Cx 2 C putative zincion-binding module (figure 4a), which is absent in the DENN superfamily. However, the following 160 amino acids have a predicted secondary structure pattern [32,37] that suggestively matches that of the prototypic longin domain fold (with a bbabbbaa topology [34]) found in the DENN1B N-terminal module. Although in folliculin there is a long loop predicted between the two C-terminal helices, HHpred [32] significantly aligns this folliculin segment with the DENN1B longin domain, suggesting that the N-terminus of folliculin might encompass a divergent longin-like fold, preceded by a split zinc-binding domain (figure 4a). Currently, efforts are underway to determine the crystal structure of this region. The presence of a longin domain would settle folliculin's kinship with the DENN superfamily, and would have implications for its function in regulation of membrane trafficking [38], as well as in structural supportper DENN1B-of Rab GEF function.
Genetically inherited syndromes offer an important insight into molecular mechanisms underlying biological processes of medical importance, such as carcinogenesis. Thirty-five years after the initial description of the inherited skin lesions and the subsequent association with the folliculin gene, we present data that address the molecular mechanism of BHD syndrome. Through structure -function analyses, we report that folliculin, which is commonly mutated in patients suffering from these malignancies, has an unusual architecture that classes it into an ancient family of proteins called the DENNs. Because most BHD symptoms are attributed to a truncated folliculin protein or its complete absence, we infer that the loss of folliculin and therefore lack of its cognate GTPase activation might be compromising the latter's regulatory function in membrane trafficking (figure 4b,c). Given the severity of the phenotype in BHD syndrome, we suggest that folliculin's GEF activity towards its GTPase might be essential for important cellular processes. In this context, it would be interesting to investigate defective endocytic transport in BHD patients.

Folliculin protein purification
The folliculin-CT was cloned into the Gateway system (Invitrogen, UK), and the recombinant protein was expressed as a thioredoxin fusion protein in BL21 (DE3) Star (Invitrogen, UK) Escherichia coli competent cells. The recombinant protein was initially purified over a 1 ml nickel-immobilized metal affinity chromatography column (Ni-IMAC). A proteolytic cleavage using tobacco etch virus protease was carried out for 1 h at room temperature to separate the affinity tag rsob.royalsocietypublishing.org Open Biol 2: 120071 from folliculin-CT. The reaction was subsequently passed over the Ni-IMAC column for removal of the tag and the remaining uncleaved fusion protein. The target protein was further purified using a Superdex 75 (GE Healthcare) size exclusion chromatography column to obtain homogeneous recombinant protein for crystallization trials. The protein was concentrated to 5 mg ml 21 using Amicon ultra filter concentrators (Millipore, UK) with a 10 000 daltons molecular weight cut-off membrane. The concentrated protein was frozen in liquid nitrogen and stored at 2808C until further use. Full-length folliculin was cloned into pOPINS (a generous gift from Dr Roger Dodd) and the recombinant protein was expressed as a SUMO fusion protein in BL21 (DE3) Star (Invitrogen, UK) E. coli competent cells. The recombinant protein was initially purified over a 6 ml Ni-IMAC. The resulting elution fractions were treated with 5 mM MgCl 2 þ 5 mM ATP, and the target protein was further purified using a Resource Q (GE Healthcare) ion exchange column. Proteolytic cleavage of the recombinant protein was performed using SUMO protease. The protein was then passed again through the Ni-IMAC, to remove the uncleaved protein and the fusion tag. The protein was concentrated to 60 mM using Amicon ultra filter concentrators (Millipore, UK) with 30 000 daltons molecular weight cut-off membrane. The concentrated protein was snap frozen in liquid nitrogen and stored at 2808C until further use.

Mutagenesis
Initial crystallization attempts with folliculin-CT wild type (corresponding to the region 341-579 aa) resulted in no significant crystals. Crystallization of the folliculin-CT required mutation of three cysteine residues, Cys 454, Cys 503 and Cys 506, to alanines. These cysteine residues could have been forming covalent intermolecular disulphide-mediated cross-links that were causing folliculin-CT to form multimers thereby inhibiting crystallization. Furthermore, the terminal 13 amino acids ( predicted to be disordered) were removed by introducing a stop codon after the residue 566 to prevent the protein from degradation. Mutagenesis of the cysteine residues to alanines in folliculin-CT was carried out using the Quick Change mutagenesis kit (Stratagene), according to the manufacturer's protocol. For mutagenesis of folliculin-CT, the PCR mixture contained 1 ml of template DNA (5 ng and 25 ng), 1 ml of forward primer (10 mM), 1 ml of reverse primer (10 mM), 1 ml of dNTPs (100 mM), 1 ml of Pfu turbo polymerase (2 U), 5 ml of 10Â reaction buffer and 40 ml of Milli Q water.

Crystallization
Initially, the crystallization trials were set up with 5 mg ml 21 folliculin-CT using 96 well crystallization plates (Griener, UK) and JCSG Crystal Screen (Molecular Dimensions, UK) and incubated at 128C. Initial crystals appeared overnight in condition H9, which corresponds to 0.2 M LiSO 4 , 0.1 M Bis-Tris pH 5.5 and 25 per cent PEG 3350 (henceforth referred to as the 'mother liquor'). The crystals were reproduced by optimizing the mother liquor using the vapour diffusion method in 24-well Linbro plates. Crystals were harvested in cryo-protectant solution containing 30 per cent polypropylene glycol (PPG; Sigma, UK) along with the original mother liquor using crystallization loops (Hampton Research Inc, USA), and stored in liquid nitrogen.

Data collection, phasing and structure refinement
Initial structure of folliculin-CT was determined by collecting MAD from selenomethionine crystals at Diamond Light source beam line I02, exploiting the anomalous signal of the incorporated selenium. Three data sets corresponding to the peak, inflection and remote energies for selenium atoms were acquired on a single folliculin-CT crystal. The data for selenium peak were collected at 12 657.42 eV (l ¼ 0.9796 Å ), the inflection data were collected at 12 656.42 eV (l ¼ 0.9797 Å ) and finally the remote data were collected at 12700.42 eV (l ¼ 0.9763 Å ). The diffraction data were processed in IMOSFLM (S1) and merged and scaled in SCALA (S2) of CCP4i (S3). The positions of the selenium atoms in the asymmetric unit were determined using AUTOSOL wizard of PHENIX (S4) software package. Fourteen selenium atom sites (seven from each molecule in the asymmetric unit) were identified and the resulting figure of merit after density modification was 0.64. The solvent-modified map calculated by AUTOSOL was interpreted by AUTOBUILD wizard, which produced an almost complete model of the structure. Refinement of the structure was carried out in REFMAC5 (S5), together with manual protein structure rebuilding in COOT (S6 , five residues in allowed regions (1.32%) and no residues in disallowed regions. All structure figures were generated using PyMOL (S10).

Guanine nucleotide exchange assay
Nucleotide loading was carried out as follows: 10 mg GSTtagged Rab was incubated in 50 mM HEPES -NaOH pH 6.8, 0.1 mg ml 21 BSA, 125 mM EDTA, 10 mM Mg-GDP and 5 mCi [ 3 H]-GDP (10 mCi ml 21 ; 5000 Ci mmol 21 ) in a total volume of 200 ml for 15 min at 308C. For standard GDPreleasing GEF assays, 100 ml of the loading reaction was mixed with 10 ml 10 mM Mg-GTP, 10-100 nM GEF protein to be tested or a buffer control, and adjusted to 120 ml final volume with assay buffer. The GEF reaction occurred for 20 min at 308C. The reaction was split into two tubes, then incubated with 500 ml ice-cold assay buffer containing 1 mM MgCl 2 and 20 ml packed glutathione -sepharose for 60 min at 48C. After washing three times with 500 ml ice-cold assay buffer the sepharose was transferred to a vial containing 4 ml scintillation fluid and counted. The amount of nucleotide exchange was calculated in pmoles of GDP released.

Acknowledgements
The authors thank Dr Julia Forman for carrying out initial bioinformatic analyses. We are grateful to Dr Mark Dodding, King's College London for discussion of WD motifs. We thank Dr Len Packman and Mr Michael Weldon in the PNAC facility at the Department of Biochemistry, University of Cambridge for help with mass spectrometry analysis and protein N-terminal sequencing of folliculin samples, respectively. The crystallographic experiments were performed in rsob.royalsocietypublishing.org Open Biol 2: 120071 the X-ray crystallographic facility at the Department of Biochemistry, University of Cambridge. We are grateful to the facility manager, Dr Dimitri Chirgadze, for his assistance in using these facilities. We thank Mr John Lester for help with DNA sequencing of folliculin clones.