Ligand-induced structural changes to maltodextrin-binding protein as studied by solution NMR spectroscopy

Solution NMR studies on the physiologically relevant ligand-free and maltotriose-bound states of maltodextrin-binding protein (MBP) are presented. Together with existing data on MBP in complex with beta-cyclodextrin (non-physiological, inactive ligand), these new results provide valuable information on changes in local structure, dynamics and global fold that occur upon ligand binding to this two-domain protein. By measuring a large number of different one-bond residual dipolar couplings, the domain conformations, critical for biological function, were investigated for all three states of MBP. Structural models of the solution conformation of MBP in a number of different forms were generated from the experimental dipolar coupling data and X-ray crystal structures using a quasi-rigid-body domain orientation algorithm implemented in the structure calculation program CNS. Excellent agreement between relative domain orientations in ligand-free and maltotriose-bound solution conformations and the corresponding crystal structures is observed. These results are in contrast to those obtained for the MBP/beta-cyclodextrin complex where the solution state is found to be approximately 10 degrees more closed than the crystalline state. The present study highlights the utility of residual dipolar couplings for orienting protein domains or macromolecules with respect to each other.     

Global folds of proteins with low densities of NOEs using residual dipolar couplings: application to the 370-residue maltodextrin-binding protein

The global fold of maltose-binding protein in complex with the substrate beta-cyclodextrin was determined by solution NMR methods. The two-domain protein is comprised of a single polypeptide chain of 370 residues, with a molecular mass of 42 kDa. Distance information in the form of H(N)-H(N), H(N)-CH(3) and CH(3)-CH(3) NOEs was recorded on (15)N, (2)H and (15)N, (13)C, (2)H-labeled proteins with methyl protonation in Val, Leu, and Ile (C(delta1) only) residues. Distances to methyl protons, critical for the structure determination, comprised 77 % of the long-range restraints. Initial structures were calculated on the basis of 1943 NOEs, 48 hydrogen bond and 555 dihedral angle restraints. A global pair-wise backbone rmsd of 5.5 A was obtained for these initial structures with rmsd values for the N and C domains of 2.4 and 3.8 A, respectively. Direct refinement against one-bond (1)H(N)-(15)N, (13)C(alpha)-(13)CO, (15)N-(13)CO, two-bond (1)H(N)-(13)CO and three-bond (1)H(N)-(13)C(alpha) dipolar couplings resulted in structures with large numbers of dipolar restraint violations. As an alternative to direct refinement against measured dipolar couplings we have developed an approach where discrete orientations are calculated for each peptide plane on the basis of the dipolar couplings described above. The orientation which best matches that in initial NMR structures calculated from NOE and dihedral angle restraints exclusively is used to refine further the structures using a new module written for CNS. Modeling studies from four different proteins with diverse structural motifs establishes the utility of the methodology. When applied to experimental data recorded on MBP the precision of the family of structures generated improves from 5.5 to 2.2 A, while the rmsd with respect to the X-ray structure (1dmb) is reduced from 5.1 to 3.3 A.     

Solution-state molecular structure of apo and oleate-liganded liver fatty acid-binding protein

Rat liver fatty acid-binding protein (LFABP) is distinctive among intracellular lipid-binding proteins (iLBPs): more than one molecule of long-chain fatty acid and a variety of diverse ligands can be bound within its large cavity, and in vitro lipid transfer to model membranes follows a mechanism that is diffusion-controlled rather than mediated by protein-membrane collisions. Because the apoprotein has proven resistant to crystallization, nuclear magnetic resonance spectroscopy offers a unique route to functionally informative comparisons of molecular structure and dynamics for LFABP in free (apo) and liganded (holo) forms. We report herein the solution-state structures determined for apo-LFABP at pH 6.0 and for holoprotein liganded to two oleates at pH 7.0, as well as the structure of the complex including locations of the ligands. 1H, 13C, and 15N resonance assignments revealed very similar types and locations of secondary structural elements for apo- and holo-LFABP as judged from chemical shift indices. The solution-state tertiary structures of the proteins were derived with the CNS/ARIA computational protocol, using distance and angular restraints based on 1H-1H nuclear Overhauser effects (NOEs), hydrogen-bonding networks, 3J(HNHA) coupling constants, intermolecular NOEs, and residual dipolar (NH) couplings. The holo-LFABP solution-state conformation is in substantial agreement with a previously reported X-ray structure [Thompson, J., Winter, N., Terwey, D., Bratt, J., and Banaszak, L. (1997) The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates, J. Biol. Chem. 272, 7140-7150], including the typical beta-barrel capped by a helix-turn-helix portal. In the solution state, the internally bound oleate has the expected U-shaped conformation and is tethered electrostatically, but the extended portal ligand can adopt a range of conformations based on the computationally refined structures, in contrast to the single conformation observed in the crystal structure. The apo-LFABP also has a well-defined beta-barrel structural motif typical of other members of the iLBP protein family, but the portal region that is thought to facilitate ligand entry and exit exhibits conformational variability and an unusual "open cap" orientation with respect to the barrel. These structural results allow us to propose a model in which ligand binding to LFABP occurs through conformational fluctuations that adjust the helix-turn-helix motif to open or close the top of the beta-barrel, and solvent accessibility to the protein cavity favors diffusion-controlled ligand transport.     

Recognition pliability is coupled to structural heterogeneity: a calmodulin intrinsically disordered binding region complex

Protein interactions within regulatory networks should adapt in a spatiotemporal-dependent dynamic environment, in order to process and respond to diverse and versatile cellular signals. However, the principles governing recognition pliability in protein complexes are not well understood. We have investigated a region of the intrinsically disordered protein myelin basic protein (MBP(145-165)) that interacts with calmodulin, but that also promiscuously binds other biomolecules (membranes, modifying enzymes). To characterize this interaction, we implemented an NMR spectroscopic approach that calculates, for each conformation of the complex, the maximum occurrence based on recorded pseudocontact shifts and residual dipolar couplings. We found that the MBP(145-165)-calmodulin interaction is characterized by structural heterogeneity. Quantitative comparative analysis indicated that distinct conformational landscapes of structural heterogeneity are sampled for different calmodulin-target complexes. Such structural heterogeneity in protein complexes could potentially explain the way that transient and promiscuous protein interactions are optimized and tuned in complex regulatory networks.     

What is the average conformation of bacteriophage T4 lysozyme in solution? A domain orientation study using dipolar couplings measured by solution NMR

Lysozyme from T4 bacteriophage is comprised of two domains that are both involved in binding substrate. Although wild-type lysozyme has been exclusively crystallized in a closed form that is similar to the peptidoglycan-bound conformation, a more open structure is thought to be required for ligand binding. To determine the relative arrangement of domains within T4 lysozyme in the solution state, dipolar couplings were measured in several different dilute liquid crystalline media by solution NMR methods. The dipolar coupling data were analyzed with a domain orientation procedure described previously that utilizes high- resolution X-ray structures. The cleft between the domains is significantly larger in the average solution structure than what is observed in the X-ray structure of the ligand-free form of the protein (approximately 17 degrees closure from solution to X-ray structures). A comparison of the solution domain orientation with X-ray-derived structures in the protein data base shows that the solution structure resembles a crystal structure obtained for the M6I mutant. Dipolar couplings were also measured on the lysozyme mutant T21C/T142C, which was oxidized to form an inter-domain disulfide bond (T4SS). In this case, the inter-domain solution structure was found to be more closed than was observed in the crystal (approximately 11 degrees). Direct refinement of lysozyme crystal structures with the measured dipolar couplings using the program CNS, establishes that this degree of closure can be accommodated whilst maintaining the inter-domain cystine bond. The differences between the average solution conformations obtained using dipolar couplings and the crystal conformations for both forms of lysozyme investigated in this study illustrate the impact that crystal packing interactions can have on the arrangement of domains within proteins and the importance of alternative methods to X-ray crystallography for evaluating inter-domain structure.     

The global conformation of the hammerhead ribozyme determined using residual dipolar couplings

The global structure of the hammerhead ribozyme was determined in the absence of Mg(2+) by solution NMR experiments. The hammerhead ribozyme motif forms a branched structure consisting of three helical stems connected to a catalytic core. The (1)H-(15)N and (1)H-(13)C residual dipolar couplings were measured in a set of differentially (15)N/(13)C-labeled ribozymes complexed with an unlabeled noncleavable substrate. The residual dipolar couplings provide orientation information on both the local and the global structure of the molecule. Analysis of the residual dipolar couplings demonstrated that the local structure of the three helical stems in solution is well modeled by an A-form conformation. However, the global structure of the hammerhead in solution in the absence of Mg(2+) is not consistent with the Y-shaped conformation observed in crystal structures of the hammerhead. The residual dipolar couplings for the helical stems were combined with standard NOE and J coupling constant NMR data from the catalytic core. The NOE data show formation of sheared G-A base pairs in domain 2. These NMR data were used to determine the global orientation of the three helical stems in the hammerhead. The hammerhead forms a rather extended structure under these conditions with a large angle between stems I and II ( approximately 153 degrees ), a smaller angle between stems II and III ( approximately 100 degrees ), and the smallest angle between stems I and III ( approximately 77 degrees ). The residual dipolar coupling data also contain information on the dynamics of the molecule and were used here to provide qualitative information on the flexibility of the helical domains in the hammerhead ribozyme-substrate complex.     

Improvement of hydrogen bond geometry in protein NMR structures by residual dipolar couplings – an assessment of the interrelation of NMR restraints

We have examined how the hydrogen bond geometry in three different proteins is affected when structural restraints based on measurements of residual dipolar couplings are included in the structure calculations. The study shows, that including restraints based solely on (1)H(N)-(15)N residual dipolar couplings has pronounced impact on the backbone rmsd and Ramachandran plot but does not improve the hydrogen bond geometry. In the case of chymotrypsin inhibitor 2 the addition of (13)CO-(13)C(alpha) and (15)N-(13)CO one bond dipolar couplings as restraints in the structure calculations improved the hydrogen bond geometry to a quality comparable to that obtained in the 1.8 A resolution X-ray structure of this protein. A systematic restraint study was performed, in which four types of restraints, residual dipolar couplings, hydrogen bonds, TALOS angles and NOEs, were allowed in two states. This study revealed the importance of using several types of residual dipolar couplings to get good hydrogen bond geometry. The study also showed that using a small set of NOEs derived only from the amide protons, together with a full set of residual dipolar couplings resulted in structures of very high quality. When reducing the NOE set, it is mainly the side-chain to side-chain NOEs that are removed. Despite of this the effect on the side-chain packing is very small when a reduced NOE set is used, which implies that the over all fold of a protein structure is mainly determined by correct folding of the backbone.     

Structural characterization of a mannose-binding protein-trimannoside complex using residual dipolar couplings

The ligand-binding properties of a 53 kDa homomultimeric trimer from mannose-binding protein (MBP) have been investigated using residual dipolar couplings (RDCs) that are easily measured from NMR spectra of the ligand and isotopically labeled protein. Using a limited set of 1H-15N backbone amide NMR assignments for MBP and orientational information derived from the RDC measurements in aligned media, an order tensor for MBP has been determined that is consistent with symmetry-based predictions of an axially symmetric system. 13C-1H couplings for a bound trisaccharide ligand, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (trimannoside) have been determined at natural abundance and used as orientational constraints. The bound ligand geometry and orientational constraints allowed docking of the trimannoside ligand in the binding site of MBP to produce a structural model for MBP-oligosaccharide interactions.     

Probing the supramodular architecture of a multidomain protein: the structure of syntenin in solution

Full understanding of the mechanism of function of multidomain proteins is dependent on our knowledge of their supramodular architecture in solution. This is a nontrivial task for both X-ray crystallography and NMR, because intrinsic flexibility makes crystallization of these proteins difficult, while their size creates a challenge for NMR. Here, we describe synergistic application of data derived from X-ray crystallography and NMR residual dipolar couplings (RDCs) to address the question of the supramodular structure of a two-domain protein, syntenin. Syntenin is a 32 kDa molecule containing two PDZ domains and is involved in cytoskeleton-membrane organization. We show that the mutual disposition of the PDZ domains clearly differs from that seen in the crystal structure, and we provide evidence that N- and C-terminal fragments of syntenin, hitherto presumed to lack ordered structure, contain folded structural elements in the full-length protein in contact with the PDZ tandem.     

Characterization of slow conformational dynamics in solids: dipolar CODEX

A solid state NMR experiment is introduced for probing relatively slow conformational exchange, based on dephasing and refocusing dipolar couplings. The method is closely related to the previously described Centerband-Only Detection of Exchange or CODEX experiment. The use of dipolar couplings for this application is advantageous because their values are known a priori from molecular structures, and their orientations and reorientations relate in a simple way to molecular geometry and motion. Furthermore the use of dipolar couplings in conjunction with selective isotopic enrichment schemes is consistent with selection for unique sites in complex biopolymers. We used this experiment to probe the correlation time for the motion of ¹³C, ¹⁵N enriched urea molecules within their crystalline lattice.     

MaxOcc: a web portal for maximum occurrence analysis

The MaxOcc web portal is presented for the characterization of the conformational heterogeneity of two-domain proteins, through the calculation of the Maximum Occurrence that each protein conformation can have in agreement with experimental data. Whatever the real ensemble of conformations sampled by a protein, the weight of any conformation cannot exceed the calculated corresponding Maximum Occurrence value. The present portal allows users to compute these values using any combination of restraints like pseudocontact shifts, paramagnetism-based residual dipolar couplings, paramagnetic relaxation enhancements and small angle X-ray scattering profiles, given the 3D structure of the two domains as input. MaxOcc is embedded within the NMR grid services of the WeNMR project and is available via the WeNMR gateway at http://py-enmr.cerm.unifi.it/access/index/maxocc . It can be used freely upon registration to the grid with a digital certificate.     

The closed and compact domain organization of the 70-kDa human cytochrome P450 reductase in its oxidized state as revealed by NMR

The NADPH cytochrome P450 reductase (CPR), a diflavin enzyme, catalyzes the electron transfer (ET) from NADPH to the substrate P450. The crystal structures of mammalian and yeast CPRs show a compact organization for the two domains containing FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), with a short interflavin distance consistent with fast ET from the NADPH-reduced FAD to the second flavin FMN. This conformation, referred as "closed", contrasts with the alternative opened or extended domain arrangements recently described for partially reduced or mutant CPR. Internal domain flexibility in this enzyme is indeed necessary to account for the apparently conflicting requirements of having FMN flavin accessible to both the FAD and the substrate P450 at the same interface. However, how interdomain dynamics influence internal and external ETs in CPR is still largely unknown. Here, we used NMR techniques to explore the global, domain-specific and residue-specific structural and dynamic properties of the nucleotide-free human CPR in solution in its oxidized state. Based on the backbone resonance assignment of this 70-kDa protein, we collected residue-specific (15)N relaxation and (1)H-(15)N residual dipolar couplings. Surprisingly and in contrast with previous studies, the analysis of these NMR data revealed that the CPR exists in a unique and predominant conformation that highly resembles the closed conformation observed in the crystalline state. Based on our findings and the previous observations of conformational equilibria of the CPR in partially reduced states, we propose that the large-scale conformational transitions of the CPR during the catalytic cycle are tightly controlled to ensure optimal electron delivery.     

A new method for the simultaneous measurement of magnitude and sign of 1DCH and 1DHH dipolar couplings in methylene groups

Heteronuclear dipolar couplings of the protein backbone have proven to have a big impact on the accuracy of protein NMR structures. H,H dipolar couplings might have the same impact on side chains. Here we present a method that combines both heteronuclear and homonuclear dipolar couplings to investigate the local conformation of methylene groups. A new pulse sequence (SPITZE-HSQC) is presented, that allows to measure the two C,H and the H,H dipolar couplings at the same time, using spin state selective transfers. The new method has been applied to the methylene groups of glycines in the protein ubiquitin. The C,H and the H,H dipolar couplings might have a key role in fast stereospecific assignment of protons in CH₂ groups.     

Structural studies of an engineered zinc biosensor reveal an unanticipated mode of zinc binding

Protein engineering was used previously to convert maltose-binding protein (MBP) into a zinc biosensor. Zn(2+) binding by the engineered MBP was thought to require a large conformational change from "open" to "closed", similar to that observed when maltose is bound by the wild-type protein. We show that although this re-designed MBP molecule binds Zn(2+) with high affinity as previously reported, it does not adopt a closed conformation in solution as assessed by small-angle X-ray scattering. High-resolution crystallographic studies of the engineered Zn(2+)-binding MBP molecule demonstrate that Zn(2+) is coordinated by residues on the N-terminal lobe only, and therefore Zn(2+) binding does not require the protein to adopt a fully closed conformation. Additional crystallographic studies indicate that this unexpected Zn(2+) binding site can also coordinate Cu(2+) and Ni(2+) with only subtle changes in the overall conformation of the protein. This work illustrates that the energetic barrier to domain closure, which normally functions to maintain MBP in an open concentration in the absence of ligand, is not easily overcome by protein design. A comparison to the mechanism of maltose-induced domain rearrangement is discussed.     

The Ig doublet Z1Z2: a model system for the hybrid analysis of conformational dynamics in Ig tandems from titin

Titin is a gigantic elastic filament that determines sarcomere ultrastructure and stretch response in vertebrate muscle. It folds into numerous Ig and FnIII domains connected in tandem. Data on interdomain arrangements and dynamics are essential for understanding the function of this filament. Here, we report a mechanistic analysis of the conformational dynamics of two Ig domains from the N terminus of titin, Z1Z2, by using X-ray crystallography, SAXS, NMR relaxation data, and residual dipolar couplings in combination. Z1Z2 preferentially adopts semiextended conformations in solution, with close-hinge arrangements representing low-probability states. Although interdomain contacts are not observed, the linker appears to acquire moderate rigidity via small, local hydrophobic interactions. Thus, Z1Z2 constitutes an adaptable modular system with restricted dynamics. We speculate that its preexistent conformation contributes to the selective recruitment of the binding partner telethonin onto the repetitive surface of the filament. The structural interconversion of four Z1Z2 conformers is analyzed.     

Application of Correlated Residual Dipolar Couplings to the Determination of the Molecular Alignment Tensor Magnitude of Oriented Proteins and Nucleic Acids

Residual dipolar couplings (RDC) between nuclear spins in partially aligned samples offer unique insights into biomacromolecular structure and dynamics. To fully benefit from the RDC data, accurate knowledge of the magnitude ( D (a)) and rhombicity ( R ) of the molecular alignment tensor, A, is important. An extended histogram method (EHM) is presented which extracts these parameters more effectively from dipolar coupling data. The method exploits the correlated nature of RDCs for structural elements of planar geometry, such as the one-bond (13)C'(i)-(13)C(i)(alpha), (13)C'(i)-(15)N(i+1), and (15)N(i+1)-(1)H(N)(i+1) couplings in peptide bonds of proteins, or suitably chosen combinations of (1) D (C1'H1'), (1) D (C2'H2'), (1) D (C1'C2'), (2) D (C2'H1'), (2) D (C1'H2'), and (3) D (H1'H2') couplings in nucleic acids, to generate an arbitrarily large number of synthetic RDCs. These synthetic couplings result in substantially improved histograms and resulting values of D (a) and R, compared with histograms generated solely from the original sets of correlated RDCs, particularly when the number of planar fragments for which couplings are available is small. An alternative method, complementary to the EHM, is also described, which uses a systematic grid search procedure, based on least-squares fitting of sets of correlated RDCs to structural elements of known geometry, and provides an unambiguous lower limit for the degree of molecular alignment.     

Structure and orientation of a G protein fragment in the receptor bound state from residual dipolar couplings

Residual dipolar couplings for a ligand that is in fast exchange between a free state and a state where it is bound to a macroscopically ordered membrane protein carry precise information on the structure and orientation of the bound ligand. The couplings originate in the bound state but can be detected on the free ligand using standard high resolution NMR. This approach is used to study an analog of the C-terminal undecapeptide of the alpha-subunit of the heterotrimeric G protein transducin when bound to photo-activated rhodopsin. Rhodopsin is the major constituent of disk-shaped membrane vesicles from rod outer segments of bovine retinas, which align spontaneously in the NMR magnet. Photo-activation of rhodopsin triggers transient binding of the peptide, resulting in measurable dipolar contributions to 1J(NH) and 1J(CH) splittings. These dipolar couplings report on the time-averaged orientation of bond vectors in the bound peptide relative to the magnetic field, i.e. relative to the membrane normal. Approximate distance restraints of the bound conformation were derived from transferred NOEs, as measured from the difference of NOESY spectra recorded prior to and after photo-activation. The N-terminal eight residues of the bound undecapeptide adopt a near-ideal alpha-helical conformation. The helix is terminated by an alpha(L) type C-cap, with Gly9 at the C' position in the center of the reverse turn. The angle between the helix axis and the membrane normal is 40 degrees (+/-4) degrees. Peptide protons that make close contact with the receptor are identified by analysis of the NOESY cross-relaxation pattern and include the hydrophobic C terminus of the peptide.     

Domain orientation and dynamics in multidomain proteins from residual dipolar couplings.

The data most commonly available for the determination of macromolecular structures in solution are NOE based distance estimates and spin-spin coupling constant based dihedral angle estimates. This information is, unfortunately, inherently short-range in nature. Thus, for many multidomain proteins, little information is available to accurately position weakly interacting domains with respect to each other. Recent studies of proteins aligned in dilute liquid crystalline solvents have shown the utility of measuring anisotropic spin interactions, such as residual dipolar couplings, to obtain unique long-range structural information. In this work, the latter approach is taken to explore the relative domain orientation in a two-domain fragment from the protein barley lectin. An approach based on singular value decomposition as opposed to simulated annealing is used to directly determine order tensors for each domain from residual (15)N-(1)H dipolar couplings, and the limitations of the two approaches are discussed. Comparison of the order tensor principal axis frames as separately determined for each domain indicates that the two domains are not oriented as in the crystal structure of wheat germ agglutinin, a highly homologous protein ( approximately 95% sequence identical). Furthermore, differences in the order tensor values suggest that the two domains are not statically positioned but are experiencing different reorientational dynamics and, to a large degree, may be considered to reorient independently. Data are also presented that suggest that a specific association occurs between one domain and the lipid bicelles comprising the liquid crystal solvent.     

Probing multiple effects on 15N, 13C alpha, 13C beta,and 13C' chemical shifts in peptides using density functional theory

We have used density functional calculations on model peptides to study conformational effects on (15)N, (13)C alpha, (13)C beta, and (13)C' chemical shifts, associated with hydrogen bonding, backbone conformation, and side-chain orientation. The results show a significant dependence on the backbone torsion angles of the nearest three residues. Contributions to (15)N chemical shifts from hydrogen bonding (up to 8 ppm), backbone conformation (up to 13 ppm), side-chain orientation and neighborhood residue effects (up to 22 ppm) are significant, and a unified theory will be required to account for their behavior in proteins. In contrast to this, the dependence on sequence and hydrogen bonding is much less for (13)C alpha and (13)C beta chemical shifts (<0.5 ppm), and moderate for carbonyl carbon shifts (<2 ppm). The effects of side-chain orientation are mainly limited to the residue itself for both nitrogen and carbon, but the chi(1) effect is also significant for the nitrogen shift of the following residue and for the (13)C' shift of the preceding residue. The calculated results are used, in conjunction with an additive model of chemical shift contributions, to create an algorithm for prediction of (15)N and (13)C shifts in proteins from their structure; this includes a model to extrapolate results to regions of torsion angle space that have not been explicitly studied by density functional theory (DFT) calculations. Crystal structures of 20 proteins with measured shifts have been used to test the prediction scheme. Root mean square deviations between calculated and experimental shifts 2.71, 1.22, 1.31, and 1.28 ppm for N, C alpha, C beta, and C', respectively. This prediction algorithm should be helpful in NMR assignment, crystal and solution structure comparison, and structure refinement.     

Kissing G Domains of MnmE Monitored by X-Ray Crystallography and Pulse Electron Paramagnetic Resonance Spectroscopy

MnmE, which is involved in the modification of the wobble position of certain tRNAs, belongs to the expanding class of G proteins activated by nucleotide-dependent dimerization (GADs). Previous models suggested the protein to be a multidomain protein whose G domains contact each other in a nucleotide dependent manner. Here we employ a combined approach of X-ray crystallography and pulse electron paramagnetic resonance (EPR) spectroscopy to show that large domain movements are coupled to the G protein cycle of MnmE. The X-ray structures show MnmE to be a constitutive homodimer where the highly mobile G domains face each other in various orientations but are not in close contact as suggested by the GDP-AlF_x structure of the isolated domains. Distance measurements by pulse double electron-electron resonance (DEER) spectroscopy show that the G domains adopt an open conformation in the nucleotide free/GDP-bound and an open/closed two-state equilibrium in the GTP-bound state, with maximal distance variations of 18 Å. With GDP and AlF_x, which mimic the transition state of the phosphoryl transfer reaction, only the closed conformation is observed. Dimerization of the active sites with GDP-AlF_x requires the presence of specific monovalent cations, thus reflecting the requirements for the GTPase reaction of MnmE. Our results directly demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle. They show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein, and they are of crucial importance for understanding the mechanistic principles of this GAD.