New insights into active site conformation dynamics of E. coli PNP revealed by combined H/D exchange approach and molecular dynamics simulation
PBN-AR
Instytucja
Wydział Fizyki (Uniwersytet Warszawski)
Informacje podstawowe
Główny język publikacji
en
Czasopismo
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
ISSN
1044-0305
EISSN
1879-1123
Wydawca
SPRINGER
DOI
URL
Rok publikacji
2016
Numer zeszytu
Strony od-do
73-82
Numer tomu
27
Link do pełnego tekstu
Identyfikator DOI
Liczba arkuszy
Słowa kluczowe
en
Allostery
Negative cooperativity
Phosphate binding site
Purine metabolism
Streszczenia
Język
en
Treść
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. When the virtually inactive Arg24Ala mutant is complexed with phosphate, all active sites are found to be in the open conformation. To understand how the sites in 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 high rigidity of the enzyme, with a few flexible regions situated at the surface and at the dimeric interface. Although H/DX was not able to distinguish single PNP active site conformations, it was able to indicate the dynamic mechanism of cross-talk between monomers. Namely, 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 the unligated state. This was not the case for the Arg24Ala mutant. Upon nucleoside binding, some release of the phosphate-induced arrest is observed for the WT, while the opposite effects occur for the Arg24Ala mutant. MD simulations confirmed that phosphate is bound tightly in the closed active sites of the WT, while it moves towards the exit of the active site when bound to the open conformation sites of the WT and to all Arg24Ala mutant conformations.
Cechy publikacji
discipline:Biofizyka – dziedzina nauk fizycznych
discipline:Biophysics – field of physical sciences
Original article
Original article presents the results of original research or experiment.
Oryginalny artykuł naukowy
Oryginalny artykuł naukowy przedstawia rezultaty oryginalnych badań naukowych lub eksperymentu.
Inne
System-identifier
PBN-R:730013