Calf spleen purine nucleoside phosphorylase: structure of its ternary complex with an N(7)-­acycloguanosine inhibitor and a phosphate anion

The calf spleen purine nucleoside phosphorylase (PNP) ternary complex with an N(7)-acycloguanosine inhibitor and a phosphate ion has been crystallized in the cubic space group P2₁3, with unit-cell parameter a = 94.11 Å and one monomer per asymmetric unit. X-ray diffraction data were collected using synchrotron radiation (Station X31, EMBL Outstation, DESY, Hamburg). The crystal structure was refined to a resolution of 2.2 Å and R and R_free values of 17.5 and 24.5%, respectively. The acyclonucleoside inhibitor is bound in the active site in an inverted (`upside-down') orientation of the purine base compared with natural substrates. The side chain of Asp243 forms two hydrogen bonds with the base ring: N^δ donates a hydrogen to N(3) and O^δ accepts a hydrogen from the guanine N(2)-amino group. N(1)—H of the base is hydrogen bonded to O^[epsilon] of Glu201, while N(9) accepts a hydrogen bond from Thr242 O^γ. In addition, a water molecule (W417) bridges the N(2)-amino group of the base and O^[epsilon] of Glu201. In the phosphate-binding site, a phosphate ion is bound to Ser33, His64, Arg84, His86, Ala116 and Ser220. The acyclic chain of the N(7)-acycloguanosine inhibitor is in a folded conformation and together with a water molecule (W388) occupies the pentose-binding site, with possible hydrogen bonds to Tyr88 O^η and His257 N^δ1. This new binding mode fully accounts for the previously observed substrate properties of 7-­β-D-ribofuranosides of hypoxanthine and guanine. It also provides a new starting point for the design of inhibitors of PNP for therapeutic and other applications.