Structural And Functional Study Of DH And PH1 Domain Of FGD6

FGD6 gene,reported as a disease-causing gene of age-related macular degeneration and autism spectrum disorder,codes a protein which is characterized as guanine nucleotide exchange factor（GEF）of Rho family small GTPase.FGD6protein is comprised of a long N-terminal domain,a Dbl homology（DH）domain,the first pleckstrin homology（PH1）domain,a FYVE domain and the second PH（PH2）domain.FGD6 protein is a member of Dbl family GEFs which contain the typical Rho GEF subdomain structure of a DH domain adjacent to a PH domain.Up to now,the characteristic of guanine nucleotide exchange activity and structure of FGD6protein remain unclear.In this study,the DH and PH1 domain of FGD6 were expressed and purified from Escherichia coli.The guanine nucleotide exchange spectrum of FGD6-DH in vitro was recorded based on mant GDP-bound Rac1 and mant GDP-bound CDC42.Isothermal titration calorimetry was employed to measure thermodynamic parameters such as enthalpy（ΔH）,Gibbs free energy（ΔG）,entropy changes（ΔS）,and binding affinity between FGD6-DH and CDC42.Ultimately,the structure of FGD6-DH was determined by X-ray crystallography to elucidate the characteristic of guanine nucleotide exchange activity of FGD6 protein.The detailed results were summarized as follows:1.The expression and purification of DH domain of human FGD6（FGD6-DH）.The coding sequence of FGD6-DH（875 aa-1059 aa）was cloned to p GEX-6P-1plasmid according to Eco RI and Xho I restriction enzyme sites,resulted a recombinant expression vector named FGD6-DH/p GEX-6P-1.It was transformed to Escherichia coli BL21（DE3）to express FGD6-DH under induction of isopropyl-β-D-thiogalactopyranoside（IPTG）.Approximately 1 mg of FGD6-DH was obtained from1 litre of cell culture after affinity chromatography,ion-exchange column chromatography and gel filtration chromatography.2.Guanine nucleotide exchange activity assay of FGD6-DH.Mant GDP-bound Rac1 and mant GDP-bound CDC42 were utilized to measure guanine nucleotide exchange activity of FGD6-DH.No detectable guanine nucleotide exchange activity of FGD6-DH against Rac1 was observed with the concentration of FGD6-DH from2.5μM to 100μM.FGD6-DH showed weak guanine nucleotide exchange activity against CDC42 with FGD6-DH concentration of 50μM and 100μM,the relative fluorescence intensity of which decreasing to 66%and 60%of initial fluorescence intensity at the time point of 1 hour.The results demonstrated that FGD6-DH recognized CDC42 specifically,however higher concentration of FGD6-DH and more time was required to accomplish the guanine nucleotide exchange of CDC42.3.The interaction of FGD6-DH and CDC42.Isothermal titration calorimetry was used to investigate binding affinity of FGD6-DH and CDC42 and to record thermodynamic parameters such as enthalpy（ΔH）,Gibbs free energy（ΔG）,entropy changes（ΔS）.Moderate binding affinity was observed between FGD6-DH and CDC42 as evidenced by the K_D value of 1.625±0.265μM.With a negativeΔH value,the interaction of FGD6-DH and CDC42 was identified as an enthalpy-driven reaction which could occur spontaneously.4.The structure of FGD6-DH and amino acids critical for guanine nucleotide exchange activity.（1）Rod-like crystals with length of 0.5 to 0.7 centimeter grew in the condition of 0.1 M HEPES p H 7.5,27%w/v PEG3350,0.1 M Mg Cl₂.The crystals,belonging to space group P4₁,were used to determine the structure of FGD6-DH reaching a final resolution of 1.5?after refinement.Similar to typical structures of other DH domains,the FGD6-DH domain displayed an oblong six-helix bundle（α1–α6）.It resembles a chaise longue with the?seat back‘created by the U-shaped loop between the fourthαhelix（α4）and the fifthαhelix（α5）.（2）A kink,formed by Ala1054-Asn1058 of FGD6,was observed in the last helix（α6）and thus occupied space for Asp63-Arg66 of CDC42.This kink impeded the interaction betweenα6 and?switch II‘domain of CDC42 and may weaken the guanine nucleotide exchange activity of FGD6-DH.（3）A superimposition of the FGD6-DH domain with the structure of intersectin bound to its substrate CDC42 revealed that FGD6-Leu1013was responsible for recognizing CDC42 via van der Waal contacts with Phe56 of CDC42（CDC42-Phe56）.Since the aromatic rings of Trp56 in Rac1（Rac1-Trp56）was bigger than that of CDC42-Phe56,the side chain of FGD6-Leu1013 would sterically hinder the contacts of FGD6 to Rac1.（4）A salt bridge between the DH domain and Asp38 of CDC42（CDC42-Asp38）was missing because the corresponding residue of FGD6 was Val889（FGD6-Val889）.The missing salt bridge may further weaken the guanine nucleotide exchange activity of FGD6-DH.FGD2 protein is predicted to have stronger guanine nucleotide exchange activity because there is an arginine at that position,which is able to form a salt bridge CDC42-Asp38.In fact,the prediction was verified by the guanine nucleotide exchange activity assay of FGD2-DH.（5）A highly conserved Glu residue of FGD6（FGD6-Glu878）was able to form several hydrogen-bonds with Tyr32/Thr35/Val36 of CDC42.FGD6-Glu878 played the most important role in the interaction with?switch I‘domain of CDC42.The substitution of Ala for Glu,a missense mutation of FGD1 causing Aarskog-Scott syndrome,indeed weakened the guanine nucleotide exchange activity of FGD6-DH.5.The role of PH1 domain on guanine nucleotide exchange activity of FGD6.The PH domains of some Rho family GEFs have no interaction with the bound small GTPase and DH domain.While some PH domains of Rho family GEFs directly contact the last helix of DH domain and small GTPase by hydrogen bonds to stabilize DH domain and enhance guanine nucleotide exchange activity of GEFs.The guanine nucleotide exchange activity curves of FGD6-DH and FGD6-DH+PH1 showed that the relative fluorescence intensity of solutions with 50μM and 100μM FGD6-DH+PH1 and CDC42 dropped to 41%and 34%of the initial fluorescence intensity respectively,comparing to 66%and 60%of samples with 50μM and 100μM FGD6-DH and CDC42.These results revealed that PH1 domain of FGD6enhanced nucleotide exchange activity of FGD6.This study systematically analyzed the characteristic of nucleotide exchange activity of FGD6 and the mechanism of it based on the structure of FGD6-DH.FGD6-DH showed weak nucleotide exchange activity to CDC42 but no nucleotide exchange activity to Rac1 in vitro.However,nucleotide exchange activity of FGD6could be enhanced by PH1 domain.Thermodynamic parameters of interaction between FGD6-DH and CDC42 were recorded for the first time,demonstrating moderate binding affinity of FGD6-DH to CDC42.It can be seen from the structure of FGD6-DH that a kink inα6 occupies space for Asp63-Arg66 of CDC42,impeding the interaction betweenα6 of FGD6-DH and?switch II‘domain of CDC42.FGD6-Val889is not able to form salt bridge with CDC42-Asp38 thus the missing salt bridge further weaken nucleotide exchange activity of FGD6-DH.At last,FGD6-Leu1013 was responsible for recognizing CDC42 via van der Waal contacts with Phe56 of CDC42（CDC42-Phe56）.But FGD6 was not able to recognize Rac1 because the side chain of FGD6-Leu1013 may sterically hinder the contacts of FGD6 to Trp56 of Rac1.This study provided a comprehensive understanding of the nucleotide exchange activity of FGD6 and shed a light on the disease causing mechanism of FGD family.