J. Mol. Biol. (2006) 364, 561–566
Dodecin Sequesters FAD in Closed Conformation from the Aqueous Solution Martin Grininger 1 , Florian Seiler 1 , Kornelius Zeth 2 and Dieter Oesterhelt 1 ⁎ 1
Max Planck Institute of Biochemistry, Department of Membrane Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany 2
Max Planck Institute for Developmental Biology, Department of Protein Evolution, Spemannstrasse 35, 72076 Tübingen, Germany
Both extensive theoretical calculations and experimental data obtained during several decades leave little doubt that flavin adenine dinucleotide (FAD) exists in an open as well as in a closed conformation in aqueous solution. However, the knowledge about the intramolecularly stacked complex of FAD is constructed on indirect methods while direct structural evidence is lacking. Recently, dodecin was reported as an unspecific flavin binding protein which exhibits the unique binding mode of incorporating stacked dimers of flavins into a single binding pocket. Here, we show that FAD is not bound in this manner, but in monomers of intramolecularly stacked conformation. As resulting from the dodecin ligand binding characteristic, this FAD stacked conformation suggests to be directly sequestered from the aqueous solution and thus to be the first X-ray structural view on a FAD solution-stacked form. Moreover, in extraordinary FAD binding, dodecin serves as a model for studying bound monomeric (FAD) versus bound dimeric (e.g. riboflavin) flavin properties. © 2006 Elsevier Ltd. All rights reserved.
Keywords: closed FAD; FAD conformation; π-stacking; intramolecular stacking; riboflavin
Introduction FAD is a physiologically relevant cofactor with redox properties that are modulated by the proteins to account for cellular demands (Figure 1). In contrast to the structurally and functionally related flavin mononucleotide (FMN), FAD possesses the unique ability to self-modulate its catalytic properties by varying the mode of intramolecular interactions between the adenine subunit and the functionally active isoalloxazine ring. This characteristic of FAD is enabled by the conformational stabilization attained when the Abbreviations used: RBF, riboflavin; Hrc-FAD, reconstituted FAD/apododecin complex; Hsk-FAD, apododecin crystals soaked with FAD; Hex-FAD, putative FAD/apododecin complex with FAD in extended (open) conformation; Trp36C2, C2-related Trp36; isoalloxazineC2, C2-related isoalloxazine. E-mail address of the corresponding author: [email protected]
aromatic moieties, isoalloxazine and adenine, stack intramolecularly to form so-called closed conformations. The intrinsic tendency of FAD for arranging in such complexes was first proposed as early as 1949 by Weber in fluorescence spectroscopic studies on FAD in aqueous solution.1–4 Time-resolved fluorescence measurements fully confirmed this early model of closed FAD.5–9 However, structural investigations have always been an object of dispute. Indeed, X-ray structural access to closed FAD is difficult due to the inherent flexibility and conformational dynamics of FAD.6 Alternating π–π interactions observed in the X-ray structural analysis of a 1:1 mixture of adenine and isoalloxazine derivatives do on the other hand lack in significance, as these could represent an artifact of packing in the crystal matrix as well.10,11 Similarly, at the high concentrations necessary for NMR structural analysis, intermolecular complexes cannot be excluded, preventing unambiguous assignments of experimental data to an intramolecularly stacked conformation of adenine and isoalloxazine.12–15
0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
The Dodecin Binding Mode
Figure 1. Molecular structure of FAD with riboflavin (RBF) and FMN sub-structures. The aromatic isoalloxazine moiety is highlighted in re-side view.
Apododecin binds FAD with changed stoichiometry Recently, we succeeded in resolving the crystal structure of dodecin.16 Dodecin is a dodecameric flavin binding protein complex, containing a pair of flavin ligands arranged in antiparallel manner within each of the six identical binding pockets (Figure 2(a)). Embedment of chromophores between Trp36 and the C2-related Trp36C2 results in fluorescence quenching and thus allows determination of binding affinities. These vary from high affinities for lumichrome (9.9( ± 3.2) nM; −45.7 kJ/mol (KD; ΔG)), lumiflavin (17.6( ± 4.0); −44.3) and riboflavin (35.8 ( ± 4.4); 42.5) to reduced affinities for FAD (439(±4); −36.3) and particularly FMN (13.7(±1.2)×103; − 27.8).17 The gain of complex stability by 8.5 kJ/mol upon elongation of the phosphoribityl chain (FMN) with AMP (FAD) is difficult to explain on the basis of the dodecin ligand binding mode. As illustrated in Figure 2(a), steric clashes in the protein C2 channel would rather predict the ligand affinity to decrease with increasing bulkiness of the aliphatic chain. However, titration curves of riboflavin and FAD shown in Figure 2(b) could clearly reveal a change in the stoichiometry of the respective complexes. While saturation of the six binding pockets of dodecin is reached with 12 riboflavin molecules, half of the ligand molecules are required for FAD, implying a change from riboflavin double to FAD single occupation of binding pockets. These results were consistent with an incorporation of either (i) a single FAD molecule in an intramolecular complexed (closed) conformation or (ii) a single FAD molecule in an extended (open) conformation, plugging the C2 channel and restricting the adoption of the second molecule. X-ray structural analysis of FAD/apododecin complexes (Hrc-FAD and Hsk-FAD) To structurally analyze this FAD binding, we performed X-ray structural investigations on FAD/ apododecin crystals which were obtained by crystallization of the reconstituted complex (Hrc-FAD) or
by soaking apododecin crystals with FAD (HskFAD), respectively (see Table 1 for data collection and refinement statistics). Although the crystallographic C2 axis through the aromatic tetrade hampers assignment of the flavin binding positions, refinement of data to 1.85 Å (Hrc-FAD) and 1.80 Å (Hsk-FAD) revealed that the dodecin binding pocket is occupied by isoalloxazine and adenine.18–20 As illustrated in Figure 3(a) and (b), the adenine complexed in the dodecin binding position is indicated by the expansion of electron density in the isoalloxazine N1 position which results from the adenine N9 aliphatic elongation. This indicates that FAD is bound in a closed conformation, with both aromatic sub-moieties occupying the binding positions of a binding pocket. While the reconstitution of apododecin with riboflavin results in Trp36-isoalloxazine-isoalloxazineC2-Trp36C2 aromatic arrangements, incorporation of FAD in its closed conformation creates Trp36-isoalloxazine-adenineTrp36C2 configured tetrades, illustrated in Figure 4. As indicated by the superposition of the Trp36 residue and the isoalloxazine ring in Figure 5, the binding positions are just marginally changed. The low plasticity of dodecin thus restricts differences of the H-RBF and the Hrc-FAD binding pocket to the isoalloxazine substituted by adenine which displays dodecin as an interesting system to study spectral properties of bound dimeric versus bound monomeric flavin properties. Dodecin sequesters closed FAD from the aqueous solution The dodecin ligand affinity derives basically from two opposing effects: (i) the stabilizing effect of the aromatic tetrade arrangement upon clamping and aligning the (iso)alloxazine moieties; and (ii) the destabilizing effect from spatial restrictions which correlates positively with the volume of the aliphatic moiety. While fixation of the isoalloxazine moiety by Trp36 and Gln55 can be regarded as a rather constant contribution to the stabilities of dodecin complexes, the destabilization by the aliphatic chain is variable and thus dictates the binding affinities in
The Dodecin Binding Mode
FAD in its closed structure results from a spatially more compatible ligand conformation which reduces the steric clashes in the protein C2 channel while retaining the aromatic tetrade stability. Indeed, the binding affinity can be assumed significantly enhanced when comparing the stability of the complex with a closed FAD ligand (Hrc-FAD) to that with an extended (open) FAD (Hex-FAD) simulated by the FMN complex (H-FMN). Correspondingly, binding of FAD in its closed conformation can be estimated to increase the complex stability by at least 8.5 kJ/mol (− 36.3 kJ/mol for Hrc-FAD versus − 27.8 kJ/mol for H-FMN). Note that the occupation of the binding pocket by a single FAD monomer in extended form would likewise reduce the steric clashes in the C2 channel, but the loss of stacking interaction by the reduction to an aromatic triade arrangement thermodynamically disfavors this protein complex. Table 1. Data collection and refinement statistic of HrcFAD (2CJC) and Hsk-FAD (2CIE) Hrc-FAD
PXI/mar225 mosaic CCD 1.0056 a = b = c = 142.39 20.0–1.85 (1.96–1.85) 74,435 (11,575) 10,961 (1714) 34.1 8.8 (66.5) 99.1 (99.8) 12.56 (2.67)
Id23-1/ ADSC Q315R 0.9763 a = b = c = 142.04 20.0–1.80 (1.94–1.80) 122,830 (19,062) 11,950 (1852) 26.3 10.6 (58.0) 99.7 (99.6) 14.58 (3.54)
F4132 20.0–1.85 (1.898–1.850) 10,330 (753) 19.37/22.23 (29.5/31.9) 543 69
F4132 20.0–1.80 (1.848–1.801) 11,369 (803) 18.69/20.89 (23.2/22.8) 543 70
Geometry rmsd of bond length (Å) Rmsd of bond angles (deg.) Mean B value (Å2)
0.018 2.169 37.0
0.016 2.146 25.1
Ramachandran Most favored (%) Additional allowed (%) Generously allowed (%) Disallowed (%)
91.5 8.5 0.0 0.0
91.5 8.5 0.0 0.0
Data set Data collection X-ray source/ detector system Wavelength (Å) Cell constants (Å) Resolution range (Å) Observations Unique reflections Wilson B-factor (Å2) a Rmerge (%) Completeness (%) I/σ(I) Refinement statistics (REFMAC) Space group Resolution range (Å)
Figure 2. (a) Adoption of riboflavin in an aromatic tetrade arrangement (C2-related residues and atoms are indexed by C2 in superscript). Dodecin comprises two flavin binding positions in a single binding pocket. Ligand binding is predominantly mediated by a two-point fixation of the aromatic isoalloxazine ring via Trp36 and Gln55. (b) Ligand emission fluorescence at 520 nm (excitation at 450 nm) is recorded at increasing apododecin molar concentrations.17 End quenching of riboflavin (black) is reached upon titration with one equivalent of flavin binding positions, while two equivalents are needed for final FAD fluorescence (gray). W36A-mutant dodecin determines the Trp36 indol rings to quench the ligand fluorescence and consequently the specific binding of a single FAD into a dodecin binding pocket (broken lines).
the rank order of lumiflavin>riboflavin>FMN.17 For FAD an anomalous behavior was observed which we propose to derive from a pre-organization of FAD in a closed conformation in solution sequestered by dodecin for the formation of a thermodynamically more stable protein complex. Consequently, the enhanced complex stability with
No. unique reflections Rcryst/cRfree (%)
No. of protein atoms No. of water molecules
The structures of Hsk-FAD and Hrc-FAD were solved with molecular replacement methods starting with the atomic coordinates of apododecin (2CC9). Water molecules generated with ARP-water upon refinement of data (REFMAC refinement software) were deleted from the binding pockets. a Rmerge = Σ|I−|/ΣI, where I is the observed intensity and the average intensity from multiple observations of symmetry-related reflections; values in parentheses correspond to the highest resolution shell. b Rcryst = Σ|(Fobs)−(Fcalc)|/Σ (Fobs). c Rfree = crystallographic R-factor based on 5% of the data withheld from the refinement for cross-validation.
The Dodecin Binding Mode
reflects a FAD solution-stacked form, depends critically on the extent of restructuring effects exerted on the FAD ligand. Basically for two reasons such effects are expected to just marginally influence the FAD conformation: (i) restructuring effects on the FAD closed conformation, as resulting from dynamics in dodecin oligomerizations, can be excluded by the Hsk-FAD structure from an apododecin crystal with the frozen dodecameric state; (ii) major rearrangements, as the reshuffling of the π–π stacking complex inside the binding pocket, are prevented for steric reasons and for reasons of a low conformational flexibility of dodecin. Dodecin has to be regarded as a rigid crystal packing matrix which provides binding pockets for key and lock binding of flavin and flavin-like ligands (lumichrome). Consequently, we propose the observed FAD conformation as a solution-stacked form of FAD frozen by the dodecin particle. This is in contrast to DNA-photolyase, the only flavoprotein reported which does not bind FAD in extended conformations. Although confirming a closed FAD conformation, DNA-photolyase structures clearly revealed the FAD aromatic moieties displaced from real π–π interactions, suggesting a dominant structuring effect of the protein and preventing from conclusions about FAD conformation in aqueous solution.21,22 Note that structural and functional data do not demonstrate that the detected isoalloxazine/adenine stacking complex represents the FAD
Figure 3. Observed 2Fobs−Fcalc electron density (blue, contoured at σ1) and Fobs−Fcalc difference electron density (red, contoured at σ3) in the dodecin binding pocket of the FAD complex (Hrc-FAD) reduced to the C2-related part (single dodecin binding position). (a) Apododecin coordinates were used for phase calculation (ligand coordinates omitted). A blue arrow highlights the electron density arising from the ribityl chain. The incorporation of closed FAD is indicated by electron density, which can not be attributed to the isoalloxazine submoiety (red arrow). (b) Upon refinement with riboflavin at an occupancy of 0.5 per binding position (adenine coordinates omitted), the Fobs−Fcalc difference electron density indicates the adenine main position and its orientation in the aromatic tetrade arrangement (pink). For clarity, riboflavin and adenine are superimposed.
Dodecin exerts little restructuring effects on ligand molecules The question to which extend the FAD closed conformation of FAD/apododecin complexes
Figure 4. Model of the closed FAD conformation in the dodecin binding pocket (one of the two C2-related FAD molecules is shown). In the π-stacking complex the adenine stacks onto the re-side of the isoalloxazine. The conformation of the linking aliphatic chain (ribitylpyrophosphate-ribofuranosyl chain) is not minimized in energy. Aromatic moieties in Figure 1 are shown with these sides pointing to the viewer, which stack in the intramolecular complex.
The Dodecin Binding Mode
Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2006.08.083
Figure 5. Superposition of the riboflavin (pink) and the FAD (cyan) isoalloxazine moiety as well as the corresponding adjacent Trp36 indol rings. Front view and side view illustrate minimal changes in aromate positions.
preferential arrangement in aqueous solution. However, as sequestered from the aqueous solution, it is a real FAD closed conformation existing in aqueous solution. Besides dodecin, serving as a model for the investigation of bound flavin dimers compared to flavin monomers, the FAD/apododecin complex structure thus provides an excellent starting model for molecular dynamic calculations on free and ligand bound FAD. Protein Data Bank accession code The atomic coordinates and structure factors have been deposited in the Protein Data Bank†, with PDB ID codes 2CJC (Hrc-FAD) and 2CIE (Hsk-FAD) as well as 2CIF for an alternative Hsk-FAD structure.
Acknowledgements We are very grateful to thank Luis Moroder for discussion and carefully reading the manuscript. Thanks to Clemens Schulze-Briese and Ehmke Pohl (SLS, Zurich) as well as Sean McSweeny (ESRF, Grenoble) for beamline assistance. M.G. also thanks Andreas Bracher for crystallographic support. † http://www.pdb.org
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The Dodecin Binding Mode
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Edited by M. Guss (Received 14 July 2006; received in revised form 25 August 2006; accepted 28 August 2006) Available online 5 September 2006