Arthur J. Rowe

Solution structure of choline binding protein A, the major adhesin of Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) remains a significant health threat worldwide, especially to the young and old. While some of the biomolecules involved in pneumococcal pathogenesis are known and understood in mechanistic terms, little is known about the molecular details of bacterium/host interactions. We report here the solution structure of the ‘repeated’ adhesion domains (domains R1 and R2) of the principal pneumococcal adhesin, choline binding protein A (CbpA). Further, we provide insights into the mechanism by which CbpA binds its human receptor, polymeric immunoglobulin receptor (pIgR). The R domains, comprised of 12 imperfect copies of the leucine zipper heptad motif, adopt a unique 3‐α‐helix, raft‐like structure. Each pair of α‐helices is antiparallel and conserved residues in the loop between Helices 1 and 2 exhibit a novel ‘tyrosine fork’ structure that is involved in binding pIgR. This and other structural features that we show are conserved in most pneumococcal strains appear to generally play an important role in bacterial adhesion to pIgR. Interestingly, pneumococcus is the only bacterium known to adhere to and invade human cells by binding to pIgR.

Transmission electron microscopy studies on pig gastric mucin and its interactions with chitosan

Transmission electron microscopy has been employed to visualize the molecular structure and organization of a highly purified preparation of pig gastric mucin (molar mass M~9 × 106g/mol, as determined by low speed sedimentation equilibrium), prepared for microscopy by two completely independent methods. Samples were prepared for imaging by air drying on mica (in the presence of 50% glycerol) as well as critical point drying in acetone/CO2. The data appear consistent with the accepted linear model for mucins, and are consistent with regions of variable degrees of glycosylation along the polypeptide backbone chain, with the overall conformation that of a loose or random coil. The behaviour of this mucin in a dilute solution mixture with the potential mucoadhesive polymer chitosan (M~1 60,000 g/mol) was then explored, and a clear interaction was demonstrated, consistent with dilute solution measurements using sedimentation velocity analysis in the analytical ultracentrifuge.

The pluripotency rheostat Nanog functions as a dimer

The defining activity of the homeodomain protein Nanog is the ability to confer cytokine-independent self-renewal upon ES (embryonic stem) cells in which it is overexpressed. However, the biochemical basis by which Nanog achieves this function remains unknown. In the present study, we show that Nanog dimerizes through a functionally critical domain. Co-immunoprecipitation of Nanog molecules tagged with distinct epitopes demonstrates that Nanog self-associates through a region in which every fifth residue is tryptophan. In vitro binding experiments establish that this region participates directly in self-association. Moreover, analytical ultracentrifugation indicates that, in solution, Nanog is in equilibrium between monomeric and dimeric forms with a K(d) of 3 muM. The functional importance of Nanog dimerization is established by ES cell colony-forming assays in which deletion of the tryptophan-repeat region eliminates the capacity of Nanog to direct LIF (leukaemia inhibitory factor)-independent self-renewal.

Subunit organisation and symmetry of pore-forming, oligomeric pneumolysin

We present a detailed analysis of the oligomeric subunit organisation of pneumolysin by the use of negative stain electron microscopy and image processing to produce a projection density map. Analysis of the rotational symmetry has revealed a large and variable subunit number, between 40–50. The projected subunit density by rotational averaging shows at least two distinct subunit domains at different radial positions. Side views of the rings reveal further details concerning the dimensions of the oligomer in the membrane. On the basis of these observations and our previous knowledge of the monomer domain structure we propose that the 4‐domain subunits are packed in a square planar arrangement to form the pneumolysin oligomer.

Serine Acetyltransferase from Escherichia coli Is a Dimer of Trimers

Equilibrium sedimentation studies show that the serine acetyltransferase (SAT) of Escherichia coli is a hexamer. The results of velocity sedimentation and quasi-elastic light scattering experiments suggest that the identical subunits are loosely packed and/or arranged in an ellipsoidal fashion. Chemical cross-linking studies indicate that the fundamental unit of quaternary structure is a trimer. The likelihood, therefore, is that in solution SAT exists as an open arrangement of paired trimers. Crystals of SAT have 32 symmetry, consistent with such an arrangement, and the cell density function is that expected for a hexamer. Electron microscopy with negative staining provides further evidence that SAT has an ellipsoidal subunit organization, the dimensions of the particles consistent with the proposed paired trimeric subunit arrangement. A bead model analysis supports the view that SAT has a low packing density and, furthermore, indicates that the monomers may have an ellipsoidal shape. Such a view is in keeping with the ellipsoidal subunit shape of trimeric LpxA, an acyltransferase with which SAT shares contiguous repeats of a hexapeptide motif.

A folded (10 S) conformer of myosin from a striated muscle and its implications for regulation of ATPase activity

Myosin from the striated adductor muscle of the scallop Pecten maximus is shown to fold into a compact 10 S conformer under relaxing conditions, as has been characterized for smooth and non-muscle myosins. The folding transition is accompanied by the trapping of nucleotide at the active site to give a species with a half-life of about an hour at 20 degrees C. Ca2+ binding to the specific, regulatory sites on a myosin head promotes unfolding to the extended 6 S conformer and activates product release by 60-fold. The unfolding transition, however, remains much slower than the contraction-relaxation cycle of scallop striated muscle and could not play a role in the regulation of these events. The dissociation of products from myosin heads in native thick filaments is Ca2(+)-regulated, but under relaxing conditions the nucleotide is released at least an order of magnitude faster than from the 10 S monomeric myosin, at a rate similar to that observed with heavy meromyosin. Thus, there is no evidence for any intermolecular interaction between neighbouring molecules in the filament analogous to the head-neck intramolecular interaction in the 10 S conformer. It is possible that the 10 S myosin state represents an inert form involved in the control of filament assembly during muscle growth and development. Removal of regulatory light chains or labelling the reactive heavy chain thiol of myosin prevents, or at least disfavours, formation of the folded 10 S conformer and allows separation of the modified protein from the native molecules.

The Central Portion of Factor H (Modules 10-15) Is Compact and Contains a Structurally Deviant CCP Module

The first eight and the last two of 20 complement control protein (CCP) modules within complement factor H (fH) encompass binding sites for C3b and polyanionic carbohydrates. These binding sites cooperate self-surface selectively to prevent C3b amplification, thus minimising complement-mediated damage to host. Intervening fH CCPs, apparently devoid of such recognition sites, are proposed to play a structural role. One suggestion is that the generally small CCPs 10–15, connected by longer-than-average linkers, act as a flexible tether between the two functional ends of fH; another is that the long linkers induce a 180° bend in the middle of fH. To test these hypotheses, we determined the NMR-derived structure of fH12–13 consisting of module 12, shown here to have an archetypal CCP structure, and module 13, which is uniquely short and features a laterally protruding helix-like insertion that contributes to a prominent electropositive patch. The unusually long fH12–13 linker is not flexible. It packs between the two CCPs that are not folded back on each other but form a shallow vee shape; analytical ultracentrifugation and X-ray scattering supported this finding. These two techniques additionally indicate that flanking modules (within fH11–14 and fH10–15) are at least as rigid and tilted relative to neighbours as are CCPs 12 and 13 with respect to one another. Tilts between successive modules are not unidirectional; their principal axes trace a zigzag path. In one of two arrangements for CCPs 10–15 that fit well with scattering data, CCP 14 is folded back onto CCP 13. In conclusion, fH10–15 forms neither a flexible tether nor a smooth bend. Rather, it is compact and has embedded within it a CCP module (CCP 13) that appears to be highly specialised given both its deviant structure and its striking surface charge distribution. A passive, purely structural role for this central portion of fH is unlikely.

Are chitosan-mucin interactions specific to different regions of the stomach? Velocity ultracentrifugation offers a clue

Previous work has shown strong interactions between pig gastric mucins and a highly deacetylated chitosan. Recently, mucins purified from different areas of the porcine stomach have been shown to differ in terms of their oligosaccharide substitution and net charge. How this regional variation may affect the properties of these mucins is of great interest in terms of the specificity of mucoadhesion with chitosan. We have investigated the interaction of a chitosan of degree of acetylation FA < 0.11 with three different mucins purified from different areas of the porcine stomach (cardia region, corpus and antrum) at two different ionic strengths. Using sedimentation velocity in the analytical ultracentrifuge equipped with a schlieren optical system coupled on line to a CCD camera, the amount of chitosan interacting with mucin was determined. The degree of interaction varies between the three mucins with those from the cardiac region displaying the highest degree of interaction; in the case of the corpus and antrum, however, the interaction increases with increase in ionic strength, implying that the interaction between mucins and chitosans may have a hydrophobic as well as an electrostatic basis. q 1999 Elsevier Science Ltd. All rights reserved.

Structure and heterogeneity of gliadin: a hydrodynamic evaluation

A study of the heterogeneity and conformation in solution [in 70% (v/v) aq. ethanol] of gliadin proteins from wheat was undertaken based upon sedimentation velocity in the analytical ultracentrifuge, analysis of the distribution coefficients and ellipsoidal axial ratios assuming quasi-rigid particles, allowing for a range of plausible time-averaged hydration values. All classical fractions (α, γ, ωslow, ωfast) show three clearly resolved components. Based on the weight-average sedimentation coefficient for each fraction and a weight-average molecular weight from sedimentation equilibrium and/or cDNA sequence analysis, all the proteins are extended molecules with axial ratios ranging from ~10 to 30 with α appearing the most extended and γ the least.

Conformation zoning of large molecules using the analytical ultracentrifuge

A substantial proportion of large molecules made naturally or by artificial means exist as linear chains. In biology this includes DNA, mRNA, many important classes of sugar polymers (polysaccharides) and denatured proteins. In physical science this includes polyethylene, Polyvinylchloride and many important polymers used in plastics and also the many new ones being explored for use in drug delivery. Crucial to how many of these large molecules function is their conformation in solution (either aqueous or organic), a realm unfortunately outside the grasp of high-resolution techniques such as X-ray crystallography. We have now however devised a quick and accessible method for identifying the conformation type or “Zone” of a molecule: Zone A (extra rigid rod type); Zone B (rigid rod type); Zone C (semi-flexible type), Zone D (completely random coil) and Zone E (compact or highly branched particle). To perform this “Conformation Zoning” requires a few milligrams of material and access to one of the new types of high-speed Centrifuge which are now proliferating in academic and industrial establishments.