Michel H. J. Koch

Determination of Domain Structure of Proteins from X-Ray Solution Scattering

An ab initio method for building structural models of proteins from x-ray solution scattering data is presented. Simulated annealing is employed to find a chain-compatible spatial distribution of dummy residues which fits the experimental scattering pattern up to a resolution of 0.5 nm. The efficiency of the method is illustrated by the ab initio reconstruction of models of several proteins, with known and unknown crystal structure, from experimental scattering data. The new method substantially improves the resolution and reliability of models derived from scattering data and makes solution scattering a useful technique in large-scale structural characterization of proteins.

Small-angle scattering studies of biological macromolecules in solution

Small-angle scattering (SAS) of x-rays and neutrons is a fundamental tool in the study of biological macromolecules. The major advantage of the method lies in its ability to provide structural information about partially or completely disordered systems. SAS allows one to study the structure of native particles in near physiological environments and to analyse structural changes in response to variations in external conditions. In this review we concentrate on SAS studies of isotropic systems, in particular, solutions of biological macromolecules, an area where major progress has been achieved during the last decade. Solution scattering studies are especially important, given the challenge of the ‘post-genomic’ era with vast numbers of protein sequences becoming available. Numerous structural initiatives aim at large-scale expression and purification of proteins for subsequent structure determination using x-ray crystallography and NMR spectroscopy. Because of the requirement of good crystals for crystallography and the low molecular mass requirement of NMR, a significant fraction of proteins cannot be analysed using these two high-resolution methods. Progress in SAS instrumentation and novel analysis methods, which substantially improve the resolution and reliability of the structural models, makes the method an important complementary tool for these initiatives. The review covers the basics of x-ray and neutron SAS, instrumentation, mathematical methods used in data analysis and major modelling techniques. Examples of applications of SAS to different types of biomolecules (proteins, nucleic acids, macromolecular complexes, polyelectrolytes) are presented. A brief account of the new opportunities offered by third and fourth generation synchrotron radiation sources (time-resolved studies, coherent scattering and single molecule scattering) is also given.

Biophysical characterization of lipopolysaccharide and lipid A inactivation by lactoferrin.

Abstract The interaction of bacterial endotoxins (LPS Re and lipid A, the endotoxic principle of LPS) with the endogenous antibiotic lactoferrin (LF) was investigated using various physical techniques and biological assays. By applying Fouriertransform infrared (FTIR) spectroscopy, we find that LF binds to the phosphate group within the lipid A part and induces a rigidification of the acyl chains of LPS. The secondary structure of the protein as monitored by the amide I band is, however, not changed. Concomitant with the IR data, scanning calorimetric data indicate a sharpening of the acyl chain phase transition. From titration calorimetric and zeta potential data, saturation of LF binding to LPS was found to lie at a : ratio of 1:3 to 1:5 M from the former and 1:10 M from the latter technique. Xray scattering data indicate a change of the lipid A aggregate structure from inverted cubic to multilamellar, and with fluorescence (FRET) spectroscopy, LF is shown to intercalate by itself into phospholipid liposomes and may also block the lipopolysaccharidebinding protein (LBP)induced intercalation of LPS. The LPSinduced cytokine production of human mononuclear cells exhibits a decrease due to LF binding, whereas the coagulation of amebocyte lysate in the Limulus test exhibited concentrationdependent changes. Based on these results, a model for the mechanisms of endotoxin inactivation by LF is proposed.

Small angle x-ray and neutron scattering from solutions of biological macromolecules

Introduction PART I. THEORY AND EXPERIMENT 1. Basics of small-angle scattering 2. X-ray and neutron scattering instruments 3. Experimental practice and data processing PART II. DATA ANALYSIS METHODS 4. Monodisperse systems 5. Polydisperse and interacting systems PART III. BIOLOGICAL APPLICATIONS OF SOLUTION SAS 6. Static structral studies 7. Kinetic and perturbation studies 8. Analysis of interparticle interactions 9. SAS in multidisciplinary studies Conclusions and future outlook Appendix 1: Waves, complex number, Fourier transforms, convolution, correlation Appendix 2: Spherical harmonics formalism Appendix 3: Interactions between spherical molecules Appendix 4: List of Web resources

The generalized endotoxic principle

Bacterial lipopolysaccharides (endotoxins, LPS) belong to the most potent immunostimulators in mammals. The endotoxic principle of LPS is located in its lipid A moiety, which for Escherichia coli‐type LPS consists of a hexaacylated diphosphoryl diglucosamine backbone. This lipid A adopts a cubic inverted aggregate structure from which a conical shape of the molecule can be deduced, whereas the tetraacyl lipid A precursor IVa adopts a cylindrical shape and is endotoxically inactive, but antagonizes active LPS. We hypothesize that non‐lipid A amphiphiles with similar physicochemical properties of amphiphilicity, charge, and shape, might mimic the respective lipid A. To test this hypothesis, phospholipid‐like amphiphiles with six acyl chains attached to a bisphosphorylated serine‐like backbone of varying length replacing the diglucosamine backbone were synthesized. The compound with a short backbone fulfills all criteria of an endotoxic agonist, and that with longbackbone fulfills those of an antagonist. This holds true for the human as well as for the murine system. Interestingly, these compounds are inactive in the Limulus amebocyte lysate test which is specific for LPS diglucosamine backbone. These results define a general endotoxic principle and, furthermore, provide new insights into an understanding of early steps of endotoxin action.

Large Differences Are Observed Between the Crystal and Solution Quaternary Structures of Allosteric Aspartate Transcarbamylase in the R State

Solution scattering curves evaluated from the crystal structures of the T and R states of the allosteric enzyme aspartate transcarbamylase from Escherichia coli were compared with the experimental x‐ray scattering patterns. Whereas the scattering from the crystal structure of the T state agrees with the experiment, large deviations reflecting a significant difference between the quaternary structures in the crystal and in solution are observed for the R state. The experimental curve of the R state was fitted by rigid body movements of the subunits in the crystal R structure which displace the latter further away from the T structure along the reaction coordinates of the T→R transition observed in the crystals. Taking the crystal R structure as a reference, it was found that in solution the distance between the catalytic trimers along the threefold axis is 0.34 nm larger and the trimers are rotated by 11° in opposite directions around the same axis; each of the three regulatory dimers is rotated by 9° around the corresponding twofold axis and displaced by 0.14 nm away from the molecular center along this axis. Proteins 27:110–117

Endotoxin-like properties of a rhamnolipid exotoxin from Burkholderia (Pseudomonas) plantarii: immune cell stimulation and biophysical characterization

Abstract Here we report on the purification, structural characterization, and biological activity of a glycolipid, 2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-α(R)-3-hydroxytetradecanoyl-(R)-3-hydroxytetradecanoate (RL-2,214) produced by Burkholderia (Pseudomonas) plantarii. RL-2,214 is structurally very similar to a rhamnolipid exotoxin from Pseudomonas aeruginosa and identical to the rhamnolipid of Burkholderia pseudomallei, the causative agent of melioidosis. Interestingly, RL-2,214 exhibits strong stimulatory activity on human mononuclear cells to produce tumor necrosis factor α, the overproduction of which is known to cause sepsis and the septic shock syndrome. Such a property has not been noted so far for rhamnolipid exotoxins, only for bacterial endotoxins (lipopolysaccharide, LPS). Consequently, we analyzed RL-2,214 with respect to its pathophysiological activities as a heat-stable extracellular toxin. Like LPS, the cell-stimulating activity of the rhamnolipid could be inhibited by incubation with polymyxin B. However, immune cell activation by RL-2,214 does nor occur via receptors that are involved in LPS (TLR4) or lipopeptide signaling (TLR2). Despite its completely different chemical structure, RL-2,214 exhibits a variety of endotoxin-related physicochemical characteristics, such as a cubic-inverted supramolecular structure. These data are in good agreement with our conformational concept of endotoxicity: intercalation of naturally originating virulence factors into the immune cell membrane leads to strong mechanical stress on integral proteins, eventually causing cell activation.

Triticum durum Metallothionein ISOLATION OF THE GENE AND STRUCTURAL CHARACTERIZATION OF THE PROTEIN USING SOLUTION SCATTERING AND MOLECULAR MODELING

A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been identified in durum wheat Triticum durum cv. Balcali85 genomic DNA. Multiple alignment analyses on the cDNA and the translated protein sequences showed that T. durum MT (dMT) can be classified as a type 1 MT. dMT has three Cys-X-Cys motifs in each of the N- and C-terminal domains and a 42-residue-long hinge region devoid of cysteines. dMT was overexpressed in Escherichia coli as a fusion protein (GSTdMT), and bacteria expressing the fusion protein showed increased tolerance to cadmium in the growth medium compared with controls. Purified GSTdMT was characterized by SDS- and native-PAGE, size exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was shown that the recombinant protein binds 4 ± 1 mol of cadmium/mol of protein and has a high tendency to form stable oligomeric structures. The structure of GSTdMT and dMT was investigated by synchrotron x-ray solution scattering and computational methods. X-ray scattering measurements indicated a strong tendency for GSTdMT to form dimers and trimers in solution and yielded structural models that were compatible with a stable dimeric form in which dMT had an extended conformation. Results of homology modeling and ab initio solution scattering approaches produced an elongated dMT structure with a long central hinge region. The predicted model and those obtained from x-ray scattering are in agreement and suggest that dMT may be involved in functions other than metal detoxification.

Physicochemical and Biological Analysis of Synthetic Bacterial Lipopeptides VALIDITY OF THE CONCEPT OF ENDOTOXIC CONFORMATION

The importance of the biological function and activity of lipoproteins from the outer or cytoplasmic membranes of Gram-positive and Gram-negative bacteria is being increasingly recognized. It is well established that they are like the endotoxins (lipopolysaccharide (LPS)), which are the main amphiphilic components of the outer membrane of Gram-negative bacteria, potent stimulants of the human innate immune system, and elicit a variety of proinflammatory immune responses. Investigations of synthetic lipopeptides corresponding to N-terminal partial structures of bacterial lipoproteins defined the chemical prerequisites for their biological activity and in particular the number and length of acyl chains and sequence of the peptide part. Here we present experimental data on the biophysical mechanisms underlying lipopeptide bioactivity. Investigation of selected synthetic diacylated and triacylated lipopeptides revealed that the geometry of these molecules (i.e. the molecular conformations and supramolecular aggregate structures) and the preference for membrane intercalation provide an explanation for the biological activities of the different lipopeptides. This refers in particular to the agonistic or antagonistic activity (i.e. their ability to induce cytokines in mononuclear cells or to block this activity, respectively). Biological activity of lipopeptides was hardly affected by the LPS-neutralizing antibiotic polymyxin B, and the biophysical interaction characteristics were found to be in sharp contrast to that of LPS with polymyxin B. The analytical data show that our concept of “endotoxic conformation,” originally developed for LPS, can be applied also to the investigated lipopeptide and suggest that the molecular mechanisms of cell activation by amphiphilic molecules are governed by a general principle.

Escherichia coli SecA shape and dimensions

SecA shape and conformational flexibility in solution were studied by small angle X‐ray scattering. Dimeric SecA is a very elongated molecule, 15 nm long and 8 nm wide. SecA is therefore four times as long as the membrane is wide. The two globular protomers are distinctly separated and share limited surface of intermolecular contacts. ATP, ADP or adenylyl‐imidodiphosphate (AMP‐PNP) binding does not alter the SecA radius of gyration. A SecA mutant that catalyzes multiple rounds of ATP hydrolysis does not undergo conformational changes detectable by small angle X‐ray scattering (SAXS). We conclude that SecA conformational alterations observed biochemically during nucleotide interaction are only small‐scale and localized. The ramifications of these findings on SecA/SecYEG interaction are discussed.