An NMR conformational study of the complexes of 13C/2H double-labelled 2′-deoxynucleosides and deoxycytidine kinase (dCK)

Abstract

The structures of the bound ¹³C/²H double-labelled [(2′R/S,5′R/S)-²H₂-1′,2′,3′,4′,5′-¹³C₅]-2′-deoxyadenosine (dAdo) and the corresponding 2′-deoxycytidine (dCyd) moieties in the complexes with human recombinant deoxycytidine kinase (dCK) have been characterized for the first time by solution NMR spectroscopy. Transferred dipole–dipole (DD) cross-correlated relaxation (CCR) and transferred NOE (TRNOE) experiments have been employed to show that the ligands (i.e. dCyd and dAdo) adopt a South-type sugar conformation when bound to dCK. The bound South-type 2′-deoxynucleosides are likely to be in a “near transition state”, such that they can be transformed into their 5′-monophosphates by transesterification from the phosphate-donor, ATP. The chemical integrity of the product, 2′-deoxynucleoside 5′-monophosphate, has been unequivocally proven by ³¹P–¹H correlation spectroscopy. It has also been observed that the aromatic–H1′ NOE crosspeaks are the same sign for the bound acceptor and products, the 5′-monophosphate of dAdo or dCyd and ADP, thereby showing that the acceptor and the products have comparable correlation times, and suggesting that they are all bound to the dCK in a ternary complex. Evidence is presented suggesting two binding sites for dAdo compared to dCyd, which has only one binding site on dCK. Our present understanding of the “near transition state” structure of the ligands in the dCK reaction complex may help the design of new nucleoside transition state therapeutics.