Structural Basis For Human Adenylate Kinase 1 Catalyzing ADP Into ATP And AMP

Human adenylate kinase 1(hAK1)plays vital roles in the energetic and metabolic regulation of cell life.The abnormal expression of hAKl is closely related with many diseases.With the presence of Mg iron,hAKl can transfer two molar ADPs into one molar ATP and one molar AMP,and activate the downstream AMP signaling.Therefore,investigation on the structural basis of hAK1 catalyzing ADP into AMP and ATP is significant to understand the pathogenic mechanism of hAK1 and to explore potent drugs.In our previous NMR chemical shift perturbation experiments,we found many residues had chemical shift perturbations with the addition of Mg2+ or ADP.In this work,we constructed hAK1 mutants of some disturbed residues.Through the enzyme activity assays,biolayer interference experiments and molecular docking,we revealed the interaction of hAK1 with Mg2+ and hAK1 with ADP,disclosed the key residues involved in the catalytic function of hAK1.According to the NMR experiments,we selected 6 perturbation sites by Mg2+titration:G22,C25,H36,T39,D93,G94.Perturbation sites by ADP titration were also selected(a total of 26):G16,G18,G20,G22,C25,L37,S38,T39,G40,R44,E62,V67,V72,G94,R132,S136,D140,D141,Q65,D93,E176,T157 and G13,V29,D78,E103.After that,mutants of these residues were constructed and relevant proteins were expressed and purified.Herein,we found that H36,G94,D93 and G22 were the key residues for hAK1 capturing Mg2+,for their point-mutation could decrease hAK1 enzymatic activity more than 50%.These residues located near the hub of phosphoryl transfer.In addition,we also explored the key residues for hAK1 catalyzing ADP.Seven hAK1 variants including G16A,G20A,G40A,R44A,G94A,D140A and D141A drastically lowered enzyme activity and ADP affinity,suggesting these residues were key residues for hAK1 capturing ADP and catalyzing ADP.Besides,BLI experiment results shown,Q65,V72,R132 and E176 residues played important roles in capturing ADP,however,they almost had no effect on the enzymatic activity of hAK1,for they were far away from the catalytic center.They located on the edge of phosphate group transfer center and were responsible for capturing the framework of ADP.Combined with previous results,it is shown that residues G22,G94 and D93 were involved in both ADP and Mg2+ binding.To some extent,Mg2+ assists ADP combination when it binds hAK1.Based on the above experimental results,the complex structure of hAK1 and ADP was constructed by molecular docking.Adenosine of ADP was mainly combined through hydrophobic effects such as pi-alkyl type,and its phosphoric acid group was mainly fixed by hydrogen bonding.Also we visual screened some lead compounds from drug library using MOE software.The interaction of these lead compounds with hAK1 and their effect on hAK1 funciton are still in the way of experimental verification.In conclusion,we disclosed the key residues for hAK1 catalyzing ADP into ATP and AMP,which are not only helpful for us to understand the molecular catalytic mechanism of hAK1,but also shed light on designing and developing inhibitors or agonists for modulating hAK1 function.