Preparation and characterization of inactivated tick-borne encephalitis virus samples for single-particle imaging at the European XFEL.

X-ray imaging of virus particles at the European XFEL could eventually allow their complete structures to be solved, potentially approaching the resolution of other structural virology methods. To achieve this ambitious goal with today's technologies, about 1 ml of purified virus suspension containing at least 1012 particles per millilitre is required. Such large amounts of concentrated suspension have never before been obtained for enveloped viruses. Tick-borne encephalitis virus (TBEV) represents an attractive model system for the development of enveloped virus purification and concentration protocols, given the availability of large amounts of inactivated virus material provided by vaccine-manufacturing facilities. Here, the development of a TBEV vaccine purification and concentration scheme is presented combined with a quality-control protocol that allows substantial amounts of highly concentrated non-aggregated suspension to be obtained. Preliminary single-particle imaging experiments were performed for this sample at the European XFEL, showing distinct diffraction patterns.

The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces.

Vibrio cholerae is a bacterial pathogen that colonizes the chitinous exoskeleton of zooplankton as well as the human gastrointestinal tract. Colonization of these different niches involves an N-acetylglucosamine binding protein (GbpA) that has been reported to mediate bacterial attachment to both marine chitin and mammalian intestinal mucin through an unknown molecular mechanism. We report structural studies that reveal that GbpA possesses an unusual, elongated, four-domain structure, with domains 1 and 4 showing structural homology to chitin binding domains. A glycan screen revealed that GbpA binds to GlcNAc oligosaccharides. Structure-guided GbpA truncation mutants show that domains 1 and 4 of GbpA interact with chitin in vitro, whereas in vivo complementation studies reveal that domain 1 is also crucial for mucin binding and intestinal colonization. Bacterial binding studies show that domains 2 and 3 bind to the V. cholerae surface. Finally, mouse virulence assays show that only the first three domains of GbpA are required for colonization. These results explain how GbpA provides structural/functional modular interactions between V. cholerae, intestinal epithelium and chitinous exoskeletons.

Essential role of the metal-ion in the IPM-assisted domain closure of 3-isopropylmalate dehydrogenase.

X-ray structures of 3-isopropylmalate dehydrogenase (IPMDH) do not provide sufficient information on the role of the metal-ion in the metal-IPM assisted domain closure. Here solution studies were carried out to test its importance. Small-angle X-ray scattering (SAXS) experiments with the Thermus thermophilus enzyme (complexes with single substrates) have revealed only a very marginal (0-5%) extent of domain closure in the absence of the metal-ion. Only the metal-IPM complex, but neither the metal-ion nor the free IPM itself, is efficient in stabilizing the native protein conformation as confirmed by denaturation experiments with Escherichia coli IPMDH and by studies of the characteristic fluorescence resonance energy transfer (FRET) signal (from Trp to bound NADH) with both IPMDHs. A possible atomic level explanation of the metal-effect is given.

Human serum albumin binding ibuprofen: a 3D description of the unfolding pathway in urea.

Small angle X-ray scattering (SAXS) technique, supported by light scattering measurements and spectroscopic data (circular dichroism and fluorescence) allowed us to restore the 3D structure at low resolution of defatted human serum albumin (HSA) in interaction with ibuprofen. The data were carried out on a set of HSA solutions with urea concentrations between 0.00 and 9.00M. The Singular Value Decomposition method, applied to the complete SAXS data set allowed us to distinguish three different states in solution. In particular a native conformation N (at 0.00M urea), an intermediate I1 (at 6.05M urea) and an unfolded structure U (at 9.00M urea) were recognized. The low-resolution structures of these states were obtained by exploiting both ab initio and rigid body fitting methods. In particular, for the protein without denaturant, a conformation recently described (Leggio et al., PCCP, 2008, 10, 6741-6750), very similar to the crystallographic heart shape, with only a slight reciprocal movement of the three domains, was confirmed. The I1 structure was instead characterized by only a closed domain (domain III) and finally, the recovered structure of the U state revealed the characteristic feature of a completely open state. A direct comparison with the free HSA pointed out that the presence of the ibuprofen provokes a shift of the equilibrium towards higher urea concentrations without changing the unfolding sequence. The work represents a type of analysis which could be exploited in future investigations on proteins in solution, in the binding of drugs or endogenous compounds and in the pharmacokinetic properties as well as in the study of allosteric effects, cooperation or anticooperation mechanisms.

Hydrophilization of Magnetic Nanoparticles with Modified Alternating Copolymers. Part 1: The Influence of the Grafting.

Iron oxide nanoparticles (NPs) with a diameter 21.6 nm were coated with poly(maleic acid-alt-1-octadecene) (PMAcOD) modified with grafted 5,000 Da poly(ethyelene glycol) (PEG) or short ethylene glycol (EG) tails. The coating procedure utilizes hydrophobic interactions of octadecene and oleic acid tails, while the hydrolysis of maleic anhydride moieties as well as the presence of hydrophilic PEG (EG) tails allows the NP hydrophilicity. The success of the NP coating was found to be independent of the degree of grafting which was varied between 20 and 80% of the -MacOD-units, but depended on the length of the grafted tail. The NP coating and hydrophilization did not occur when the modified copolymer contained 750 Da PEG tails independently of the grafting degree. To explain this phenomenon the micellization of the modified PMAcOD copolymers in water was analyzed by small angle x-ray scattering (SAXS). The PMAcOD molecules with the grafted 750 Da PEG tails form compact non-interacting disk-like micelles, whose stability apparently allows for no interactions with the NP hydrophobic shells. The PMAcOD containing the 5,000 Da PEG and EG tails form much larger aggregates capable of an efficient coating of the NPs. The coated NPs were characterized using transmission electron microscopy, dynamic light scattering, ζ-potential measurements, and thermal gravimetry analysis. The latter method demonstrated that the presence of long PEG tails in modified PMAcOD allows the attachment of fewer macromolecules (by a factor of ~20) compared to the case of non-modified or EG modified PMAcOD, emphasizing the importance of PEG tails in NP hydrophilization. The NPs coated with PMAcOD modified with 60% (towards all -MAcOD- units) of the 5,000 PEG tails bear a significant negative charge and display good stability in buffers. Such NPs can be useful as magnetic cores for virus-like particle formation.

Hydrophilization of Magnetic Nanoparticles with Modified Alternating Copolymers. Part 2: Behavior in solution.

Aqueous solutions of iron oxide nanoparticles (NPs) stabilized by poly(maleic acid-alt-1-octadecene) (PMAcOD) modified with the 5,000 Da poly(ethylene glycol) (PEG) or the short ethylene glycol (EG) tails were analyzed by small-angle X-ray scattering (SAXS). Advanced SAXS data analysis methods were employed to systematically characterize the structure and interactions between the NPs. Depending on the type of the grafted tail and the grafting density all NPs can be separated into three groups. All the samples contain mixtures of individual nanoparticles, their dynamic clusters and aggregates, and the fractions of these species are different in the different groups. The first group consists of NPs coated with PMAcOD modified with the long PEG tails with the maximal grafting density, and the content of dynamic clusters and aggregates in the samples of this group does not exceed 4%. The samples from the second group with less dense coatings demonstrate a larger amount (5-7%) of the aggregates and dynamic clusters. The samples from the third group consisting of the NPs protected by EG modified PMAcOD contain mostly individual NPs and some amount of dumbbell dimers without noticeable aggregation. Importantly, the solution behavior of the NPs is independent on the iron oxide core size. Our results therefore provide means of predicting stabilization and avoiding aggregation of NPs based on the type of a protective shell.

Urea-induced denaturation process on defatted human serum albumin and in the presence of palmitic acid.

We report a study on the unfolding behavior of the most abundant protein contained in plasma, human serum albumin. The unfolding mechanisms in denaturing conditions induced by urea are studied for the defatted form (HSA) and for the palmitic acid:albumin (HSAPalm) complex. We employed the singular value decomposition method to determine the minimum number of structural states present in the unfolding processes. Low-resolution three-dimensional structures are reconstructed from the one-dimensional small-angle X-ray scattering patterns and are correlated with the parameters obtained from static and dynamic light scattering experiments. The unfolding process is pointed out by both ab initio and rigid body fitting methods that highlight a stepwise evolution of the protein structure toward open conformations. The superimpositions of the 3D structures provided independently by the two methods show very good agreements. The hydrodynamic radii estimated for the protein best fitting conformations are in satisfactory agreement with the experimental ones. The results show that the HSA unfolding process is consistent with previous spectroscopic studies that suggest a multistep unfolding pathway. In particular, a scheme in which domains I and II are opened in sequence and the presence of two intermediates are evidenced is presented. The opening sequence is different from that found using guanidine hydrochloride as denaturant agent. The stabilizing role of the fatty acids in the urea denaturation process is evident. The palmitic acid ligand strongly stabilizes the protein, which remains in the native form up to high denaturant concentrations. In this case, the unfolding process is characterized by a single-step mechanism.

Atomic structures of two novel immunoglobulin-like domain pairs in the actin cross-linking protein filamin.

Filamins are actin filament cross-linking proteins composed of an N-terminal actin-binding domain and 24 immunoglobulin-like domains (IgFLNs). Filamins interact with numerous proteins, including the cytoplasmic domains of plasma membrane signaling and cell adhesion receptors. Thereby filamins mechanically and functionally link the cell membrane to the cytoskeleton. Most of the interactions have been mapped to the C-terminal IgFLNs 16-24. Similarly, as with the previously known compact domain pair of IgFLNa20-21, the two-domain fragments IgFLNa16-17 and IgFLNa18-19 were more compact in small angle x-ray scattering analysis than would be expected for two independent domains. Solution state NMR structures revealed that the domain packing in IgFLNa18-19 resembles the structure of IgFLNa20-21. In both domain pairs the integrin-binding site is masked, although the details of the domain-domain interaction are partly distinct. The structure of IgFLNa16-17 revealed a new domain packing mode where the adhesion receptor binding site of domain 17 is not masked. Sequence comparison suggests that similar packing of three tandem filamin domain pairs is present throughout the animal kingdom, and we propose that this packing is involved in the regulation of filamin interactions through a mechanosensor mechanism.

Structural and biophysical characterization of the proteins interacting with the herpes simplex virus 1 origin of replication.

The C terminus of the herpes simplex virus type 1 origin-binding protein, UL9ct, interacts directly with the viral single-stranded DNA-binding protein ICP8. We show that a 60-amino acid C-terminal deletion mutant of ICP8 (ICP8DeltaC) also binds very strongly to UL9ct. Using small angle x-ray scattering, the low resolution solution structures of UL9ct alone, in complex with ICP8DeltaC, and in complex with a 15-mer double-stranded DNA containing Box I of the origin of replication are described. Size exclusion chromatography, analytical ultracentrifugation, and electrophoretic mobility shift assays, backed up by isothermal titration calorimetry measurements, are used to show that the stoichiometry of the UL9ct-dsDNA15-mer complex is 2:1 at micromolar protein concentrations. The reaction occurs in two steps with initial binding of UL9ct to DNA (Kd approximately 6 nM) followed by a second binding event (Kd approximately 0.8 nM). It is also shown that the stoichiometry of the ternary UL9ct-ICP8DeltaC-dsDNA15-mer complex is 2:1:1, at the concentrations used in the different assays. Electron microscopy indicates that the complex assembled on the extended origin, oriS, rather than Box I alone, is much larger. The results are consistent with a simple model whereby a conformational switch of the UL9 DNA-binding domain upon binding to Box I allows the recruitment of a UL9-ICP8 complex by interaction between the UL9 DNA-binding domains.

Kinetic and mechanistic characterization of Mycobacterium tuberculosis glutamyl-tRNA synthetase and determination of its oligomeric structure in solution.

Mycobacterium tuberculosis glutamyl-tRNA synthetase (Mt-GluRS), encoded by Rv2992c, was overproduced in Escherichia coli cells, and purified to homogeneity. It was found to be similar to the other well-characterized GluRS, especially the E. coli enzyme, with respect to the requirement for bound tRNA(Glu) to produce the glutamyl-AMP intermediate, and the steady-state kinetic parameters k(cat) (130 min(-1)) and K(M) for tRNA (0.7 microm) and ATP (78 microm), but to differ by a one order of magnitude higher K(M) value for L-Glu (2.7 mm). At variance with the E. coli enzyme, among the several compounds tested as inhibitors, only pyrophosphate and the glutamyl-AMP analog glutamol-AMP were effective, with K(i) values in the mum range. The observed inhibition patterns are consistent with a random binding of ATP and L-Glu to the enzyme-tRNA complex. Mt-GluRS, which is predicted by genome analysis to be of the non-discriminating type, was not toxic when overproduced in E. coli cells indicating that it does not catalyse the mischarging of E. coli tRNA(Gln) with L-Glu and that GluRS/tRNA(Gln) recognition is species specific. Mt-GluRS was significantly more sensitive than the E. coli form to tryptic and chymotryptic limited proteolysis. For both enzymes chymotrypsin-sensitive sites were found in the predicted tRNA stem contact domain next to the ATP binding site. Mt-GluRS, but not Ec-GluRS, was fully protected from proteolysis by ATP and glutamol-AMP. Small-angle X-ray scattering showed that, at variance with the E. coli enzyme that is strictly monomeric, the Mt-GluRS monomer is present in solution in equilibrium with the homodimer. The monomer prevails at low protein concentrations and is stabilized by ATP but not by glutamol-AMP. Inspection of small-angle X-ray scattering-based models of Mt-GluRS reveals that both the monomer and the dimer are catalytically active. By using affinity chromatography and His(6)-tagged forms of either GluRS or glutamyl-tRNA reductase as the bait it was shown that the M. tuberculosis proteins can form a complex, which may control the flux of Glu-tRNA(Glu) toward protein or tetrapyrrole biosynthesis.

Intrinsic structural disorder of mouse proNGF.

The unprocessed precursor of the Nerve Growth Factor (NGF), proNGF, has additional functions, besides its initially described role as a chaperone for NGF folding. The precursor protein endows apoptotic and/or neurotrophic properties, in contrast to the mature part. The structural and molecular basis for such distinct activities are presently unknown. Aiming to gain insights into the specific molecular interactions that govern rm-proNGF biological activities versus those of its mature counterpart, a structural study by synchrotron small angle X-ray scattering (SAXS) in solution was carried out. The different binding properties of the two proteins were investigated by surface plasmon resonance (SPR) using, as structural probes, a panel of anti-NGF antibodies and the soluble forms of TrkA and p75(NTR) receptors. SAXS measurements revealed the rm-proNGF to be dimeric and anisometric, with the propeptide domain being intrinsically unstructured. Ab initio reconstructions assuming twofold symmetry generated two types of structural models, a globular "crab-like" and an elongated shape that resulted in equally good fits of the scattering data. A novel method accounting for possible coexistence of different conformations contributing to the experimental scattering pattern, with no symmetry constraints, suggests the "crab-like" to be a more likely proNGF conformation. To exploit the potential of chemical stabilizers affecting the existing conformational protein populations, SAXS data were also collected in the presence of ammonium sulphate. An increase of the proNGF compactness was observed. SPR data pinpoints that the propeptide of proNGF may act as an intrinsically unstructured protein domain, characterized by a molecular promiscuity in the interaction/binding to multiple partners (TrkA and p75(NTR) receptors and a panel of neutralizing anti-NGF antibodies) depending on the physiological conditions of the cell. These data provide a first insight into the structural basis for the selectivity of mouse short proNGF, versus NGF, towards its binding partners.

Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra.

Bark of elderberry (Sambucus nigra) contains a galactose (Gal)/N-acetylgalactosamine (GalNAc)-specific lectin (SNA-II) corresponding to slightly truncated B-chains of a genuine Type-II ribosome-inactivating protein (Type-II RIPs, SNA-V), found in the same species. The three-dimensional X-ray structure of SNA-II has been determined in two distinct crystal forms, hexagonal and tetragonal, at 1.90 A and 1.35 A, respectively. In both crystal forms, the SNA-II molecule folds into two linked beta-trefoil domains, with an overall conformation similar to that of the B-chains of ricin and other Type-II RIPs. Glycosylation is observed at four sites along the polypeptide chain, accounting for 14 saccharide units. The high-resolution structures of SNA-II in complex with Gal and five Gal-related saccharides (GalNAc, lactose, alpha1-methylgalactose, fucose, and the carcinoma-specific Tn antigen) were determined at 1.55 A resolution or better. Binding is observed in two saccharide-binding sites for most of the sugars: a conserved aspartate residue interacts simultaneously with the O3 and O4 atoms of saccharides. In one of the binding sites, additional interactions with the protein involve the O6 atom. Analytical gel filtration, small angle X-ray scattering studies and crystal packing analysis indicate that, although some oligomeric species are present, the monomeric species predominate in solution.

Communication between the nucleotide site and the main molecular hinge of 3-phosphoglycerate kinase.

3-Phosphoglycerate kinase is a hinge-bending enzyme with substrate-assisted domain closure. However, the closure mechanism has not been described in terms of structural details. Here we present experimental evidence of the participation of individual substrate binding side chains in the operation of the main hinge which is distant from the substrate binding sites. The combined mutational, kinetic, and structural (DSC and SAXS) data for human 3-phosphoglycerate kinase have shown that catalytic residue R38, which also binds the substrate 3-phosphoglycerate, is essential in inducing domain closure. Similarly, residues K219, N336, and E343 which interact with the nucleotide substrates are involved in the process of domain closure. The other catalytic residue, K215, covers a large distance during catalysis but has no direct role in domain closure. The transmission path of the nucleotide effect toward the main hinge of PGK is described for the first time at the level of interactions existing in the tertiary structure.

Dynamics in a pure encounter complex of two proteins studied by solution scattering and paramagnetic NMR spectroscopy.

In the general view of protein-complex formation, a transient and dynamic encounter complex proceeds to form a more stable, well-defined, and active form. In weak protein complexes, however, the encounter state can represent a significant population of the complex. The redox proteins adrenodoxin (Adx) and cytochrome c (C c) associate to form such a weak and short-lived complex, which is nevertheless active in electron transfer. To study the conformational freedom within the protein complex, the native complex has been compared to a cross-linked counterpart by using solution scattering and NMR spectroscopy. Oligomerization behavior of the native complex in solution revealed by small-angle X-ray scattering indicates a stochastic nature of complex formation. For the cross-linked complex, interprotein paramagnetic effects are observed, whereas for the native complex, extensive averaging occurs, consistent with multiple orientations of the proteins within the complex. Simulations show that C c samples about half of the surface area of adrenodoxin. It is concluded that the complex of Adx/C c is entirely dynamic and can be considered as a pure encounter complex.

Structural properties of AMP-activated protein kinase: dimerization, molecular shape, and changes upon ligand binding.

Heterotrimeric AMP-activated protein kinase (AMPK) is crucial for energy homeostasis of eukaryotic cells and organisms. Here we report on (i) bacterial expression of untagged mammalian AMPK isoform combinations, all containing gamma(1), (ii) an automated four-dimensional purification protocol, and (iii) biophysical characterization of AMPK heterotrimers by small angle x-ray scattering in solution (SAXS), transmission and scanning transmission electron microscopy (TEM, STEM), and mass spectrometry (MS). AMPK in solution at low concentrations (~1 mg/ml) largely consisted of individual heterotrimers in TEM analysis, revealed a precise 1:1:1 stoichiometry of the three subunits in MS, and behaved as an ideal solution in SAXS. At higher AMPK concentrations, SAXS revealed concentration-dependent, reversible dimerization of AMPK heterotrimers and formation of higher oligomers, also confirmed by STEM mass measurements. Single particle reconstruction and averaging by SAXS and TEM, respectively, revealed similar elongated, flat AMPK particles with protrusions and an indentation. In the lower AMPK concentration range, addition of AMP resulted in a significant decrease of the radius of gyration by approximately 5% in SAXS, which indicates a conformational switch in AMPK induced by ligand binding. We propose a structural model involving a ligand-induced relative movement of the kinase domain resulting in a more compact heterotrimer and a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr(172), thus positively affecting AMPK activity.

Role of side-chains in the operation of the main molecular hinge of 3-phosphoglycerate kinase.

The single mutants (F165A, E192A, F196A, S392A, T393A) at and near the main hinge (beta-strand L) of human 3-phosphoglycerate kinase (hPGK) exhibit variously reduced enzyme activity, indicating the cumulative effects of these residues in regulating domain movements. The residues F165 and E192 are also essential in maintaining the conformational integrity of the whole molecule, including the hinge-region. Shortening of betaL by deleting T393 has led to a dramatic activity loss and the concomitant absence of domain closure (as detected by small angle X-ray scattering), demonstrating the role of betaL in functioning of hPGK. The role of each residue in the conformational transmission is described.

Interaction of human 3-phosphoglycerate kinase with L-ADP, the mirror image of D-ADP.

l-Nucleoside-analogues, mirror images of the natural d-nucleosides, are a new class of antiviral and anticancer agents. In the cell they have to be phosphorylated to pharmacologically active triphosphate forms, the last step seems to involve human 3-phosphoglycerate kinase (hPGK). Here we present a steady state kinetic and biophysical study of the interaction of the model compound l-MgADP with hPGK. l-MgADP is a good substrate with k(cat) and K(m) values of 685s(-1) and 0.27mM, respectively. Double inhibition studies suggest that l-MgADP binds to the specific adenosine-binding site and protects the conformation of hPGK molecule against heat denaturation, as detected by microcalorimetry. Structural details of the interaction in the enzyme active site are different for the d- and l-enantiomers (e.g. the effect of Mg(2+)), but these differences do not prevent the occurrence of the catalytic cycle, which is accompanied by the hinge-bending domain closure, as indicated by SAXS measurements.

Hydrophilic Monodisperse Magnetic Nanoparticles Protected by an Amphiphilic Alternating Copolymer.

Iron oxide nanoparticles (NPs) with diameters of 16.1, 20.5, and 20.8 nm prepared from iron oleate precursors were coated with poly(maleic acid-alt-1-octadecene) (PMAcOD). The coating procedure exploited hydrophobic interactions of octadecene and oleic acid tails while hydrolysis of maleic anhydride moieties allowed the NP hydrophilicity. The PMAcOD nanostructure in water and the PMAcOD-coated NPs were studied using transmission electron microscopy, zeta-potential measurements, small-angle X-ray scattering, and fluorescence measurements. The combination of several techniques suggests that independently of the iron oxide core and oleic acid shell structures, PMAcOD encapsulates NPs, forming stable hydrophilic shells which withstand absorption of hydrophobic molecules, such as pyrene, without shell disintegration. Moreover, the PMAcOD molecules are predominantly attached to a single NP instead of self-assembling into the PMAcOD disklike nanostructures or attachment to several NPs. This leads to highly monodisperse aqueous samples with only a small fraction of NPs forming large aggregates due to cross-linking by the copolymer macromolecules.

Crystal structure and activity of Kunjin virus NS3 helicase; protease and helicase domain assembly in the full length NS3 protein.

Flaviviral NS3 is a multifunctional protein displaying N-terminal protease activity in addition to C-terminal helicase, nucleoside 5'-triphosphatase (NTPase), and 5'-terminal RNA triphosphatase (RTPase) activities. NS3 is held to support the separation of RNA daughter and template strands during viral replication. In addition, NS3 assists the initiation of replication by unwinding the RNA secondary structure in the 3' non-translated region (NTR). We report here the three-dimensional structure (at 3.1 A resolution) of the NS3 helicase domain (residues 186-619; NS3:186-619) from Kunjin virus, an Australian variant of the West Nile virus. As for homologous helicases, NS3:186-619 is composed of three domains, two of which are structurally related and held to host the NTPase and RTPase active sites. The third domain (C-terminal) is involved in RNA binding/recognition. The NS3:186-619 construct occurs as a dimer in solution and in the crystals. We show that NS3:186-619 displays both ATPase and RTPase activities, that it can unwind a double-stranded RNA substrate, being however inactive on a double-stranded DNA substrate. Analysis of different constructs shows that full length NS3 displays increased helicase activity, suggesting that the protease domain plays an assisting role in the RNA unwinding process. The structural interaction between the helicase and protease domain has been assessed using small angle X-ray scattering on full length NS3, disclosing that the protease and helicase domains build a rather elongated molecular assembly differing from that observed in the NS3 protein from hepatitis C virus.

Fine-tuning of intrinsic N-Oct-3 POU domain allostery by regulatory DNA targets.

The 'POU' (acronym of Pit-1, Oct-1, Unc-86) family of transcription factors share a common DNA-binding domain of approximately 160 residues, comprising so-called 'POUs' and 'POUh' sub-domains connected by a flexible linker. The importance of POU proteins as developmental regulators and tumor-promoting agents is due to linker flexibility, which allows them to adapt to a considerable variety of DNA targets. However, because of this flexibility, it has not been possible to determine the Oct-1/Pit-1 linker structure in crystallographic POU/DNA complexes. We have previously shown that the neuronal POU protein N-Oct-3 linker contains a structured region. Here, we have used a combination of hydrodynamic methods, DNA footprinting experiments, molecular modeling and small angle X-ray scattering to (i) structurally interpret the N-Oct-3-binding site within the HLA DRalpha gene promoter and deduce from this a novel POU domain allosteric conformation and (ii) analyze the molecular mechanisms involved in conformational transitions. We conclude that there might exist a continuum running from free to 'pre-bound' N-Oct-3 POU conformations and that regulatory DNA regions likely select pre-existing conformers, in addition to molding the appropriate DBD structure. Finally, we suggest that a specific pair of glycine residues in the linker might act as a major conformational switch.