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

ABSTRACT: AlkB homolog 5 (Alkbh5) is one of nine members of the AlkB family, which are nonheme Fe2+/?-ketoglutarate-dependent dioxygenases that catalyze the oxidative demethylation of modified nucleotides and amino acids. Alkbh5 is highly selective for the N6-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 15N and 13Cmethyl relaxation dispersion and 15N-R1 and R1? 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 pf1 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 Alkbh5 that is fully consistent with the experimental NMR data. Our structural model reveals that Alkbh5 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 ?-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 AlkB proteins. The structural ensemble reported in this work provides the first atomic-resolution model of an AlkB protein in its disordered conformational state to our knowledge.

SUBMITTER: Purslow JA 

PROVIDER: S-EPMC6303414 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature