New Insights into Active Site Conformation Dynamics of E. coli PNP Revealed by Combined H/D Exchange Approach and Molecular Dynamics Simulations

Abstract

Abstract. The biologically active form of purine nucleoside phosphorylase (PNP) from Escherichia coli (EC 2.4.2.1) is a homohexamer unit, assembled as a trimer of dimers. Upon binding of phosphate, neighboring monomers adopt different active site conformations, described as open and closed. To get insight into the functions of the two distinctive active site conformations, virtually inactive Arg24Ala mutant is complexed with phosphate; all active sites are found to be in the open conformation. To understand how the sites of neighboring monomers communicate with each other, we have combined H/D exchange (H/DX) experiments with molecular dynamics (MD) simulations. Both methods point to the mobility of the enzyme, associated with a few flexible regions situated at the surface and within the dimer interface. Although H/ DX provides an average extent of deuterium uptake for all six hexamer active sites, it was able to indicate the dynamic mechanism of cross-talk between monomers, allostery. Using this technique, it was found that phosphate binding to the wild type (WT) causes arrest of the molecular motion in backbone fragments that are flexible in a ligand-free state. This was not the case for the Arg24Ala mutant. Upon nucleoside substrate/inhibitor binding, some release of the phosphate-induced arrest is observed for the WT, whereas the opposite effects occur for the Arg24Ala mutant. MD simulations confirmed that phosphate is bound tightly in the closed active sites of the WT; conversely, in the open conformation of the active site of the WT phosphate is bound loosely moving towards the exit of the active site. In Arg24Ala mutant binary complex P i is bound loosely, too.

Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features

“…However, during MD simulations, significant movements of P i ion from its initial position towards the exit of the binding site were observed in 14 of 24 active sites considering all systems with P i present inside the binding site of the starting structure for MD simulation. Unlike during the simulations of PNP from E. coli , where P i movement was noticed only for active sites in open conformations , in the case of HpPNP, movement of P i ion was also observed for active sites in closed conformation. However, it was more often observed for active sites in open conformation, and it occurred on a shorter time scale compared to closed active sites.…”

“…For many years we have been studying a homohexameric enzyme purine nucleoside phosphorylase (PNP, purine nucleoside orthophosphate ribosyl transferase, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2.4.2.1.html) and the conformational changes of its six active sites upon ligand binding, with the final goal of understanding the highly complex mechanism of this enzyme action . PNP catalyses the reversible phosphorolytic cleavage of the glycosidic bond of purine ribo‐ and deoxyribonucleosides:…”

Crystallographic snapshots of ligand binding to hexameric purine nucleoside phosphorylase and kinetic studies give insight into the mechanism of catalysis

“…3 ) is found just in two peptide fragments, p22 and p23, both holding a mutation site (Asp204Ala and Arg217Ala) and covering sequence regions of the WT E. coli PNP 202 VSDHIRTHEQTTAAE 216 for p22 and 217 RQTTFNDM 224 for p23. In all other fragments, changes induced by phosphate and FA binding are the same for both enzyme forms, WT 4 and DM. Namely, phosphate binding induces a significant reduction in the conformational dynamics of the binary complex in those structure regions that contain amino acid residues establishing strong H-bonds with P i in the active site.…”

“…After FA binding, the ribose part of the molecule establishes H-bonds with the side chain of Glu181, fixing motion of that part of the protein structure and causing a lower deuterium uptake for peptide 19 in both enzyme forms, WT 4 and DM (not shown).…”

“…In addition to the phosphorolysis being a two-substrate and two-product reversible reaction, with communication between monomers forming a dimer 1 , the mechanism is further complicated by the higher order communication, i.e . an allosteric cross-talk between dimers forming the hexamer 4 , 5 . The way in which this allosteric communication is achieved is still unclear, but the complexity is reflected in an ever increasing number of different arrangements of active site conformations between monomers in the available crystal structures of hexameric PNPs 6 .…”