Flexibility of KorA, a plasmid-encoded, global transcription regulator, in the presence and the absence of its operator.

The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53.

Different Myosin Head Conformations in Bony Fish Muscles Put into Rigor at Different Sarcomere Lengths.

At a resting sarcomere length of approximately 2.2 µm bony fish muscles put into rigor in the presence of BDM (2,3-butanedione monoxime) to reduce rigor tension generation show the normal arrangement of myosin head interactions with actin filaments as monitored by low-angle X-ray diffraction. However, if the muscles are put into rigor using the same protocol but stretched to 2.5 µm sarcomere length, a markedly different structure is observed. The X-ray diffraction pattern is not just a weaker version of the pattern at full overlap, as might be expected, but it is quite different. It is compatible with the actin-attached myosin heads being in a different conformation on actin, with the average centre of cross-bridge mass at a higher radius than in normal rigor and the myosin lever arms conforming less to the actin filament geometry, probably pointing back to their origins on their parent myosin filaments. The possible nature of this new rigor cross-bridge conformation is discussed in terms of other well-known states such as the weak binding state and the 'roll and lock' mechanism; we speculate that we may have trapped most myosin heads in an early attached strong actin-binding state in the cross-bridge cycle on actin.

P13, the EMBL macromolecular crystallography beamline at the low-emittance PETRA III ring for high- and low-energy phasing with variable beam focusing.

The macromolecular crystallography P13 beamline is part of the European Molecular Biology Laboratory Integrated Facility for Structural Biology at PETRA III (DESY, Hamburg, Germany) and has been in user operation since mid-2013. P13 is tunable across the energy range from 4 to 17.5 keV to support crystallographic data acquisition exploiting a wide range of elemental absorption edges for experimental phase determination. An adaptive Kirkpatrick-Baez focusing system provides an X-ray beam with a high photon flux and tunable focus size to adapt to diverse experimental situations. Data collections at energies as low as 4 keV (λ = 3.1 Å) are possible due to a beamline design minimizing background and maximizing photon flux particularly at low energy (up to 1011 photons s-1 at 4 keV), a custom calibration of the PILATUS 6M-F detector for use at low energies, and the availability of a helium path. At high energies, the high photon flux (5.4 × 1011 photons s-1 at 17.5 keV) combined with a large area detector mounted on a 2θ arm allows data collection to sub-atomic resolution (0.55 Å). A peak flux of about 8.0 × 1012 photons s-1 is reached at 11 keV. Automated sample mounting is available by means of the robotic sample changer `MARVIN' with a dewar capacity of 160 samples. In close proximity to the beamline, laboratories have been set up for sample preparation and characterization; a laboratory specifically equipped for on-site heavy atom derivatization with a library of more than 150 compounds is available to beamline users.

Fusion of Legionella pneumophila outer membrane vesicles with eukaryotic membrane systems is a mechanism to deliver pathogen factors to host cell membranes.

The formation and release of outer membrane vesicles (OMVs) is a phenomenon observed in many bacteria, including Legionella pneumophila. During infection, this human pathogen primarily invades alveolar macrophages and replicates within a unique membrane-bound compartment termed Legionella-containing vacuole. In the current study, we analysed the membrane architecture of L. pneumophila OMVs by small-angle X-ray scattering and biophysically characterized OMV membranes. We investigated the interaction of L. pneumophila OMVs with model membranes by Förster resonance energy transfer and Fourier transform infrared spectroscopy. These experiments demonstrated the incorporation of OMV membrane material into liposomes composed of different eukaryotic phospholipids, revealing an endogenous property of OMVs to fuse with eukaryotic membranes. Cellular co-incubation experiments showed a dose- and time-dependent binding of fluorophore-labelled OMVs to macrophages. Trypan blue quenching experiments disclosed a rapid internalization of OMVs into macrophages at 37 and 4 °C. Purified OMVs induced tumour necrosis factor-α production in human macrophages at concentrations starting at 300 ng ml(-1). Experiments on HEK293-TLR2 and TLR4/MD-2 cell lines demonstrated a dominance of TLR2-dependent signalling pathways. In summary, we demonstrate binding, internalization and biological activity of L. pneumophila OMVs on human macrophages. Our data support OMV membrane fusion as a mechanism for the remote delivery of virulence factors to host cells.

BioSAXS Sample Changer: a robotic sample changer for rapid and reliable high-throughput X-ray solution scattering experiments.

Small-angle X-ray scattering (SAXS) of macromolecules in solution is in increasing demand by an ever more diverse research community, both academic and industrial. To better serve user needs, and to allow automated and high-throughput operation, a sample changer (BioSAXS Sample Changer) that is able to perform unattended measurements of up to several hundred samples per day has been developed. The Sample Changer is able to handle and expose sample volumes of down to 5 µl with a measurement/cleaning cycle of under 1 min. The samples are stored in standard 96-well plates and the data are collected in a vacuum-mounted capillary with automated positioning of the solution in the X-ray beam. Fast and efficient capillary cleaning avoids cross-contamination and ensures reproducibility of the measurements. Independent temperature control for the well storage and for the measurement capillary allows the samples to be kept cool while still collecting data at physiological temperatures. The Sample Changer has been installed at three major third-generation synchrotrons: on the BM29 beamline at the European Synchrotron Radiation Facility (ESRF), the P12 beamline at the PETRA-III synchrotron (EMBL@PETRA-III) and the I22/B21 beamlines at Diamond Light Source, with the latter being the first commercial unit supplied by Bruker ASC.

Tropoelastin bridge region positions the cell-interactive C terminus and contributes to elastic fiber assembly.

The tropoelastin monomer undergoes stages of association by coacervation, deposition onto microfibrils, and cross-linking to form elastic fibers. Tropoelastin consists of an elastic N-terminal coil region and a cell-interactive C-terminal foot region linked together by a highly exposed bridge region. The bridge region is conveniently positioned to modulate elastic fiber assembly through association by coacervation and its proximity to dominant cross-linking domains. Tropoelastin constructs that either modify or remove the entire bridge and downstream regions were assessed for elastogenesis. These constructs focused on a single alanine substitution (R515A) and a truncation (M155n) at the highly conserved arginine 515 site that borders the bridge. Each form displayed less efficient coacervation, impaired hydrogel formation, and decreased dermal fibroblast attachment compared to wild-type tropoelastin. The R515A mutant protein additionally showed reduced elastic fiber formation upon addition to human retinal pigmented epithelium cells and dermal fibroblasts. The small-angle X-ray scattering nanostructure of the R515A mutant protein revealed greater conformational flexibility around the bridge and C-terminal regions. This increased flexibility of the R515A mutant suggests that the tropoelastin R515 residue stabilizes the structure of the bridge region, which is critical for elastic fiber assembly.

The TR-icOS setup at the ESRF: time-resolved microsecond UV-Vis absorption spectroscopy on protein crystals.

The technique of time-resolved macromolecular crystallography (TR-MX) has recently been rejuvenated at synchrotrons, resulting in the design of dedicated beamlines. Using pump-probe schemes, this should make the mechanistic study of photoactive proteins and other suitable systems possible with time resolutions down to microseconds. In order to identify relevant time delays, time-resolved spectroscopic experiments directly performed on protein crystals are often desirable. To this end, an instrument has been built at the icOS Lab (in crystallo Optical Spectroscopy Laboratory) at the European Synchrotron Radiation Facility using reflective focusing objectives with a tuneable nanosecond laser as a pump and a microsecond xenon flash lamp as a probe, called the TR-icOS (time-resolved icOS) setup. Using this instrument, pump-probe spectra can rapidly be recorded from single crystals with time delays ranging from a few microseconds to seconds and beyond. This can be repeated at various laser pulse energies to track the potential presence of artefacts arising from two-photon absorption, which amounts to a power titration of a photoreaction. This approach has been applied to monitor the rise and decay of the M state in the photocycle of crystallized bacteriorhodopsin and showed that the photocycle is increasingly altered with laser pulses of peak fluence greater than 100 mJ cm-2, providing experimental laser and delay parameters for a successful TR-MX experiment.

Variations of subunit {varepsilon} of the Mycobacterium tuberculosis F1Fo ATP synthase and a novel model for mechanism of action of the tuberculosis drug TMC207.

The subunit ε of bacterial F(1)F(O) ATP synthases plays an important regulatory role in coupling and catalysis via conformational transitions of its C-terminal domain. Here we present the first low-resolution solution structure of ε of Mycobacterium tuberculosis (Mtε) F(1)F(O) ATP synthase and the nuclear magnetic resonance (NMR) structure of its C-terminal segment (Mtε(103-120)). Mtε is significantly shorter (61.6 Å) than forms of the subunit in other bacteria, reflecting a shorter C-terminal sequence, proposed to be important in coupling processes via the catalytic ß subunit. The C-terminal segment displays an α-helical structure and a highly positive surface charge due to the presence of arginine residues. Using NMR spectroscopy, fluorescence spectroscopy, and mutagenesis, we demonstrate that the new tuberculosis (TB) drug candidate TMC207, proposed to bind to the proton translocating c-ring, also binds to Mtε. A model for the interaction of TMC207 with both ε and the c-ring is presented, suggesting that TMC207 forms a wedge between the two rotating subunits by interacting with the residues W15 and F50 of ε and the c-ring, respectively. T19 and R37 of ε provide the necessary polar interactions with the drug molecule. This new model of the mechanism of TMC207 provides the basis for the design of new drugs targeting the F(1)F(O) ATP synthase in M. tuberculosis.

Recombinant production of Yersinia enterocolitica pyruvate kinase isoenzymes PykA and PykF.

The glycolytic enzyme pyruvate kinase (PK) generates ATP from ADP through substrate-level phosphorylation powered by the conversion of phosphoenolpyruvate to pyruvate. In contrast to other bacteria, Enterobacteriaceae, such as pathogenic yersiniae, harbour two pyruvate kinases encoded by pykA and pykF. The individual roles of these isoenzymes are poorly understood. In an attempt to make the Yersinia enterocolitica pyruvate kinases PykA and PykF amenable to structural and functional characterisation, we produced them untagged in Escherichia coli and purified them to near homogeneity through a combination of ion exchange and size exclusion chromatography, yielding more than 180 mg per litre of batch culture. The solution structure of PykA and PykF was analysed through small angle X-ray scattering which revealed the formation of PykA and PykF tetramers and confirmed the binding of the allosteric effector fructose-1,6-bisphosphate (FBP) to PykF but not to PykA.

Ligand binding induces an ammonia channel in 2-amino-2-desoxyisochorismate (ADIC) synthase PhzE.

PhzE utilizes chorismate and glutamine to synthesize 2-amino-2-desoxyisochorismate (ADIC) in the first step of phenazine biosynthesis. The PhzE monomer contains both a chorismate-converting menaquinone, siderophore, tryptophan biosynthesis (MST) and a type 1 glutamine amidotransferase (GATase1) domain connected by a 45-residue linker. We present here the crystal structure of PhzE from Burkholderia lata 383 in a ligand-free open and ligand-bound closed conformation at 2.9 and 2.1 Å resolution, respectively. PhzE arranges in an intertwined dimer such that the GATase1 domain of one chain provides NH(3) to the MST domain of the other. This quaternary structure was confirmed by small angle x-ray scattering. Binding of chorismic acid, which was found converted to benzoate and pyruvate in the MST active centers of the closed form, leads to structural rearrangements that establish an ammonia transport channel approximately 25 Å in length within each of the two MST/GATase1 functional units of the dimer. The assignment of PhzE as an ADIC synthase was confirmed by mass spectrometric analysis of the product, which was also visualized at 1.9 Å resolution by trapping in crystals of an inactive mutant of PhzD, an isochorismatase that catalyzes the subsequent step in phenazine biosynthesis. Unlike in some of the related anthranilate synthases, no allosteric inhibition was observed in PhzE. This can be attributed to a tryptophan residue of the protein blocking the potential regulatory site. Additional electron density in the GATase1 active center was identified as zinc, and it was demonstrated that Zn(2+), Mn(2+), and Ni(2+) reduce the activity of PhzE.

Solution structure of subunit F (Vma7p) of the eukaryotic V(1)V(O) ATPase from Saccharomyces cerevisiae derived from SAXS and NMR spectroscopy.

Vacuolar ATPases use the energy derived from ATP hydrolysis, catalyzed in the A(3)B(3) sector of the V(1) ATPase to pump protons via the membrane-embedded V(O) sector. The energy coupling between the two sectors occurs via the so-called central stalk, to which subunit F does belong. Here we present the first low resolution structure of recombinant subunit F (Vma7p) of a eukaryotic V-ATPase from Saccharomyces cerevisiae, analyzed by small angle X-ray scattering (SAXS). The protein is divided into a 5.5nm long egg-like shaped region, connected via a 1.5nm linker to a hook-like segment at one end. Circular dichroism spectroscopy revealed that subunit F comprises of 43% α-helix, 32% ß-sheet and a 25% random coil arrangement. To determine the localization of the N- and C-termini in the protein, the C-terminal truncated form of F, F(1-94) was produced and analyzed by SAXS. Comparison of the F(1-94) shape with the one of subunit F showed the missing hook-like region in F(1-94), supported by the decreased D(max) value of F(1-94) (7.0nm), and indicating that the hook-like region consists of the C-terminal residues. The NMR solution structure of the C-terminal peptide, F(90-116), was solved, displaying an α-helical region between residues 103 and 113. The F(90-116) solution structure fitted well in the hook-like region of subunit F. Finally, the arrangement of subunit F within the V(1) ATPase is discussed.

Solution structure of subunit a, a104₋363, of the Saccharomyces cerevisiae V-ATPase and the importance of its C-terminus in structure formation.

The 95 kDa subunit a of eukaryotic V-ATPases consists of a C-terminal, ion-translocating part and an N-terminal cytosolic domain. The latter's N-terminal domain (~40 kDa) is described to bind in an acidification-dependent manner with cytohesin-2 (ARNO), giving the V-ATPase the putative function as pH-sensing receptor. Recently, the solution structure of the very N-terminal segment of the cytosolic N-terminal domain has been solved. Here we produced the N-terminal truncated form SCa104₋363 of the N-terminal domain (SCa1₋363) of the Saccharomyces cerevisiae V-ATPase and determined its low resolution solution structure, derived from SAXS data. SCa104₋363 shows an extended S-like conformation with a width of about 3.88 nm and a length of 11.4 nm. The structure has been superimposed into the 3D reconstruction of the related A1A0 ATP synthase from Pyrococcus furiosus, revealing that the SCa104₋363 fits well into the density of the collar structure of the enzyme complex. To understand the importance of the C-terminus of the protein SCa1₋363, and to determine the localization of the N- and C-termini in SCa104₋363, the C-terminal truncated form SCa106₋324 was produced and analyzed by SAXS. Comparison of the SCa104₋363 and SCa106₋324 shapes showed that the additional loop region in SCa104₋363 consists of the C-terminal residues. Whereas SCa104₋363 is monomeric in solution, SCa106₋324 forms a dimer, indicating the importance of the very C-terminus in structure formation. Finally, the solution structure of SCa104₋363 and SCa106₋324 will be discussed in terms of the topological arrangement of subunit a and cytoheisn-2 in V-ATPases.

Role of dimerization and substrate exclusion in the regulation of bone morphogenetic protein-1 and mammalian tolloid.

The bone morphogenetic protein (BMP)-1/tolloid metalloproteinases are evolutionarily conserved enzymes that are fundamental to dorsal-ventral patterning and tissue morphogenesis. The lack of knowledge regarding how these proteinases recognize and cleave their substrates represents a major hurdle to understanding tissue assembly and embryonic patterning. Although BMP-1 and mammalian tolloid (mTLD) are splice variants, it is puzzling why BMP-1, which lacks 3 of the 7 noncatalytic domains present in all other family members, is the most effective proteinase. Using a combination of single-particle electron microscopy, small-angle X-ray scattering, and other biophysical measurements in solution, we show that mTLD, but not BMP-1, forms a calcium-ion-dependent dimer under physiological conditions. Using a domain deletion approach, we provide evidence that EGF2, which is absent in BMP-1, is critical to the formation of the dimer. Based on a combination of structural and functional data, we propose that mTLD activity is regulated by a substrate exclusion mechanism. These results provide a mechanistic insight into how alternative splicing of the Bmp1 gene produces 2 proteinases with differing biological activities and have broad implications for regulation of BMP-1/mTLD and related proteinases during BMP signaling and tissue assembly.

Structural insight into the glycosylphosphatidylinositol transamidase subunits PIG-K and PIG-S from yeast.

The addition of glycosylphosphatidylinositol (GPI) anchors to eukaryotic proteins in the lumen of the endoplasmic reticulum is catalyzed by the transamidase complex, composed of at least five subunits (PIG-K, PIG-S, PIG-T, PIG-U and GPAA1). Here PIG-K(24-337) and PIG-S(38-467) from yeast, including the residues 24-337 and 38-467 of the entire 411 and 534 residue protein, respectively, was produced in Escherichia coli and purified to homogeneity. Analysis of secondary structure by circular dichroism spectroscopy showed that yPIG-K(24-377) comprises 52% α-helix and 12% ß-sheet, whereas yPIG-S(38-467) involves 58% α-helix and 18% ß-sheet. The radius of gyration (R(g)) and the maximum size (D(max)) of both proteins have been analyzed by small angle X-ray scattering (SAXS) and determined to be 2.64±0.3 and 10.3±0.1 nm (yPIG-K(24-377)) as well as 3.06±0.02 nm (R(g)) and 16.9±0.4 nm (D(max)) in the case of yPIG-S(38-467), respectively. Using an ab initio approach, the first low-resolution solution structures of both proteins were restored. yPIG-K(24-377) is an elongated particle consisting of an egg-like portion and a small globular segment linked together by an 1.9 nm long stalk. yPIG-S(38-467) forms an elongated molecule in solution with a larger domain of 10.1 nm in length, a diameter of 9.1 nm and a smaller domain of 6.7 nm in length and 3.4 nm in width. The two domains of yPIG-S(38-467) are tilted relative to each other. Finally, the arrangements of PIG-K and PIG-S inside the ensemble of the transamidase complex are discussed.

Probing the existence of non-thermal Terahertz radiation induced changes of the protein solution structure.

During the last decades discussions were taking place on the existence of global, non-thermal structural changes in biological macromolecules induced by Terahertz (THz) radiation. Despite numerous studies, a clear experimental proof of this effect for biological particles in solution is still missing. We developed a setup combining THz-irradiation with small angle X-ray scattering (SAXS), which is a sensitive method for detecting the expected structural changes. We investigated in detail protein systems with different shape morphologies (bovine serum albumin, microtubules), which have been proposed to be susceptible to THz-radiation, under variable parameters (THz wavelength, THz power densities up to 6.8 mW/cm2, protein concentrations). None of the studied systems and conditions revealed structural changes detectable by SAXS suggesting that the expected non-thermal THz-induced effects do not lead to alterations of the overall structures, which are revealed by scattering from dissolved macromolecules. This leaves us with the conclusion that, if such effects are present, these are either local or outside of the spectrum and power range covered by the present study.

The effect of NBD-Cl in nucleotide-binding of the major subunit alpha and B of the motor proteins F1FO ATP synthase and A1AO ATP synthase.

Subunit alpha of the Escherichia coli F(1)F(O) ATP synthase has been produced, and its low-resolution structure has been determined. The monodispersity of alpha allowed the studies of nucleotide-binding and inhibitory effect of 4-Chloro-7-nitrobenzofurazan (NBD-Cl) to ATP/ADP-binding. Binding constants (K ( d )) of 1.6 microM of bound MgATP-ATTO-647N and 2.9 microM of MgADP-ATTO-647N have been determined from fluorescence correlation spectroscopy data. A concentration of 51 microM and 55 microM of NBD-Cl dropped the MgATP-ATTO-647N and MgADP-ATTO-647N binding capacity to 50% (IC(50)), respectively. In contrast, no effect was observed in the presence of N,N'-dicyclohexylcarbodiimide. As subunit alpha is the homologue of subunit B of the A(1)A(O) ATP synthase, the interaction of NBD-Cl with B of the A-ATP synthase from Methanosarcina mazei Gö1 has also been shown. The data reveal a reduction of nucleotide-binding of B due to NBD-Cl, resulting in IC(50) values of 41 microM and 42 microM for MgATP-ATTO-647N and MgADP-ATTO-647N, respectively.

A helical structural nucleus is the primary elongating unit of insulin amyloid fibrils.

Although amyloid fibrillation is generally believed to be a nucleation-dependent process, the nuclei are largely structurally uncharacterized. This is in part due to the inherent experimental challenge associated with structural descriptions of individual components in a dynamic multi-component equilibrium. There are indications that oligomeric aggregated precursors of fibrillation, and not mature fibrils, are the main cause of cytotoxicity in amyloid disease. This further emphasizes the importance of characterizing early fibrillation events. Here we present a kinetic x-ray solution scattering study of insulin fibrillation, revealing three major components: insulin monomers, mature fibrils, and an oligomeric species. Low-resolution three-dimensional structures are determined for the fibril repeating unit and for the oligomer, the latter being a helical unit composed of five to six insulin monomers. This helical oligomer is likely to be a structural nucleus, which accumulates above the supercritical concentration used in our experiments. The growth rate of the fibrils is proportional to the amount of the helical oligomer present in solution, suggesting that these oligomers elongate the fibrils. Hence, the structural nucleus and elongating unit in insulin amyloid fibrillation may be the same structural component above supercritical concentrations. A novel elongation pathway of insulin amyloid fibrils is proposed, based on the shape and size of the fibrillation precursor. The distinct helical oligomer described in this study defines a conceptually new basis of structure-based drug design against amyloid diseases.

Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation.

Bacteria have evolved elaborate communication strategies to co-ordinate their group activities, a process termed quorum sensing (QS). Pseudomonas aeruginosa is an opportunistic pathogen that utilizes QS for diverse activities, including disease pathogenesis. P. aeruginosa has evolved a novel communication system in which the signal molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal, PQS) is trafficked between cells via membrane vesicles (MVs). Not only is PQS packaged into MVs, it is required for MV formation. Although MVs are involved in important biological processes aside from signalling, the molecular mechanism of MV formation is unknown. To provide insight into the molecular mechanism of MV formation, we examined the interaction of PQS with bacterial lipids. Here, we show that PQS interacts strongly with the acyl chains and 4'-phosphate of bacterial lipopolysaccharide (LPS). Using PQS derivatives, we demonstrate that the alkyl side-chain and third position hydroxyl of PQS are critical for these interactions. Finally, we show that PQS stimulated purified LPS to form liposome-like structures. These studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non-signalling functions.

Metal-mediated self-assembly of a beta-sandwich protein.

The beta-sandwich cupredoxin Plastocyanin (Pc) was found to self-assemble in the presence of Zn(2+), a known mediator of protein-protein interfaces. Diffraction-quality crystals of Pc grew from solutions containing zinc acetate as the sole precipitant. Di- and trinuclear zinc sites contribute to the crystal contacts in this structure. A different crystal form, also involving numerous zinc bridging ions, was obtained in the presence of poly(ethylene glycol) 8 000. Comparison of the two crystal forms reveals the effect of macromolecular crowding on self-assembly. Solution-state structural characterisation of the Zn(2+)-mediated Pc oligomers was performed by using a combination of chemical shift perturbation mapping and small-angle X-ray scattering. The data indicate the formation of dimers in solution. The implications for metal-mediated assembly and crystallisation are discussed.

Structural features of the full-length adaptor protein GADS in solution determined using small-angle X-ray scattering.

The Grb2-related adaptor protein GADS plays a central role during the initial phases of signal transduction in T lymphocytes. GADS possesses N- and C-terminal Src homology 3 (SH3) domains flanking a central Src homology 2 (SH2) domain and a 126-residue region rich in glutamine and proline residues, presumed to be largely unstructured. The SH2 domain of GADS binds the adaptor protein LAT; the C-terminal SH3 domain pairs GADS to the adaptor protein SLP-76, whereas the function of the central region is unknown. High-resolution three-dimensional models are available for the isolated SH2 and C-terminal SH3 domains in complex with their respective binding partners, LAT and SLP-76. However, in part because of its intrinsic instability, there is no structural information for the entire GADS molecule. Here, we report the low-resolution structure of full-length GADS in solution using small-angle x-ray scattering (SAXS). Based on the SAXS data, complemented by gel filtration experiments, we show that full-length GADS is monomeric in solution and that its overall structural parameters are smaller than those expected for a protein with a long unstructured region. Ab initio and rigid body modeling of the SAXS data reveal that full-length GADS is a relatively compact molecule and that the potentially unstructured region retains a significant degree of structural order. The biological function of GADS is discussed based on its overall structure.