Integrative Structural Biology Studies Of Toxoplasma Gondii MIC2-M2AP Complex

Toxoplasmosis,caused by Toxoplasma gondii infection,is prone to cause death in severely immunocompromised populations such as patients with malignant tumors.Toxoplasma gondii relies on numerous secreted proteins to achieve rapid invasion of host cells,and microneme protein 2（MIC2）is an important protein secreted by microneme that is related to parasite gliding and invasion of host cells.MIC2 is expressed in all stages of growth and development and usually binds stably to MIC2-associated protein（M2AP）.It is known that the central β domain of M2 AP interacts with the sixth thrombospondin repeat domain（TSR）of MIC2,but the specific binding mode remains elusive.Therefore,we are aimed to explore the binding mode of MIC2 and M2 AP through a series of structural biology methods.In order to obtain the structure of the complex,we used a prokaryotic expression system to obtain samples of the key domains of MIC2 and M2 AP.Although we are not able to crystallize the complex directly by mixing the two proteins with ratio 1:1or designing a linker to connect the two fragments,the crystal structure of the central domain of M2 AP was finally successfully crystallized and solved.Furthermore,the fragment of TSR6 can also be crystallized but the phase was not accessible with neither MR nor experimental phasing,we thus employed the software Alpha Fold2 to predict structure of the sixth TSR domain of MIC2,and then tried docking with the crystallized M2 AP.To verify the accuracy of the docking complexes,we designed 9single point mutations in the MIC2-TSR6 and tested the affinity of the MIC2 mutant proteins with M2 AP.We found that the H620 plays a key role and the H620 A mutant decreases the affinity by ～15000 fold.Also,T636 and F637 of MIC2 interact with L125 and W134 of M2 AP to stabilize the complex.In addition,we performed a small-angle scattering experiment on the MIC2-M2 AP complex to get the envelop of the complex,and used it to search for the most favorable docking model.We ultimately identified a reliable binding model of the MIC2-M2 AP complex using the mutagenesis data and SAXS envelop,which helped us to understand the binding mode of MIC2 and M2 AP at the molecular level.Meanwhile,we hypothesized that an imidazole analog of the H620 side chain may be used to break the binding of MIC2 and M2AP,which may potentially be developed to block toxoplasma gondii infection.