The crystal structure of recombinant human triosephosphate isomerase (hTIM) has been determined complexed with the transition-state analogue 2-phosphoglycolate at a resolution of 2.8 A. After refinement, the R-factor is 16.7% with good geometry. The asymmetric unit contains 1 complete dimer of 53,000 Da, with only 1 of the subunits binding the inhibitor. The so-called flexible loop, comprising residues 168-174, is in its "closed" conformation in the subunit that binds the inhibitor, and in the "open" conformation in the other subunit. The tips of the loop in these 2 conformations differ up to 7 A in position. The RMS difference between hTIM and the enzyme of Trypanosoma brucei, the causative agent of sleeping sickness, is 1.12 A for 487 C alpha positions with 53% sequence identity. Significant sequence differences between the human and parasite enzymes occur at about 13 A from the phosphate binding site. The chicken and human enzymes have an RMS difference of 0.69 A for 484 equivalent residues and about 90% sequence identity. Complementary mutations ensure a great similarity in the packing of side chains in the core of the beta-barrels of these 2 enzymes. Three point mutations in hTIM have been correlated with severe genetic disorders ranging from hemolytic disorder to neuromuscular impairment. Knowledge of the structure of the human enzyme provides insight into the probable effect of 2 of these mutations, Glu 104 to Asp and Phe 240 to Ile, on the enzyme. The third mutation reported to be responsible for a genetic disorder, Gly 122 to Arg, is however difficult to explain. This residue is far away from both catalytic centers in the dimer, as well as from the dimer interface, and seems unlikely to affect stability or activity. Inspection of the 3-dimensional structure of trypanosomal triosephosphate isomerase, which has a methionine at position 122, only increased the mystery of the effects of the Gly to Arg mutation in the human enzyme.

Human triosephosphate isomerase (hTIM), a dimeric enzyme, was altered by site-directed mutagenesis in order to determine whether it can be dissociated into monomers. Two hTIM mutants were produced, in which a glutamine residue was substituted for either Met14 or Arg98, both of which are interface residuces. These substitutions strongly interfere with TIM subunit association, since these mutant TIMs appear to exist as compact monomers in dynamic equilibrium with dimers. In kinetic studies, the M14Q mutant exhibits significant catalytic activity, while the R98Q enzyme is inactive. The M14Q enzyme is nevertheless much less active than unmutated hTIM. Moreover, its specific activity is concentration dependent, suggesting a dissociation process in which the monomers are inactive. In order to determine the conformational stability of the wild-type and mutant hTIMs, unfolding of all three enzymes was monitored by circular dichroism and tryptophan fluorescence spectroscopy. In each case, protein stability is concentration dependent, and the unfolding reaction is compatible with a two-state model involving the native dimer and unfolded monomers. The conformational stability of hTIM, as estimated according to this model, is 19.3 (+/-0.4) kcal/mol. The M14Q and R98Q replacements significantly reduce enzyme stability, since the free energies of unfolding are 13.8 and 13.5 (+/- 0.3) kcal/mol respectively, for the mutants, A third mutant, in which the M14Q and R98Q replacements are cumulated, behaves like a monomer. The stability of this mutant is not concentration-dependent, and the unfolding reaction is assigned to a transition from a folded monomer to an unfolded monomer. The conformational stability of this double mutant is estimated 2.5 (+/-0.1) kcal/mol. All these data combined suggest that TIM monomers are thermodynamically unstable. This might explain why TIM occurs only as a dimer.

Neisseria meningitidis serogroup B is a major cause of bacterial meningitis in younger populations. The available vaccines are based on outer membrane vesicles obtained from wild-type strains. In children less than 2 years old they confer protection only against strains expressing homologous PorA, a major, variable outer membrane protein (OMP). We genetically modified a strain in order to eliminate PorA and to overproduce one or several minor and conserved OMPs. Using a mouse model mimicking children's PorA-specific bactericidal activity, it was demonstrated that overproduction of more than one minor OMP is required to elicit antibodies able to induce complement-mediated killing of strains expressing heterologous PorA. It is concluded that a critical density of bactericidal antibodies needs to be reached at the surface of meningococci to induce complement-mediated killing. With minor OMPs, this threshold is reached when more than one antigen is targeted, and this allows cross-protection.

The structure of triosephosphate isomerase (TIM) from the organism Escherichia coli has been determined at a resolution of 2.6 A. The structure was solved by the molecular replacement method, first at 2.8 A resolution with a crystal grown by the technique of hanging-drop crystallization from a mother liquor containing the transition-state analogue 2-phosphoglycolate (2PG). As a search model in the molecular replacement calculations, the refined structure of TIM from Trypanosoma brucei, which has a sequence identity of 46% compared to the enzyme from E. coli, was used. An E. coli TIM crystal grown in the absence of 2PG, diffracting to 2.6 A resolution, was later obtained by application of the technique of macro-seeding using a seed crystal grown from a mother liquor without 2PG. The final 2.6 A model has a crystallographic R factor of 11.9%, and agrees well with standard stereochemical parameters. The structure of E. coli TIM suggests the importance of residues which favour helix initiation for the formation of the TIM fold. In addition, TIM from E. coli shows peculiarities in its dimer interface, and in the packing of core residues within the /3-barrel.

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Human triosephosphate isomerase (hTIM) is a dimeric enzyme of identical subunits, adopting the alpha/beta-barrel fold. In a previous work, a monomeric mutant of hTIM was engineered in which Met14 and Arg98, two interface residues, were changed to glutamine. Analysis of equilibrium denaturation of this monomeric mutant, named M14Q/R98Q, revealed that its conformational stability, 2.5kcal/mol, is low as compared to the stability of dimeric hTIM (19.3 kcal/mol). The fact that this value is also lower than the conformational stabilities usually found for monomeric proteins suggests that the hTIM monomers are thermodynamically unstable. In the present work, we attempted to stabilize the M14Q/R98Q mutant by introducing stabilizing mutations in alpha-helices of the protein. Five mutations were proposed, designed to increase alpha-helix propensity by introducing alanines at solvent-exposed sites (Q179A, K193A), to introduce favorable interactions with helix dipoles (Q179D, S105D), or to reduce the conformational entropy of unfolding by introducing proline residues at the "N-cap" position of alpha-helices (A215P). Three replacements (Q179D, K193A, and A215P) were found to increase the stability of the native dimeric hTIM and the monomeric M14Q/R98Q. These results suggest that the monomeric hTIM mutant can be stabilized to a considerable extent by following well-established rules for protein stabilization. A comparison of the stabilizing effect performed by the mutations on the dimeric hTIM and the monomeric M14Q/R98Q allowed us to reinforce a model of equilibrium denaturation proposed for both proteins.

We have attempted to construct an artificial polypeptide that folds like the eight-stranded parallel beta-barrel structures. Our approach consists of repeating eight times a unit peptide designed to adopt a 'beta-strand/alpha-helix' pattern. A first 'test' sequence for this structural unit was deduced from a series of parameters defined after an analysis of three natural alpha/beta-barrel proteins and including principally the lengths of the secondary structure elements, the alpha/beta packing and the fitting on average Garnier profiles. The gene encoding this structural unit was synthesized, cloned and expressed in Escherichia coli either as a monomer or as direct repeats of 2-12 units. Preliminary structural characterization of the 7-, 8- and 9-fold unit polypeptides by circular dichroism measurements indicates the presence of the predicted amount of alpha-helix in the three proteins. Further analysis by urea-gradient gel electrophoresis demonstrates that, in the conditions tested, only the 8-fold unit polypeptide forms a compact structure through a cooperative and rapid two-state folding transition involving long-range molecular interactions.

Currently available Neisseria meningitidis serogroup B (MenB) vaccines are based on outer membrane vesicles (OMVs) that are obtained from wild-type strains. They are purified with the aim of decreasing the lipooligosaccharide (LOS) content and hence reduce the reactogenicity of the vaccine even though LOS is a potential protective antigen. In <2-year-old children, these MenB vaccines confer protection only against strains expressing homologous PorA, a major and variable outer membrane protein. Our objective was to develop a safe LOS-based vaccine against MenB. To this end, we used modified porA knockout strains expressing genetically detoxified (msbB gene-deleted) L2 and L3,7 LOSs, allowing the production of LOSenriched OMVs. The vaccine-induced antibodies were found to be bactericidal against nearly all invasive strains, irrespective of capsular serogroup. In addition, we have also demonstrated that LOS lacking the terminal galactose (with a lgtB mutation; truncated L3 LOS), but not LOS produced without the galE gene, induced a bactericidal antibody response in mice similar to that seen for LOS containing the full lacto-Nneotetraose (L3,7 LOS). In conclusion, a bivalent detoxified LOS OMV-based vaccine demonstrated the potential to afford a broad cross-protection against meningococcal disease.The gram-negative Neisseria meningitidis serogroup B (MenB) is a major cause of bacterial meningitis in younger populations. The disease is associated with high morbidity and mortality rates, despite the availability of optimized treatments. Therefore, disease prevention by vaccination is considered a better approach than treatment. Currently available MenB vaccines are based on outer membrane vesicles (OMVs) that are obtained from wild-type strains and purified with the aim of decreasing the endotoxin lipooligosaccharide (LOS) content, hence reducing the reactogenicity of the vaccine. However, in Ͻ2-year-old children, these MenB vaccines confer protection only against strains expressing the homologous PorA, a major and variable outer membrane protein (OMP) present on the vaccine OMVs (25,34,43). Other vaccine approaches able to afford a larger cross-protection, among which the development of a safe LOS-based vaccine might be a valuable option, seem necessary. Indeed, antibodies to LOS have been shown to be bactericidal, both in humans (4) and in monkeys (45), and natural bacterial clearance in humans appears to be linked with anti-LOS activity (5). These two observations point out LOS as a potential vaccine candidate.LOS is composed of a lipid A anchor to the bacterial outer membrane and a variable glycan moiety. Although LOS has endotoxin properties, it behaves like an exotoxin due to the capacity of N. meningitidis to secrete large amounts of its outer membrane in the form of blebs or OMVs. Moreover, the fulminant cases of meningococcemia relate to a great extent to the amount of circulating LOS (2).Original immunotyping allowed the determination of 11 distinct LOS types among meningococcal strains, designated L1 to L11...