Solution Small-angle X-ray Research Articles

Structural stability of E. coli trigger factor studied by synchrotron small-angle X-ray scattering

Solution small-angle X-ray scattering (SAXS) is an effective technique for quantitatively measuring the compactness and shape of proteins. We use SAXS to study the structural characteristics and unfolding transitions induced by urea for full length Escherichia coli trigger factor (TF) and a series of truncation mutants, obtaining and comparing the radiuses of gyration (Rg), the distance-distribution function (P(r) function) and integrated intensity of TF variants in native and unfolding states. The C-terminal 72-residue truncated mutant TF360 exhibited dramatic structural differences and reduced stability compared with the whole TF molecule, while the N-domain truncated mutant MC maintained its compact structure with reduced stability. These results indicate that the C-terminal region of TF plays an important role in the structural and conformational stabilities of the TF molecule, while the N-domain is relatively independent.

Neuronal porosome – The secretory portal at the nerve terminal: Its structure–function, composition, and reconstitution

Cup-shaped secretory portals at the cell plasma membrane called porosomes mediate secretion from cells. Membrane bound secretory vesicles transiently dock and fuse at the cytosolic compartment of the porosome base to expel intravesicular contents to the outside during cell secretion. In the past decade, the structure, isolation, composition, and functional reconstitution of the neuronal porosome complex has been accomplished providing a molecular understanding of its structure–function. Neuronal porosomes are 15nm cup-shaped lipoprotein structures composed of nearly 40 proteins; compared to the 120 nm nuclear pore complex comprised of over 500 protein molecules composed of 30 different proteins. Being a membrane-associated supramolecular complex has precluded determination of the atomic structure of the porosome. However recent studies using small-angle X-ray solution scattering (SAXS), provide at sub-nanometer resolution, the native 3D structure of the neuronal porosome complex associated with docked synaptic vesicle at the nerve terminal. Additionally, results from the SAXS study and earlier studies using atomic force microscopy, provide the possible molecular mechanism involved in porosome-mediated neurotransmitter release at the nerve terminal.

High-resolution modeling of protein structures based on flexible fitting of low-resolution structural data.

To circumvent the difficulty of directly solving high-resolution biomolecular structures, low-resolution structural data from Cryo-electron microscopy (EM) and small angle solution X-ray scattering (SAXS) are increasingly used to explore multiple conformational states of biomolecular assemblies. One promising venue to obtain high-resolution structural models from low-resolution data is via data-constrained flexible fitting. To this end, we have developed a new method based on a coarse-grained Cα-only protein representation, and a modified form of the elastic network model (ENM) that allows large-scale conformational changes while maintaining the integrity of local structures including pseudo-bonds and secondary structures. Our method minimizes a pseudo-energy which linearly combines various terms of the modified ENM energy with an EM/SAXS-fitting score and a collision energy that penalizes steric collisions. Unlike some previous flexible fitting efforts using the lowest few normal modes, our method effectively utilizes all normal modes so that both global and local structural changes can be fully modeled with accuracy. This method is also highly efficient in computing time. We have demonstrated our method using adenylate kinase as a test case which undergoes a large open-to-close conformational change. The EM-fitting method is available at a web server (http://enm.lobos.nih.gov), and the SAXS-fitting method is available as a pre-compiled executable upon request.

Molecular structure and dynamics of the dimeric human small heat shock protein HSPB6

ATP-independent small heat-shock proteins (sHSPs) are an essential component of the cellular chaperoning machinery. Under both normal and stress conditions, sHSPs bind partially unfolded proteins and prevent their irreversible aggregation. Canonical vertebrate sHSPs, such as the α-crystallins, form large polydisperse oligomers from which smaller, functionally active subspecies dissociate. Here we focus on human HSPB6 which, despite having considerable homology to the α-crystallins in both the N-terminal region and the signature α-crystallin domain (ACD), only forms dimers in solution that represent the basic chaperoning subspecies. We addressed the three-dimensional structure and functional properties of HSPB6 in a hybrid study employing X-ray crystallography, solution small-angle X-ray scattering (SAXS), mutagenesis, size-exclusion chromatography and chaperoning assays. The crystal structure of a proteolytically stable fragment reveals typical ACD dimers which further form tetrameric assemblies as a result of extensive inter-dimer patching of the β4/β8 grooves. The patching is surprisingly mediated by tripeptide motifs, found in the N-terminal domain directly adjacent to the ACD, that are resembling but distinct from the canonical IxI sequence commonly binding this groove. By combining the crystal structure with SAXS data for the full-length protein, we derive a molecular model of the latter. In solution, HSPB6 shows a strong attractive self-interaction, a property that correlates with its chaperoning activity. Both properties are dictated by the unstructured yet compact N-terminal domain, specifically a region highly conserved across vertebrate sHSPs.

Nucleotide-induced asymmetry within ATPase activator ring drives σ54–RNAP interaction and ATP hydrolysis

It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54-RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase and the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.

Modeling the structure of RNA molecules with small-angle X-ray scattering data.

We propose a novel fragment assembly method for low-resolution modeling of RNA and show how it may be used along with small-angle X-ray solution scattering (SAXS) data to model low-resolution structures of particles having as many as 12 independent secondary structure elements. We assessed this model-building procedure by using both artificial data on a previously proposed benchmark and publicly available data. With the artificial data, SAXS-guided models show better similarity to native structures than ROSETTA decoys. The publicly available data showed that SAXS-guided models can be used to reinterpret RNA structures previously deposited in the Protein Data Bank. Our approach allows for fast and efficient building of de novo models of RNA using approximate secondary structures that can be readily obtained from existing bioinformatic approaches. We also offer a rigorous assessment of the resolving power of SAXS in the case of small RNA structures, along with a small multimetric benchmark of the proposed method.

Solution Small Angle X-ray Scattering (SAXS) Studies of RecQ from Deinococcus radiodurans and Its Complexes with Junction DNA Substrates

RecQ helicases, essential enzymes for maintaining genome integrity, possess the capability to participate in a wide variety of DNA metabolisms. They can initiate the homologous recombination repair pathway by unwinding damaged dsDNA and suppress hyper-recombination by promoting Holliday junction (HJ) migration. To learn how DrRecQ participates in the homologous recombination repair pathway, solution structures of Deinococcus radiodurans RecQ (DrRecQ) and its complexes with DNA substrates were investigated by small angle x-ray scattering. We found that the catalytic core and the most N-terminal HRDC (helicase and RNase D C-terminal) domain (HRDC1) undergo a conformational change to a compact state upon binding to a junction DNA. Furthermore, models of DrRecQ in complexes with two kinds of junction DNA (fork junction and HJ) were built based on the small angle x-ray scattering data, and together with the EMSA results, possible binding sites were proposed. It is demonstrated that two DrRecQ molecules bind to the opposite arms of HJ. This architecture is similar to the RuvAB complex and is hypothesized to be highly conserved in the other HJ migration proteins. This work provides us new clues to understand the roles DrRecQ plays in the RecFOR pathway.

Difference between conformations of immunoglobulin M and human rheumatoid factor based on small-angle X-ray solution scattering data

Radial distribution functions calculated from molecular models point to the difference between conformations of immunoglobulin M and human rheumatoid factor, based on small-angle X-ray solution scattering data.

X-ray solution structure of the native neuronal porosome-synaptic vesicle complex: Implication in neurotransmitter release

Nanoportals at the cell plasma membrane called porosomes, mediate secretion from cells. In neurons porosomes are 15nm cup-shaped lipoprotein structure composed of nearly 40 proteins. The size and complexity of the porosome has precluded determination of its atomic structure. Here we report at nanometer resolution the native 3D structure of the neuronal porosome-synaptic vesicle complex within isolated nerve terminals using small-angle X-ray solution scattering. In addition to furthering our understanding of the porosome structure, results from the study suggests the molecular mechanism involved in neurotransmitter release at the nerve terminal.

Global structures of IgG isotypes expressing identical variable regions

Until relatively recently the immunoglobulin molecule was viewed as composed of two independent domains comprised of the variable (V) and constant (C) regions. However, recent work has established that the C region mediates allosteric changes in the V region that can influence specificity and affinity. To further explore cross-domain interrelationship in murine IgG structure we carried out solution small angle X-ray scattering (SAXS) measurements for four V region identical IgG isotypes. SAXS analysis revealed elongated Y-shaped structures in solution with significantly different, isotype-dependent domain orientations. To further explore local C region effects on the V region, the IgG3 Fab crystal structure from the same family was determined to 2.45Å resolution. The IgG3 Fab crystal structure differs from a closely related previously solved IgG1 Fab revealing significant structural differences, which may account for isotype-related specificity differences in V region identical Abs. Among the four murine isotypes, IgG3 was the most different in solution with regards to overall structure as well as aggregate formation in solution suggesting that the greater apparent affinity of this isotype resulted from polyvalent complexes with enhanced avidity. Our results provide additional evidence that Ig V and C domains influence each other structurally and suggest that V region structure can have significant effects on overall Ig structure.

Small-angle X-ray Solution Scattering Study of the Multi-aminoacyl-tRNA Synthetase Complex Reveals an Elongated and Multi-armed particle

In animal cells, nine aminoacyl-tRNA synthetases are associated with the three auxiliary proteins p18, p38, and p43 to form a stable and conserved large multi-aminoacyl-tRNA synthetase complex (MARS), whose molecular mass has been proposed to be between 1.0 and 1.5 MDa. The complex acts as a molecular hub for coordinating protein synthesis and diverse regulatory signal pathways. Electron microscopy studies defined its low resolution molecular envelope as an overall rather compact, asymmetric triangular shape. Here, we have analyzed the composition and homogeneity of the native mammalian MARS isolated from rabbit liver and characterized its overall internal structure, size, and shape at low resolution by hydrodynamic methods and small-angle x-ray scattering in solution. Our data reveal that the MARS exhibits a much more elongated and multi-armed shape than expected from previous reports. The hydrodynamic and structural features of the MARS are large compared with other supramolecular assemblies involved in translation, including ribosome. The large dimensions and non-compact structural organization of MARS favor a large protein surface accessibility for all its components. This may be essential to allow structural rearrangements between the catalytic and cis-acting tRNA binding domains of the synthetases required for binding the bulky tRNA substrates. This non-compact architecture may also contribute to the spatiotemporal controlled release of some of its components, which participate in non-canonical functions after dissociation from the complex.

Structural Determinants and Mechanism of Mammalian CRM1 Allostery

Proteins carrying nuclear export signals cooperatively assemble with the export factor CRM1 and the effector protein RanGTP. In lower eukaryotes, this cooperativity is coupled to CRM1 conformational changes; however, it is unknown if mammalian CRM1 maintains its compact conformation or shows similar structural flexibility. Here, combinations of small-angle X-ray solution scattering and electron microscopy experiments with molecular dynamics simulations reveal pronounced conformational flexibility in mammalian CRM1 and demonstrate that RanGTP binding induces association of its N- and C-terminal regions to form a toroid structure. The CRM1 toroid is stabilized mainly by local interactions between the terminal regions, rather than by global strain. The CRM1 acidic loop is key in transmitting the effect of this RanGTP-induced global conformational change to the NES-binding cleft by shifting its population to the open state, which displays enhanced cargo affinity. Cooperative CRM1 export complex assembly thus constitutes a highly dynamic process, encompassing an intricate interplay of global and local structural changes.

Structure, Dynamics, and Specificity of Endoglucanase D from Clostridium cellulovorans

The enzymatic degradation of cellulose is a critical step in the biological conversion of plant biomass into an abundant renewable energy source. An understanding of the structural and dynamic features that cellulases utilize to bind a single strand of crystalline cellulose and hydrolyze the β-1,4-glycosidic bonds of cellulose to produce fermentable sugars would greatly facilitate the engineering of improved cellulases for the large-scale conversion of plant biomass. Endoglucanase D (EngD) from Clostridium cellulovorans is a modular enzyme comprising an N-terminal catalytic domain and a C-terminal carbohydrate-binding module, which is attached via a flexible linker. Here, we present the 2.1-Å-resolution crystal structures of full-length EngD with and without cellotriose bound, solution small-angle X-ray scattering (SAXS) studies of the full-length enzyme, the characterization of the active cleft glucose binding subsites, and substrate specificity of EngD on soluble and insoluble polymeric carbohydrates. SAXS data support a model in which the linker is flexible, allowing EngD to adopt an extended conformation in solution. The cellotriose-bound EngD structure revealed an extended active-site cleft that contains seven glucose-binding subsites, but unlike the majority of structurally determined endocellulases, the active-site cleft of EngD is partially enclosed by Trp162 and Tyr232. EngD variants, which lack Trp162, showed a significant reduction in activity and an alteration in the distribution of cellohexaose degradation products, suggesting that Trp162 plays a direct role in substrate binding.

Heptyl α‐D‐Mannosides Grafted on a β‐Cyclodextrin Core To Interfere with Escherichia coli Adhesion: An In Vivo Multivalent Effect

n-Heptyl α-D-mannoside (HM) has previously been identified as a nanomolar FimH antagonist able to prevent Escherichia coli adhesion. We have designed mono- and heptavalent glycoconjugates in which HM is tethered to β-cyclodextrin (β-CD) through short and long spacers. One-pot click or co-clicking procedures were developed to directly obtain the glycoconjugates from unprotected HM and β-CD precursors. These FimH antagonists were examined biophysically and in vivo. Reverse titrations by isothermal calorimetry led to trapping of the short-tethered heptavalent β-CD in a complex with three FimH lectins. Combined dynamic light scattering and small-angle X-ray solution scattering data allowed the construction of a model of the FimH trimer. The heptavalent β-CDs were shown to capture and aggregate living bacteria in solution and are therefore also able to aggregate FimH when attached to different bacteria pili. The first in vivo evaluation of multivalent FimH inhibitors has been performed. The heptavalent β-CDs proved to be much more effective anti-adhesive agents than monovalent references with doses of around 2 μg instilled in the mouse bladder leading to a significantly decreased E. coli load. Intravenously injected radiolabeled glycoconjugates can rapidly reach the mouse bladder and >2 μg concentrations can easily be retained over 24 h to prevent fluxing bacteria from rebinding.

All-Atom Ensemble Modeling to Analyze Small-Angle X-Ray Scattering of Glycosylated Proteins

The flexible and heterogeneous nature of carbohydrate chains often renders glycoproteins refractory to traditional structure determination methods. Small-angle X-ray scattering (SAXS) can be a useful tool for obtaining structural information of these systems. All-atom modeling of glycoproteins with flexible glycan chains was applied to interpret the solution SAXS data for a set of glycoproteins. For simpler systems (single glycan, with a well-defined protein structure), all-atom modeling generates models in excellent agreement with the scattering pattern and reveals the approximate spatial occupancy of the glycan chain in solution. For more complex systems (several glycan chains, or unknown protein substructure), the approach can still provideinsightful models, though the orientations of glycans become poorly determined. Ab initio shape reconstructions appear to capture the global morphology of glycoproteins but in most cases offer littleinformation about glycan spatial occupancy. The all-atom modeling methodology is available as a web server at http://salilab.org/allosmod-foxs.

The Functional Dependence of the Intrinsically Disordered Domains of Tau

Hyperphosphorylated tau is the precursor to neurofibrillary tangles, one of the hallmark symptoms of Alzheimer's disease [1]. In its non-diseased state, the microtubule-associated protein tau promotes the assembly of tubulin into microtubules and regulates axonal transport. However, dysfunction of tau has been implicated in a class of diseases known as tauopathies, of which Alzheimer's is the most pervasive [2]. Characterization of tau has proven to be difficult, due to the presence of intrinsically disordered domains in the N- and C-termini of tau which precludes protein crystallography [3]. Using solution small-angle x-ray scattering, we examined tau-induced bundles of microtubules in cell-free conditions. By this method, we directly measured the inter-microtubule spacing by tau, the magnitude of the attractive force of tau, and the domains of the N- and C- termini that are responsible for the bundling of microtubules.The six isoforms of tau are the result of alternative splicing and are commonly described as sequentially having an N-terminal (consisting of a projection domain and proline-rich region), microtubule-binding repeats, and a C-terminal. To examine the functional dependence of the projection domain, proline-rich region, and C-terminal, distinct tau constructs were expressed with deleted domains. Using the wild-type isoforms, the inter-microtubule spacing was found to be modulated by the length of the projection domain. Surprisingly, the use of the deletion constructs showed that neither the projection-domain or proline-rich region is necessary for bundling, in conflict with existing models [4].Funded by DOE-BES-DOE-DE-FG02-06ER46314 and NSF-DMR-11019001. J.G. Wood et. al. PNAS. 83; 4040 (1986).2. B. Esmaeli-Azad, J.H. McCarty, and S.C. Feinstein. J. Cell Sci. 107; 869 (1994).3. H. Wille, G. Drewes, J. Biernat, E.-M. Mandelkow, and E. Mandelkow. JCB. 188; 573 (1992).4. Y. Kanai et. al. JCB. 109; 1173 (1989).

1SEP-02 X線溶液散乱と分子動力学シミュレーションで探る蛋白質の構造揺らぎ(1SEP 新学術領域「天然変性タンパク質の分子認識機構と機能発現-生理的準安定状態を捉える新技術-」共催,相関構造生物学とX線溶液散乱,シンポジウム,日本生物物理学会第51回年会(2013年度))

1SEP-02 X線溶液散乱と分子動力学シミュレーションで探る蛋白質の構造揺らぎ(1SEP 新学術領域「天然変性タンパク質の分子認識機構と機能発現-生理的準安定状態を捉える新技術-」共催,相関構造生物学とX線溶液散乱,シンポジウム,日本生物物理学会第51回年会(2013年度))

X線溶液散乱と分子動力学シミュレーションで探るタンパク質の構造揺らぎ

The combination of small-angle X-ray solution scattering (SAXS) experiments and molecular dynamics (MD) simulations is now becoming a powerful tool to study protein dynamics in solution at an atomic resolution. We have developed a method called MD-SAXS, in which this combination is used with explicit evaluation of X-ray scattering from water molecules hydrating protein molecules. This method offers a link between low-resolution structural information from SAXS and the three-dimensional high-resolution structure. The study using MD-SAXS method revealed the importance of intrinsic dynamics of DNA-binding protein EcoO109I in its function.

MD–SAXS method with nonspherical boundaries

Small-angle X-ray solution scattering (SAXS) experiments provide information about protein structures in solution, albeit at low resolution. The molecular dynamics (MD)–SAXS method is capable of linking SAXS data and high-resolution atomic structures of proteins including hydrating water molecules. The MD–SAXS method, which was previously limited to spherical boundary conditions, is here extended to nonspherical boundary conditions while retaining accuracy. The method presented here is computationally effective, particularly for elongated proteins, because a number of water molecules can be eliminated compared to the original formulation.

Solution Small-Angle X-ray Scattering as a Screening and Predictive Tool in the Fabrication of Asymmetric Block Copolymer Membranes.

Small-angle X-ray scattering (SAXS) analysis of the diblock copolymer poly(styrene-b-(4-vinyl)pyridine) in a ternary solvent system of 1,4-dioxane, tetrahydrofuran, and N,N-dimethylformamide, and the triblock terpolymer poly(isoprene-b-styrene-b-(4-vinyl)pyridine) in a binary solvent system of 1,4-dioxane and tetrahydrofuran, reveals a concentration-dependent onset of ordered structure formation. Asymmetric membranes fabricated from casting solutions with polymer concentrations at or slightly below this ordering concentration possess selective layers with the desired nanostructure. In addition to rapidly screening possible polymer solution concentrations, solution SAXS analysis also predicts hexagonal and square pore lattices of the final membrane surface structure. These results suggest solution SAXS as a powerful tool for screening casting solution concentrations and predicting surface structure in the fabrication of asymmetric ultrafiltration membranes from self-assembled block copolymers.