Mutagenesis, Structure, Stability and in Silico Studies on Fibroblast Growth Factor Homologous Factor 2

Abstract

Fibroblast growth factor homologous factors (FHFs) belong to a subclass of Fibroblast Growth Factor (FGF) family owing to their high sequence and structural similarities with FGFs. However, despite these similarities, there are properties which set them apart from FGFs. FHFs lack the secretion signal sequence unlike other FGF members, except FGF1 and 2. It may be possible that FGF1 partially unfolds and secretes outside the cell, suggesting that lower stability of protein may be trade-off for secretion. Unlike FGFs, FHFs are not able to bind to FGF receptors (FGFRs) and trigger a cascade of cellular events. FHFs have been implicated in development of mammalian nervous system by binding to intracellular domains of voltage-gated sodium channels (VGSCs), neuronal MAP kinase scaffold protein and islet-brain-2 (IB2). The two amino acids Arg52 and Val95 are conserved in all FHFs and mutation of these residues lead to its inability to bind with VGSC/IB2. However, it is not clear whether the loss of binding is due to destabilization of the protein on mutation leading to partial denaturation or due to involvement of Arg52 and Val95 in conferring functionality to FHFs. The aim of the study is to mutate the two conserved residues of FHF2 (Arg52 and Val95) with its corresponding FGF counterpart amino acids and to study the effects of the single and double mutations on the overall structure, stability and functionality of FHF2. The single and double mutations were prepared using site directed mutagenesis approach and were confirmed by DNA sequencing. The wild type and recombinant proteins were expressed in E. coli by inducing with IPTG and were purified to high yield using a novel two step method involving nickel affinity followed by heparin affinity column chromatography. Biophysical characterisation studies of the proteins using methods like fluorescence intrinsic and extrinsic spectroscopy, circular dichroism spectroscopy, FTIR analysis, trypsin digestions studies etc. revealed that while V95N mutation remarkably destabilizes FHF2, R52G mutation exhibits slight stabilising effect on FHF2, whereas, the double mutant exhibited no effect on stability of FHF2. Thus, it can be assumed that the contradictory effects of the two different mutations on the protein stability, nullify each other in the double mutant. In silico studies on the wild type and mutant proteins using docking analysis, site directed mutator (SDM) tool, protein interaction calculator (PIC) and molecular dynamic simulation studies seem to corroborate with the findings of the biophysical studies. Docking studies reveal that R52 is directly involved in stable interactions with VGSC whereas, V95 though not directly involved in interaction with VGSC but plays an important role in imparting stability to the protein and hence its mutation leads to significant destabilization of FHF2. Overall these studies shed light towards the role of specific amino acids in determining protein stability and functionality and will provide the rationale for different function of FHFs despite being highly similar to FGFs in terms of sequence and structure.