Structural investigations of E. Coli dihydrolipoamide dehydrogenase in solution: Small-angle X-ray scattering and molecular docking

Dihydrolipoamide dehydrogenase from Escherichia coli (LpD) is a bacterial enzyme that is involved in the central metabolism and shared in common between the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes. In the crystal structure, E. coli LpD is known to exist as a dimer. The present work is focused on analyzing the solution structure of LpD by small-angle X-ray scattering, molecular docking, and analytical ultracentrifugation. It was shown that in solution LpD exists as an equilibrium mixture of a dimer and a tetramer. The presence of oligomeric forms is determined by the multifunctionality of LpD in the cell, in particular, the required stoichiometry in the complexes

BILMIX : a new approach to restore the size polydispersity and electron density profiles of lipid bilayers from liposomes using small-angle X-ray scattering data

Small-angle X-ray scattering (SAXS) is one of the major tools for the study of model membranes, but interpretation of the scattering data remains non-trivial. Current approaches allow the extraction of some structural parameters and the electron density profile of lipid bilayers. Here it is demonstrated that parametric modelling can be employed to determine the polydispersity of spherical or ellipsoidal vesicles and describe the electron density profile across the lipid bilayer. This approach is implemented in the computer program BILMIX. BILMIX delivers a description of the electron density of a lipid bilayer from SAXS data and simultaneously generates the corresponding size distribution of the unilamellar lipid vesicles

Spatial organization of Dps and DNA–Dps complexes

DNA co-crystallization with Dps family proteins is a fundamental mechanism, which preserves DNA in bacteria from harsh conditions. Though many aspects of this phenomenon are well characterized, the spatial organization of DNA in DNA–Dps co-crystals is not completely understood, and existing models need further clarification. To advance in this problem we have utilized atomic force microscopy (AFM) as the main structural tool, and small-angle X-scattering (SAXS) to characterize Dps as a key component of the DNA-protein complex. SAXS analysis in the presence of EDTA indicates a significantly larger radius of gyration for Dps than would be expected for the core of the dodecamer, consistent with the N-terminal regions extending out into solution and being accessible for interaction with DNA. In AFM experiments, both Dps protein molecules and DNA–Dps complexes adsorbed on mica or highly oriented pyrolytic graphite (HOPG) surfaces form densely packed hexagonal structures with a characteristic size of about 9 nm. To shed light on the peculiarities of DNA interaction with Dps molecules, we have characterized individual DNA–Dps complexes. Contour length evaluation has confirmed the non-specific character of Dps binding with DNA and revealed that DNA does not wrap Dps molecules in DNA–Dps complexes. Angle analysis has demonstrated that in DNA–Dps complexes a Dps molecule contacts with a DNA segment of ~6 nm in length. Consideration of DNA condensation upon complex formation with small Dps quasi-crystals indicates that DNA may be arranged along the rows of ordered protein molecules on a Dps sheet

X-Ray Solution Scattering Study of Four Escherichia coli Enzymes Involved in Stationary-Phase Metabolism

The structural analyses of four metabolic enzymes that maintain and regulate the stationary growth phase of Escherichia coli have been performed primarily drawing on the results obtained from solution small angle X-ray scattering (SAXS) and other structural techniques. The proteins are (i) class I fructose-1,6-bisphosphate aldolase (FbaB); (ii) inorganic pyrophosphatase (PPase); (iii) 5-keto-4-deoxyuronate isomerase (KduI); and (iv) glutamate decarboxylase (GadA). The enzyme FbaB, that until now had an unknown structure, is predicted to fold into a TIM-barrel motif that form globular protomers which SAXS experiments show associate into decameric assemblies. In agreement with previously reported crystal structures, PPase forms hexamers in solution that are similar to the previously reported X-ray crystal structure. Both KduI and GadA that are responsible for carbohydrate (pectin) metabolism and acid stress responses, respectively, form polydisperse mixtures consisting of different oligomeric states. Overall the SAXS experiments yield additional insights into shape and organization of these metabolic enzymes and further demonstrate the utility of hybrid methods, i.e., solution SAXS combined with X-ray crystallography, bioinformatics and predictive 3D-structural modeling, as tools to enrich structural studies. The results highlight the structural complexity that the protein components of metabolic networks may adopt which cannot be fully captured using individual structural biology techniques

The dimeric ectodomain of the alkali-sensing insulin receptor–related receptor (ectoIRR) has a droplike shape

Insulin receptor–related receptor (IRR) is a receptor tyrosine kinase of the insulin receptor family and functions as an extracellular alkali sensor that controls metabolic alkalosis in the regulation of the acid–base balance. In the present work, we sought to analyze structural features of IRR by comparing them with those of the insulin receptor, which is its closest homolog but does not respond to pH changes. Using small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM), we investigated the overall conformation of the recombinant soluble IRR ectodomain (ectoIRR) at neutral and alkaline pH. In contrast to the well-known inverted U-shaped (or λ-shaped) conformation of the insulin receptor, the structural models reconstructed at different pH values revealed that the ectoIRR organization has a “droplike” shape with a shorter distance between the fibronectin domains of the disulfide-linked dimer subunits within ectoIRR. We detected no large-scale pH-dependent conformational changes of ectoIRR in both SAXS and AFM experiments, an observation that agreed well with previous biochemical and functional analyses of IRR. Our findings indicate that ectoIRR's sensing of alkaline conditions involves additional molecular mechanisms, for example engagement of receptor juxtamembrane regions or the surrounding lipid environment

Influenza virus Matrix Protein M1 preserves its conformation with pH, changing multimerization state at the priming stage due to electrostatics

Influenza A virus matrix protein M1 plays an essential role in the virus lifecycle, but its functional and structural properties are not entirely defined. Here we employed small-angle X-ray scattering, atomic force microscopy and zeta-potential measurements to characterize the overall structure and association behavior of the full-length M1 at different pH conditions. We demonstrate that the protein consists of a globular N-terminal domain and a flexible C-terminal extension. The globular N-terminal domain of M1 monomers appears preserved in the range of pH from 4.0 to 6.8, while the C-terminal domain remains flexible and the tendency to form multimers changes dramatically. We found that the protein multimerization process is reversible, whereby the binding between M1 molecules starts to break around pH 6. A predicted electrostatic model of M1 self-assembly at different pH revealed a good agreement with zeta-potential measurements, allowing one to assess the role of M1 domains in M1-M1 and M1-lipid interactions. Together with the protein sequence analysis, these results provide insights into the mechanism of M1 scaffold formation and the major role of the flexible and disordered C-terminal domain in this process

Superposition of the active site residues of Class I Fba.

<p>The predicted active site topology of the <i>E</i>. <i>coli</i> enzyme (derived from I-Tasser, Model 1) is shown in dark green on both figure panels. <b>A.</b> Spatial alignment of <i>E</i>. <i>coli</i> FbaB and the X-ray crystal structure of FbaB from <i>Thermoproteus tenax</i> (PDB: 1W8R [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156105#pone.0156105.ref052" target="_blank">52</a>]). <b>B.</b> The alignment between <i>E</i>. <i>coli</i> FbaB relative to <i>Oryctolagus cuniculus</i> FbAB (PDB: 1ZAI [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156105#pone.0156105.ref009" target="_blank">9</a>]). The substrate fructose-1,6-bisphosphate (FBP), taken from the 1W8R crystal structure, is represented as a ball-and-stick in panel A. The amino acid numbers of the corresponding proteins are given in black for the X-ray crystal structures and in green for the I-Tasser <i>E</i>.<i>coli</i> model.</p