New Insights into Active Site Conformation Dynamics of E. coli PNP Revealed by Combined H/D Exchange Approach and Molecular Dynamics Simulations.
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 Pi is bound loosely, too.
Division of Physical Chemistry, Ruđer Bošković Institute, Zagreb, Croatia. kazazic@irb.hr.
Saša
http://orcid.org/0000-0002-6185-2229
S
Kazazić
Division of Physical Chemistry, Faculty of Science at University of Zagreb, Zagreb, Croatia. bbertosa@chem.pmf.hr.
Branimir
B
Bertoša
Division of Physical Chemistry, Ruđer Bošković Institute, Zagreb, Croatia.
Marija
M
Luić
Division of Physical Chemistry, Ruđer Bošković Institute, Zagreb, Croatia.
Goran
G
Mikleušević
Institute of Biochemistry and Biophysics Department, Polish Academy of Science, Warsaw, Poland.
Krzysztof
K
Tarnowski
Institute of Biochemistry and Biophysics Department, Polish Academy of Science, Warsaw, Poland.
Michal
M
Dadlez
Division of Biophysics, Institute of Experimental Physics, University of Warsaw, Warsaw, Poland.
Marta
M
Narczyk
Division of Biophysics, Institute of Experimental Physics, University of Warsaw, Warsaw, Poland.
Agnieszka
A
Bzowska
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D041961:Deuterium Exchange Measurement / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology*; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D010710:Phosphates; D011683:Purine-Nucleoside Phosphorylase
Allostery; Negative cooperativity; Phosphate binding site; Purine metabolism; Purine nucleoside phosphorylase