The Rational Design Of Iron-sulfur Cluster Binding Site In Magnetoreceptor MagR

Objective: Iron-sulfur proteins play essential roles in a wide variety of cellular processes such as respiration,photosynthesis,nitrogen fixation and electron transfer.In 2015,a highly conserved iron-sulfur protein,Isc A,was identified as a putative animal magnetoreceptor and named MagR.MagR senses external magnetic field by forming a macromolecular rod-like complex with photoreceptor cryptochrome(Cry)and possesses intrinsic magnetic moment.Based on this,a biocompass model of animal magnetoreceptor was proposed.Furthermore,two different types of iron-sulfur clusters,[2Fe-2S] and [3Fe-4S],have been identified in MagR and may serve as a magnetic switch to modulate the magnetic property of MagR.Iron-sulfur proteins are well suited to various practical applications as molecular redox sensors or molecular “wires” for electron transfer.Various technologies have been developed recently using one particular iron-sulfur protein,MagR,as a magnetic tag.The stability of iron-sulfur clusters varies significantly between anaerobic and aerobic conditions due to their intrinsic sensitivity to oxygen.When they are harbored in proteins,the stability is primarily dependent on the microenvironments within a biomolecular structure,such as the oxygen accessibility to the clusters,and coordination bonds of the clusters,et al.The limited protein stability and low magnetic sensitivity of MagR hindered its wide application.Therefore,the iron-sulfur binding site of clMagR was rationally re-designed in this experiment to strengthen or stabilize the iron-sulfur cluster and promote its better function.Methods: To further stabilize the iron-sulfur cluster in clMagR,here in this study,Modeller software was used for homology modeling to analyze the iron-sulfur cluster binding sites of clMagR.We also analyzed the known iron-sulfur cluster binding protein structures from RCSB Protein Data Bank and performed sequence alignment for the CX2 C motif of iron-sulfur binding sites based on known structures(PDB ID: 3ZXS,4Z3 Y,5C4I,4UNF,4S23,1DUR).Finally,the iron-sulfur cluster binding site of clMagR was rationally re-designed by site-directed mutagenesis.The conformation,type of iron-sulfur cluster,chemical reconstitution,total iron content and thermostability of the clMagR mutant proteins were analyzed by using size-exclusion chromatography,ultraviolet-visible(UV-vis)absorption spectrum,circular dichroism(CD)spectrum,electron paramagnetic resonance(EPR)spectroscopy to screen for suitable mutants.Results: The results of size-exclusion chromatography and SDS-PAGE showed that the conformation and molecular weight of all mutants were similar to wild type clMagR.The results of UV-vis spectrum,CD spectrum and EPR spectroscopy showed that all mutants did not affect the type of iron-sulfur cluster clMagR bound.All mutants except clMagRT57 C decreased the iron-sulfur binding efficiency and iron content in clMagR.At room temperature,wild type clMagR protein is losing its iron-sulfur clusters significantly faster than clMagRT57 C.In addition,the analysis of far-UV circular dichroism results showed that the melting point(Tm)of clMagRT57 C was significantly higher than that of wild type clMagR,indicating that clMagRT57 C prolonged the thermostability.Conclusions: The ultimate goal of protein engineering of MagR is to provide a molecular tool for magnetogenetics in the future with improved stability at ambient temperature and hypersensitivity to external magnetic field changes.The binding site of clMagR was rationally redesigned based on the 3D structural model of clMagR and the sequence alignment of some known iron-sulfur proteins.One such mutation,clMagRT57 C,showed improved iron-sulfur binding efficiency and higher iron content,as well as prolonged thermostability.Thus,clMagRT57 C can serve as a prototype for further design of more stable and sensitive magnetic toolbox for magnetogenetics in the future.