핵자기공명법을 이용한 AKmtSM(A11S)의 구조 및 동역학연구

Abstract

The adenylate kinase from mycobacterium tuberculosis (AKmt), a protein of 181 residues plays an essential role for the bacterial survival, by reversible transferring of γ-phosphate from ATP to AMP forming ADP. Crystal and NMR solution structural studies shown that AKmt enzyme have a high flexible site called P-loop (7-13 residues) which is strongly binds with ATP ligand. In p-loop by site directed mutagenesis substituted alanine residue with serine at position 11 (Ala Ser) was made in this study. AKmtA11S was cloned, expressed in Escherichia coli and purified as a 15N labeled protein for NMR studies. Wild and mutant AKmt were studied in free and complex forms with binding ligands (AMP, ATP & Mg+2 ion) in vice versa and with its strong inhibitor (Ap5A) by using 15N HSQC titration and relaxation NMR experiments. AMP bound binary complex of mutant and wild type AKmt shows similar binding affinity. On the other hand the ATP binary complex of mutant enzyme shows 10-fold increase in binding affinity than it wild type. The AMP and ATP bound binary complexes of mutant were titrated against ATP and AMP ligand respectively, the dissociation constant (Kd) results shown that AMP strongly inhibit ATP binding towards the AMPbd in their ternary complex while the ATP ligand enhanced the AMP binding (2-4 fold) towards both binding domains in its ternary complex, compare to the AMP bound binary complexes of wild and mutant AKmt. The binary complex of inhibitor bound mutant enzyme shows that Ap5A bounds to both binding domains undergo conformational change quite different for the ternary complexes (AKmtA11S+AMP+ATP) and (AKmtA11S+ATP+AMP) closed both binding domains. The 15N relaxation data enables us to obtain R1, R2, NOE and global rotational correlation time (τm) for both wild and mutant enzyme in free and complex forms. For the secondary structure study relaxation data were fitted by using modelfree analysis, calculating order parameter (S2) and internal correlation time (te) of amide vectors along the polypeptide sequence. The S2 shows slightly decrease for some of side chain and backbone residues upon ligand binding, suggesting that the immobilization of a residue upon ligand binding resulted in an entropy penalty and the mobilization of other residues contributed to compensate for this penalty. On the basis of previous and our present solution NMR experimental data suggested open/close conformation for binary complex (AMP & ATP) and fully close conformation for ternary complexes like inhibitor binary complex.