Active Site Breathing of Human Alkbh5 Revealed by Solution NMR and Accelerated Molecular Dynamics

AIkB homolog 5 (Alkbh5) is one of nine members of the AIkB family, which are nonheme Fe2+/alpha-ketoglutaratedependent dioxygenases that catalyze the oxidative demethylation of modified nucleotides and amino acids. Alkbh5 is highly selective for the N-6 -methyladenosine modification, an epigenetic mark that has spawned significant biological and pharmacological interest because of its involvement in important physiological processes, such as carcinogenesis and stem cell differentiation. Herein, we investigate the structure and dynamics of human Alkbh5 in solution. By using N-15 and C-13(methyl) relaxation dispersion and N-15-R-1 and R-1 rho, NMR experiments, we show that the active site of apo Alkbh5 experiences conformational dynamics on multiple timescales. Consistent with this observation, backbone amide residual dipolar couplings measured for Alkbh5 in phage pf 1 are inconsistent with the static crystal structure of the enzyme. We developed a simple approach that combines residual dipolar coupling data and accelerated molecular dynamics simulations to calculate a conformational ensemble of AlkbhS that is fully consistent with the experimental NMR data. Our structural model reveals that AlkbhS is more disordered in solution than what is observed in the crystal state and undergoes breathing motions that expand the active site and allow access to alpha-ketoglutarate. Disordered-to-ordered conformational changes induced by sequential substrate/cofactor binding events have been often invoked to interpret biochemical data on the activity and specificity of AIkB proteins. The structural ensemble reported in this work provides the first atomic-resolution model of an AIkB protein in its disordered conformational state to our knowledge.