J Mol Model (2014) 20:2400 DOI 10.1007/s00894-014-2400-8
ORIGINAL PAPER
Molecular dynamics comparison of E. coli WrbA apoprotein and holoprotein David Reha & Balasubramanian Harish & Dhiraj Sinha & Zdenek Kukacka & James McSally & Olga Ettrichova & Petr Novak & Jannette Carey & Rüdiger Ettrich
Received: 29 December 2013 / Accepted: 23 July 2014 / Published online: 26 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract 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.
This paper belongs to Topical Collection MIB 2013 (Modeling Interactions in Biomolecules VI)
Keywords Global motions . Force field parametrization . Binding site volume . Electrostatic potential surface . NAD(P)H:quinone oxidoreductase
Electronic supplementary material The online version of this article (doi:10.1007/s00894-014-2400-8) contains supplementary material, which is available to authorized users.
Introduction
D. Reha (*) : D. Sinha : O. Ettrichova : R. Ettrich Institute of Nanobiology and Structural Biology, Global Change Research Center, Academy of Sciences of the Czech Republic, Zamek 136, 373 33 Nove Hrady, Czech Republic e-mail: [email protected] D. Reha : D. Sinha : R. Ettrich Faculty of Sciences, University of South Bohemia in Ceske Budejovice, Zamek 136, 373 33 Nove Hrady, Czech Republic B. Harish : J. McSally : J. Carey Chemistry Department, Princeton University, Princeton, NJ 08544-1009, USA Z. Kukacka : P. Novak Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Praha 4, Czech Republic Z. Kukacka : P. Novak Faculty of Science, Charles University in Prague, Albertov 6, 128 43 Praha 2, Czech Republic
E. coli protein WrbA ([1]; Fig. 1) is the founding member of a class of novel flavoproteins conserved from bacteria to higher plants [2] whose exact functional role is still unknown [3]. Like the flavodoxins to which it is distantly related, WrbA can use its physiological cofactor FMN [4] to transfer electrons [5], but unlike the flavodoxins WrbA transfers two electrons at a time [6] from NADH to quinones. This function is similar to that of the mammalian FAD-dependent NAD (P) H:quinone oxidoreductases [7, 8] to which WrbA is structurally related, suggesting a role in quinone detoxification and oxidativestress defense. Unlike both these related proteins, WrbA in solution participates in a dimer-tetramer equilibrium [4]. It has thus been suggested [9, 10] that WrbA represents a structural and functional bridge between the monomeric FMNdependent bacterial flavodoxins and the dimeric FADdependent mammalian NAD (P) H-quinone oxidoreductases.
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binding site, a result that was confirmed by mass spectrometry. This finding raised the possibility that Met10 could act as an electron sink, with either a specific or nonspecific role in WrbA function. In an effort to evaluate whether this oxidation may have a role in WrbA physiology, the present analyses were undertaken using molecular dynamics simulations in combination with further mass spectrometry of the protein during its purification from bacterial cells. The iterative application of MD simulations and wet experiments is part of a research approach that has been usefully applied to several other systems under study in ongoing collaborative work [12–15]. The results reported here indicate that Met10 is rapidly oxidized to the sulfoxide in an FMN-dependent reaction; heterogeneous secondary oxidations are also detected that cannot be assigned to unique residues. Despite this fast, sitespecific reaction when apoWrbA is reconstituted with FMN, Met10 is found in the non-oxidized state during purification of the holoprotein. Together these findings rule out many artifactual origins for Met10 sulfoxide (Met10SO) in both the mass spectrometric and crystallographic results, and they suggest that methionine sulfoxide is unlikely to play a role in WrbA function in vivo. The simulations reported here appear to be the first on the WrbA system. They capture a change in quaternary structure within the tetrameric assembly that is the major difference between apo- and holoWrbA crystal structures, allowing its origins to be examined.
Methods Fig. 1 WrbA. a. Tetrahedral arrangement of subunits in the holoprotein tetramer. The polypeptide chains are shown as tubes, and FMN as skeletal models. Throughout this work the analysis uses the color code for subunits shown here: subunit A, gray (or black in some figures); B, red; C, green; D, blue. FMN in each binding site is colored to match its subunit. The large interfaces between subunits referred to in the text are the gray-green and red-blue interfaces; the small interfaces are gray-red and blue-green. Each FMN binding site comprises residues from three subunits; e.g., the gray FMN contacts residues from the gray, red and blue subunits. Arrows numbered 1 to 4 and orange coloring on the red subunit correspond to the flexible regions marked on Fig. 1b. b. Detail of one subunit (red in panel a), showing an overlay of the holoWrbA crystal structure (gray) and a snapshot (cyan) from the apoWrbA simulation after formation of the salt bridge discussed in the text between active-site residues Arg78 and Asp168. The protein backbone is shown as ribbons in helical segments, arrows in strand segments, and tubes in irregular segments, with FMN shown as a skeletal model. Arg78 and Asp168 are labeled and shown as ball-and-stick models, and the position of Met10 is indicated by yellow on the gray backbone. The viewpoint is one commonly used for the related flavodoxins, with the FMN-binding site at the top, resulting from rotation of the red monomer of panel a by∼-90 deg about the X axis and by∼90 deg about the Y axis; numbered arrows 1 to 4 point to the same flexible regions as in panel a, and can be used for orientation
A recent near-atomic resolution (1.2 Å) X-ray crystal structure of reconstituted holoWrbA [11] resolved methionine sulfoxide with full occupancy at residue Met10 in the FMN-
Protein expression, purification, and holoprotein reconstitution WrbA was expressed in E. coli BL21 (λDE3) cells and purified as previously described [4] using DEAE Sepharose (Highprep, Sigma) followed by Affi-Blue Gel (BioRad) column chromatography. Initiator methionine is cleaved from mature E. coli WrbA [4], and residue numbering here starts with Ala1. The concentration of purified apoprotein was determined by UV absorption spectroscopy using the monomer extinction coefficient ε280 =22,831 M−1 cm−1 [4]. Mass spectrometry FMN (700 μM final concentration) was added to apoWrbA (200 μM monomer) in 20 mM HEPES buffer pH 7.2 and vortexed for 15 s. Aliquots corresponding to 10 μg WrbA were collected at the indicated times and WrbA protein was isolated using Microtrap columns (Michrom Bioresources, CA). Samples were loaded on the columns in 1 % acetic acid, washed with 1 % acetic acid/10 % acetonitrile, eluted with 1 % acetic acid/80 % acetonitrile, and diluted 20-fold in 0.1 % formic acid/50 % methanol. Mass spectra were acquired in
J Mol Model (2014) 20:2400
positive-ion mode on an APEX-Ultra FTMS instrument equipped with a 9.4 T superconducting magnet and a Dual II electrospray ionization ion source (Bruker Daltonics, Billerica, MA). The cell was opened for 1.6 msec, and accumulation time was set at 0.4 s for MS experiments and at 3.0 s for MS/ MS experiments, where one experiment consists of the average of eight spectra for MS and 64 spectra for MS/MS. MS/ MS experiments fragmented the most intense charge states of the parent ions. The isolation window was set at three atomic mass units (amu) and the collision energy at 7 V. The acquisition data set size was 512 k points with the mass range starting at m/z 300 amu, resulting in a resolution of 200,000 m/z (full width at half-height) at m/z 400. The instrument was externally calibrated using clusters of arginine resulting in mass accuracy
ORIGINAL PAPER
Molecular dynamics comparison of E. coli WrbA apoprotein and holoprotein David Reha & Balasubramanian Harish & Dhiraj Sinha & Zdenek Kukacka & James McSally & Olga Ettrichova & Petr Novak & Jannette Carey & Rüdiger Ettrich
Received: 29 December 2013 / Accepted: 23 July 2014 / Published online: 26 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract 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.
This paper belongs to Topical Collection MIB 2013 (Modeling Interactions in Biomolecules VI)
Keywords Global motions . Force field parametrization . Binding site volume . Electrostatic potential surface . NAD(P)H:quinone oxidoreductase
Electronic supplementary material The online version of this article (doi:10.1007/s00894-014-2400-8) contains supplementary material, which is available to authorized users.
Introduction
D. Reha (*) : D. Sinha : O. Ettrichova : R. Ettrich Institute of Nanobiology and Structural Biology, Global Change Research Center, Academy of Sciences of the Czech Republic, Zamek 136, 373 33 Nove Hrady, Czech Republic e-mail: [email protected] D. Reha : D. Sinha : R. Ettrich Faculty of Sciences, University of South Bohemia in Ceske Budejovice, Zamek 136, 373 33 Nove Hrady, Czech Republic B. Harish : J. McSally : J. Carey Chemistry Department, Princeton University, Princeton, NJ 08544-1009, USA Z. Kukacka : P. Novak Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Praha 4, Czech Republic Z. Kukacka : P. Novak Faculty of Science, Charles University in Prague, Albertov 6, 128 43 Praha 2, Czech Republic
E. coli protein WrbA ([1]; Fig. 1) is the founding member of a class of novel flavoproteins conserved from bacteria to higher plants [2] whose exact functional role is still unknown [3]. Like the flavodoxins to which it is distantly related, WrbA can use its physiological cofactor FMN [4] to transfer electrons [5], but unlike the flavodoxins WrbA transfers two electrons at a time [6] from NADH to quinones. This function is similar to that of the mammalian FAD-dependent NAD (P) H:quinone oxidoreductases [7, 8] to which WrbA is structurally related, suggesting a role in quinone detoxification and oxidativestress defense. Unlike both these related proteins, WrbA in solution participates in a dimer-tetramer equilibrium [4]. It has thus been suggested [9, 10] that WrbA represents a structural and functional bridge between the monomeric FMNdependent bacterial flavodoxins and the dimeric FADdependent mammalian NAD (P) H-quinone oxidoreductases.
2400, Page 2 of 14
J Mol Model (2014) 20:2400
binding site, a result that was confirmed by mass spectrometry. This finding raised the possibility that Met10 could act as an electron sink, with either a specific or nonspecific role in WrbA function. In an effort to evaluate whether this oxidation may have a role in WrbA physiology, the present analyses were undertaken using molecular dynamics simulations in combination with further mass spectrometry of the protein during its purification from bacterial cells. The iterative application of MD simulations and wet experiments is part of a research approach that has been usefully applied to several other systems under study in ongoing collaborative work [12–15]. The results reported here indicate that Met10 is rapidly oxidized to the sulfoxide in an FMN-dependent reaction; heterogeneous secondary oxidations are also detected that cannot be assigned to unique residues. Despite this fast, sitespecific reaction when apoWrbA is reconstituted with FMN, Met10 is found in the non-oxidized state during purification of the holoprotein. Together these findings rule out many artifactual origins for Met10 sulfoxide (Met10SO) in both the mass spectrometric and crystallographic results, and they suggest that methionine sulfoxide is unlikely to play a role in WrbA function in vivo. The simulations reported here appear to be the first on the WrbA system. They capture a change in quaternary structure within the tetrameric assembly that is the major difference between apo- and holoWrbA crystal structures, allowing its origins to be examined.
Methods Fig. 1 WrbA. a. Tetrahedral arrangement of subunits in the holoprotein tetramer. The polypeptide chains are shown as tubes, and FMN as skeletal models. Throughout this work the analysis uses the color code for subunits shown here: subunit A, gray (or black in some figures); B, red; C, green; D, blue. FMN in each binding site is colored to match its subunit. The large interfaces between subunits referred to in the text are the gray-green and red-blue interfaces; the small interfaces are gray-red and blue-green. Each FMN binding site comprises residues from three subunits; e.g., the gray FMN contacts residues from the gray, red and blue subunits. Arrows numbered 1 to 4 and orange coloring on the red subunit correspond to the flexible regions marked on Fig. 1b. b. Detail of one subunit (red in panel a), showing an overlay of the holoWrbA crystal structure (gray) and a snapshot (cyan) from the apoWrbA simulation after formation of the salt bridge discussed in the text between active-site residues Arg78 and Asp168. The protein backbone is shown as ribbons in helical segments, arrows in strand segments, and tubes in irregular segments, with FMN shown as a skeletal model. Arg78 and Asp168 are labeled and shown as ball-and-stick models, and the position of Met10 is indicated by yellow on the gray backbone. The viewpoint is one commonly used for the related flavodoxins, with the FMN-binding site at the top, resulting from rotation of the red monomer of panel a by∼-90 deg about the X axis and by∼90 deg about the Y axis; numbered arrows 1 to 4 point to the same flexible regions as in panel a, and can be used for orientation
A recent near-atomic resolution (1.2 Å) X-ray crystal structure of reconstituted holoWrbA [11] resolved methionine sulfoxide with full occupancy at residue Met10 in the FMN-
Protein expression, purification, and holoprotein reconstitution WrbA was expressed in E. coli BL21 (λDE3) cells and purified as previously described [4] using DEAE Sepharose (Highprep, Sigma) followed by Affi-Blue Gel (BioRad) column chromatography. Initiator methionine is cleaved from mature E. coli WrbA [4], and residue numbering here starts with Ala1. The concentration of purified apoprotein was determined by UV absorption spectroscopy using the monomer extinction coefficient ε280 =22,831 M−1 cm−1 [4]. Mass spectrometry FMN (700 μM final concentration) was added to apoWrbA (200 μM monomer) in 20 mM HEPES buffer pH 7.2 and vortexed for 15 s. Aliquots corresponding to 10 μg WrbA were collected at the indicated times and WrbA protein was isolated using Microtrap columns (Michrom Bioresources, CA). Samples were loaded on the columns in 1 % acetic acid, washed with 1 % acetic acid/10 % acetonitrile, eluted with 1 % acetic acid/80 % acetonitrile, and diluted 20-fold in 0.1 % formic acid/50 % methanol. Mass spectra were acquired in
J Mol Model (2014) 20:2400
positive-ion mode on an APEX-Ultra FTMS instrument equipped with a 9.4 T superconducting magnet and a Dual II electrospray ionization ion source (Bruker Daltonics, Billerica, MA). The cell was opened for 1.6 msec, and accumulation time was set at 0.4 s for MS experiments and at 3.0 s for MS/ MS experiments, where one experiment consists of the average of eight spectra for MS and 64 spectra for MS/MS. MS/ MS experiments fragmented the most intense charge states of the parent ions. The isolation window was set at three atomic mass units (amu) and the collision energy at 7 V. The acquisition data set size was 512 k points with the mass range starting at m/z 300 amu, resulting in a resolution of 200,000 m/z (full width at half-height) at m/z 400. The instrument was externally calibrated using clusters of arginine resulting in mass accuracy
