Preparation,Crystal Growth And Structure Analysis Of Arabidopsis Thaliana VSP1 Seleniumprotein Derivatives

Arabidopsi thaliana vegetative storage proteins are mainly accumulated in flowers and buds.VSPs transcripts can be induced by mechanical wound,insect herbivory and jasmonic acid(JA),etc.Arabidopsis thaliana vsps(VSP1andVSP2)are acid phosphatases belonging to the haloacid dehalogenase(HAD)superfamily.The acid phosphatase activities of these two enzymes require divalent metal ions and were enhanced mostly by copper ion.Arabidopsi thaliana VSPs are likely to participate in plant development control and defence.Recombinant Arabidopsis VSP2 is an anti-insect protein,correlated with its acid phosphatase acitivity.Anomalous X-ray scattering of selenium is used to provide phase information.VSP1 contains no methionine in the middle of its primary sequence.VSP1-Met-mutant was constructed with three rounds of site-directed mutagenesis reactions were mutated to methionines.The mutated expression plasmid was introduced into E.coli strain B834.The host cells were grown in M9 minimal medium supplemented with selenomethionine.The Se-Met-labeled VSP1 crystals was found in solution containing 0.1 M sodium citrate tribasic dihydrate(pH 5.6),5%(v/v)2-propanol and 18%(w/v)polyethylene glycol 4,000.After optimization,crystals for data collection were obtained.A SAD data set at 0.9789 A with a selenomethionyl VSP1 crystal was collected and was processed to 2.5A.Combined with a native data,the crystal structure of VSP1 was obtained.The crystal structure of VSP1 is belonging to the C2 space group,exsiting with two monomers per asymmetric unit.Each monomer has approximate dimensions of 54 X 31 X 30 A3.Similar to most HAD superfamily members,VSP1 is a two-domain architecture,one consisting a core of a central five-stranded parallel ?-sheets flanked by three a-helices on one side and two a-helices on the other side,the other one is an?-helical domain containing four helices that is in a cap-like shape on the top of the?/? core domain.The size and location of the cap domain suggests that VSP1 belongs to the classical C1 cap type of the HAD superfamily.The core domain of VSP1 contains four catalytic loops,possessing the residues,binding to metal ion in the active site,which are responsible for catalysis.A magnesium ion is located at the catalytic center,coordinating Asp 124,Asp 126,Asp245,Tyr266 and three water molecules.In details,the carbonyl oxygen of Asp 126,the carboxyl oxygens of Asp 124 and Asp 245,and the hydroxyl oxygen of Tyr266 participate in the coordination.The studies on the substrate specificity and inhibitor effects of the acid phosphatase activity of Arabidopsis VSP1 and VSP2 found that:pNPP and MUP were the most readiy substrates of VSPs;EDTA(mainly chelating divalent metal ions),NAD and NADP(competitive inhibitors)had significant inhibitory effect on VSP1.