Expression And Purification Of RF6 From Rice And Structural Study Of RF6 Interactor OsHXK6

Cytoplasmic male sterility（CMS）is a widespread natural phenomenon in plants.The anthers of CMS plants develop abnormally and fail to produce functional male gametes,while the female organ is normal and can be fertilized to produce seeds.CMS fertility can be restored by nuclear genes namely restorer of fertility（Rf）.Honglian-type CMS（HL-CMS）belongs to the gametophytic type,the fertility of which can be restored by two dominant Rf genes,Rf5 and Rf6.Both of them belong to the PPR gene family.Rf6 encodes a P-class PPR protein with 894 amino acids and is consisted of twenty PPR motifs,in which the PPR3,PPR4 and PPR5 motifs are duplicated in the same place as a unit.However,this duplication is not happened in rf6 encoded by its allele in CMS materials.Previous studies demonstrated that the duplication of that three PPR motifs is essential to the fertility restoration of HL-CMS.Thus,it is speculated that RF6 and rf6are greatly different in their structures.At the same time,it was reported that rice hexokinase 6（OsHXK6）is a member of the fertility restoration complex of HL-CMS,and it interacts with RF6 both in vivo and in vitro.In addition,hexokinases have determined to be multifunction proteins.They are the only proteins that can phosphorylate glucose in plants,and have an impact on the whole life cycle of plants from seed germination to senescence.This paper attempts to provide the molecular basis for the mechanism research on fertility restoration of HL-CMS,and the catalytic mechanism of hexokinases by structural studies.The main results are as fowllowing:1.All the 33 cysteine residues in RF6 were mutated to serine by one-step PCR technology,and then the mutated sequence was modified in eight different ways on their N-terminal and/or C-terminal to facilitate its expression and purification.Our results showed that the two-terminal sequence of RF6 has a great influence on its expression and purification.The expression and purification of some modified RF6were significantly improved,especially the second modification,which was named as RF6-2.Then RF6-2 was highly expressed both in Escherichia coli and eukaryotic cell SF9,and finally we obtained protein GST-RF6-2 with high purity.Crystals of RF6-2have been screened with the purified GST-RF6-2.Meanwhile,the purified protein was applied to the functional study of the fertility restoration mechanism of HL-CMS.The method we have explored to modify and purify RF6 can provide new ideas for expressing and purifing other important PPR proteins.2.OsHXK6（27-506）was successfully expressed in Escherichia coli,and a large scale of OsHXK6 with high purity and uniform conformation was obtained.After crystal screening and optimization,we obtained high quality crystals.But we only collected data with resolution below 3.5?after X-ray diffraction.Then we designed an OsHXK6truncation from residue 35 to 506 according to its predicted secondary structure,which suggests a random coil at the N-ternimal after its transmembrane sequence,and we finally obtained the diffraction data with resolution below 3.0?.These results indicate that the sequence at both ternimals of the protein have a great influence on its crystal growth and quality.At the same time,the OsHXK6 truncation was co-crystallized with its substrate:glucose and ATP.Finally,we resolved four structures of OsHXK6truncation with different substrates,OsHXK6-apo,OsHXK6-Glc,OsHXK6-ADP-PO₄-Mg²⁺and OsHXK6-Glc-ADP-PO₄-Mg²⁺.3.Among these four structures,the OsHXK6-apo is in a“open”state conformation,while the other three are in a“closed”state conformation,and exhibit the almost same structures.The structure of OsHXK6-Glc reveals a classical“induced-fit”mechanism.The structure of OsHXK6-ADP-PO₄-Mg²⁺complex is a novel intermediate state during the glucose phosphorylation reaction catalyzed by hexokinase which we trapped for the first time.It demonstrates for the first time that the conformation of hexokinase with ADP-PO₄-Mg²⁺binding is in a"closed"state,indicating that the binding of ADP-PO₄could induce a conformational change of hexokinase.Structural superposition of OsHXK6-ADP-PO₄-Mg²⁺and OsHXK6-Glc reveals a similar mechanism of ADP-PO₄and Glc in the conformational change of hexokinase.4.Two of our structures in ADP-PO₄ binding form provide the binding mode of ATP to plant hexokinase for the first time.The adenine ring of ADP forms hydrogen bond with Ser369;the ribose forms hydrogen bonds with Thr261 and Leu452;theα-phosphoryl group forms hydrogen bonds with Asn114 and Gly451;theβ-phosphoryl group forms hydrogen bonds with Gly112,Thr113,and Thr261;and theγ-phosphorylate forms hydrogen bonds with Asp109,Ser185,Asp449,Asp486,and Ser488.Site-directed mutagenesis and enzyme activity assay in vitro demonstrate that Gly112,Thr261,Gly262,and Gly450 are essential for the binding of OsHXK6 with ADP-PO₄.5.The sequence alignment of hexokinases in rice,maize and Arabidopsis reveales that the binding sites of catalytic hexokinases to substrates are highly conserved.However,some of these binding sites were mutated in the eight naturally non-catalytic hexokinases,particularly at the Thr261 residue site.Six of the eight non-catalytic hexokinases are mutated at this site,suggesting that Thr261 is important for maintaining the binding of ATP to catalytic hexokinase.We hypothesize that mutation in these substrate binding sites is one of the causes of losing their catalytic ability.Structural comparison of OsHXK6-Glc-ADP-PO₄-Mg²⁺and hexokinase-ADP/AMP-PNP binding complexes in human and bacterial reveals a high conservation on the substrates binding in bacterial,human and plants.