Germline mutations of the folliculin gene are normally responsible for Birt–Hogg–Dubé (BHD) syndrome. The 3D structure of the C-terminal domain of folliculin (FLCN), folliculin-CT, has been previously determined (Nookala et al., 2012). FLCN is a tumor suppressor and a guanine nucleotide exchange factor (GEF) for Rab35. GEF activity of FLCN towards its GTPase might be essential for cellular processes. Most of the reported FLCN mutations lead to the BHD phenotype (Lim et al., 2010) and to loss of GEF activity which triggers carcinogenesis (Nookala et al., 2012). A new study by Verma et al. (2016) examines the effect of FLCN mutations on the protein conformation and in loss of function. Authors performed molecular dynamics (MD) simulation on mutated protein variants to predict the protein conformation which is associated with BHD phenotype.
Three folliculin-CT pathogenic mutations – Ser386Asp, Leu418Trp, and His429Pro – were selected for the study. Root mean square deviation (RMSD) trajectory of wild FLCN and mutants was calculated to assess the conformational stability of the proteins during the simulations. RMSD profile was found to be always very low for all the variants of FLCN, indicating the stability of the system, however all the mutant FLCN forms showed a higher RMSD profile compared to the wild form. In addition, all the mutants showed a significantly lower total energy profile as compared to the wild form indicating that mutant forms are more stable than the wild form. When analysing proteins, the radius of gyration (Rg) indicates the level of compaction in the protein structure. All FLCN mutant forms showed decrease in the Rg indicating their folding during MD simulation. All three mutations lead to a more compact structure of folliculin with Ser386Asp and Leu418Trp being the more compact. Hydrogen bonds were similar in all studied forms of FLCN. The root mean square fluctuation (RMSF) profile was recorded and revealed that dDENN domain residues in all mutant forms studied had much higher fluctuation than the wild FLCN with the Leu418Trp mutant showing the highest fluctuation in dDENN domain. These results suggest that mutations induce conformational changes in dDENN domain of FLCN. Comparison of the initial modelled structure with the average structure of all FLCN forms revealed that the wild form was very similar to the initial structure, while all mutant forms showed remarkable differences in structure. A recent study has demonstrated that folliculin interacting proteins 1 and 2 (FNIP1 and FNIP2) double-knockout mice developed kidney cancer analogous to the heterozygous FLCN-knockout mouse model. These findings support the idea that interaction with FNIPs is essential for the tumor-suppressive function of FLCN, and kidney tumorigenesis in BHD may be triggered by loss of interactions between FLCN and the FNIPs (Hasumi et al., 2015). The present study suggests that mutations in FLCN bring significant conformational changes, especially in the d-DENN region, which may be the reason for loss of FLCN GEF activity or interaction with Rab35 and partner proteins FNIP1/2. The MD simulation trajectory was inspected with principal components analysis to better understand the conformational changes of the all FLCN forms. Correlated motion plots were used to show how atoms move relative to each other. Covariance analyses revealed that most of the dominant motions were in d-DENN part of the FLCN protein. The wild FLCN showed anti-correlated motions in the DENN domain, while all the mutant forms showed both correlated and anti-correlated motions. These motions are responsible for the structural changes in FLCN mutant forms showed by the RMSF profile.
In summary, this study predicts that the FLCN-CT mutations, Ser386Asp, Leu418Trp, and His429Pro, result in significant change in the protein structure, especially in d-DENN domain. This structural variation is likely to occur due to mutation that might lead to less affinity for Rab35 and for the binding proteins. The FLCN–Rab35 complex structure is crucial to completely understand the whole interaction scenario and mechanism of FLCN GEF activity. The molecular insight into the FLCN structural conformations in the presence and absence of mutations provides valuable information about the interactions essential for normal functioning of cellular process and the molecular basis of BHD syndrome.
- Verma S, Tyagi C, Goyal S, Pandey B, Jamal S, Singh A, & Grover A (2016). Mutations induce conformational changes in folliculin C-terminal domain: possible cause of loss of guanine exchange factor activity and Birt-Hogg-Dubé syndrome. Journal of biomolecular structure & dynamics, 1-6 PMID: 27484154