TY - JOUR
T1 - Molecular dynamics comparison of E. coli WrbA apoprotein and holoprotein
AU - Reha, David
AU - Harish, Balasubramanian
AU - Sinha, Dhiraj
AU - Kukacka, Zdenek
AU - McSally, James
AU - Ettrichova, Olga
AU - Novak, Petr
AU - Carey, Jannette
AU - Ettrich, Rüdiger
N1 - Funding Information:
We gratefully acknowledge support from the Czech Science Foundation (P207/10/1934) and joint Czech - US National Science Foundation International Research Cooperation (OISE08-53423). Access to the computing and storage facilities owned by parties and projects contributing to the National Grid Infrastructure MetaCentrum, provided under the programme “Projects of Large Infrastructure for Research, Development, and Innovations” (LM2010005), is acknowledged.
Publisher Copyright:
© 2014, Springer-Verlag Berlin Heidelberg.
PY - 2014/9/1
Y1 - 2014/9/1
N2 - WrbA is a novel multimeric flavodoxin-like protein of unknown function. A recent high-resolution X-ray crystal structure of E. coli WrbA holoprotein revealed a methionine sulfoxide residue with full occupancy in the FMN-binding site, a finding that was confirmed by mass spectrometry. In an effort to evaluate whether methionine sulfoxide may have a role in WrbA function, the present analyses were undertaken using molecular dynamics simulations in combination with further mass spectrometry of the protein. Methionine sulfoxide formation upon reconstitution of purified apoWrbA with oxidized FMN is fast as judged by kinetic mass spectrometry, being complete in ∼5 h and resulting in complete conversion at the active-site methionine with minor extents of conversion at heterogeneous second sites. Analysis of methionine oxidation states during purification of holoWrbA from bacterial cells reveals that methionine is not oxidized prior to reconstitution, indicating that methionine sulfoxide is unlikely to be relevant to the function of WrbA in vivo. Although the simulation results, the first reported for WrbA, led to no hypotheses about the role of methionine sulfoxide that could be tested experimentally, they elucidated the origins of the two major differences between apo- and holoWrbA crystal structures, an alteration of inter-subunit distance and a rotational shift within the tetrameric assembly.
AB - WrbA is a novel multimeric flavodoxin-like protein of unknown function. A recent high-resolution X-ray crystal structure of E. coli WrbA holoprotein revealed a methionine sulfoxide residue with full occupancy in the FMN-binding site, a finding that was confirmed by mass spectrometry. In an effort to evaluate whether methionine sulfoxide may have a role in WrbA function, the present analyses were undertaken using molecular dynamics simulations in combination with further mass spectrometry of the protein. Methionine sulfoxide formation upon reconstitution of purified apoWrbA with oxidized FMN is fast as judged by kinetic mass spectrometry, being complete in ∼5 h and resulting in complete conversion at the active-site methionine with minor extents of conversion at heterogeneous second sites. Analysis of methionine oxidation states during purification of holoWrbA from bacterial cells reveals that methionine is not oxidized prior to reconstitution, indicating that methionine sulfoxide is unlikely to be relevant to the function of WrbA in vivo. Although the simulation results, the first reported for WrbA, led to no hypotheses about the role of methionine sulfoxide that could be tested experimentally, they elucidated the origins of the two major differences between apo- and holoWrbA crystal structures, an alteration of inter-subunit distance and a rotational shift within the tetrameric assembly.
KW - Binding site volume
KW - Electrostatic potential surface
KW - Force field parametrization
KW - Global motions
KW - NAD(P)H:quinone oxidoreductase
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U2 - 10.1007/s00894-014-2400-8
DO - 10.1007/s00894-014-2400-8
M3 - Article
C2 - 25152065
AN - SCOPUS:84952330382
SN - 1610-2940
VL - 20
SP - 1
EP - 14
JO - Journal of Molecular Modeling
JF - Journal of Molecular Modeling
IS - 9
M1 - 2400
ER -