Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent

Abstract

E. coli purine nucleoside phosphorylase is a homohexamer, which structure, in the apo form, can be described as a trimer of dimers. Earlier studies suggested that ligand binding and kinetic properties are well described by two binding constants and two sets of kinetic constants. However, most of the crystal structures of this enzyme complexes with ligands do not hold the three-fold symmetry, but only two-fold symmetry, as one of the three dimers is different (both active sites in the open conformation) from the other two (one active site in the open and one in the closed conformation). Our recent detailed studies conducted over broad ligand concentration range suggest that protein–ligand complex formation in solution actually deviates from the two-binding-site model. To reveal the details of interactions present in the hexameric molecule we have engineered a single tryptophan Y160W mutant, responding with substantial intrinsic fluorescence change upon ligand binding. By observing various physical properties of the protein and its various complexes with substrate and substrate analogues we have shown that indeed three-binding-site model is necessary to properly describe binding of ligands by both the wild type enzyme and the Y160W mutant. Thus we have pointed out that a symmetrical dimer with both active sites in the open conformation is not forced to adopt this conformation by interactions in the crystal, but most probably the dimers forming the hexamer in solution are not equivalent as well. This, in turn, implies that an allosteric cooperation occurs not only within a dimer, but also among all three dimers forming a hexameric molecule.

The structure of His-tagged Geobacillus stearothermophilus purine nucleoside phosphorylase reveals a `spanner in the works'

“…2a). The hexameric structure shows threefold symmetry through the ring-shaped hexamer and His-GsePNPase can be viewed as being a trimer of dimers (a/b, a 0 /b 0 and c/c 0 ), like other hexameric PNPases (Narczyk et al, 2021). The observed electron density for each chain is continuous, apart from residues 209-210 in chain c and portions of the N-terminal tag.…”

“…of 0-2.5 A ˚) in a PDBeFold search (Krissinel & Henrick, 2004a,b, 2005Krissinel et al, 2004). Its structure is most like those of Helicobacter pylori PNPase (Narczyk et al, 2018) and the well studied EcPNPase (Narczyk et al, 2021), with r.m.s.d.s of 0.42-0.51 A ˚. The subunits of His-GsePNPase are similar to each other, with r.m.s.d.s of 0.136-0.160 A ˚on pairwise comparison.…”

Chemo-Enzymatic Generation of Highly Fluorescent Nucleoside Analogs Using Purine-Nucleoside Phosphorylase

“…This variability was not observed for canonical purines, with the only exception of the recently reported synthesis of xanthine-N7-β-D-riboside [ 97 ]. The recent paper of Narczyk et al [ 98 ] suggesting the functional non-equivalence of PNP subunits for the E. coli PNP may provide a partial explanation of this phenomenon.…”

The Interaction of Heptakis (2,6-di-O-Methyl)-β-cyclodextrin with Mianserin Hydrochloride and Its Influence on the Drug Toxicity

“…A mathematical model called "One-sets of sites" [17,18] was used, selected from the options implemented in the ORIGIN 7 program. This model has been chosen because of the smallest errors of the fitted parameters [19] in comparison to the "Two-sets of sites model" [19,20] or the "Sequential model" [20,21]. The association constant value for MIA and DM-β-CD molecules represents a moderate strength of interaction, since it is greater than 10 M −1 and less than 10 7 M −1 [17].…”