Genetically modified L3,7 and L2 lipooligosaccharides from Neisseria meningitidis serogroup B confer a broad cross-bactericidal response.

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 LOS-enriched 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-N-neotetraose (L3,7 LOS). In conclusion, a bivalent detoxified LOS OMV-based vaccine demonstrated the potential to afford a broad cross-protection against meningococcal disease.

Three hTIM mutants that provide new insights on why TIM is a dimer.

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.

Cloning and overexpression of the triosephosphate isomerase genes from psychrophilic and thermophilic bacteria. Structural comparison of the predicted protein sequences.

We focused on the temperature adaptation of triosephosphate isomerase (TIM; E.C. 5.3.1.1.) by comparing the structure of TIMs isolated from bacterial organisms living in either cold or hot environments. The TIM gene from psychrophilic bacteria Moraxella sp. TA137 was cloned and its nucleotide sequence determined. Its deduced amino acid sequence revealed 34% identity with the thermophilic bacteria Bacillus stearothermophilus TIM. Expression vectors were constructed and recombinant Moraxella TA137 and Bacillus stearothermophilus TIMs were overproduced and purified to homogeneity. Recombinant TIM inactivation constants (Ki), measured at various temperatures, compared to those of the mesophilic Escherichia coli recombinant TIM clearly show that Moraxella TA137 and B. stearothermophilus TIMs have respectively psychrophilic and thermophilic characteristics. To try to elucidate the structure-thermolability and structure-thermostability relationship, factors affecting the overall stability of these two TIMs were examined, based on the alignment with the mesophilic chicken TIM, the three-dimensional structure of which is already known. From this comparison, it appears that the adaptability of TIM to high temperature is favored by better stabilizing residues for the helix dipole as well as better helix-forming residues whereas the adaptability of TIM to low temperature seems to reside in the nature of helix-capping residues.

Crystal structure of recombinant human triosephosphate isomerase at 2.8 A resolution. Triosephosphate isomerase-related human genetic disorders and comparison with the trypanosomal enzyme.

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.

Synthesis, purification and initial structural characterization of octarellin, a de novo polypeptide modelled on the alpha/beta-barrel 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.

Stabilization of human triosephosphate isomerase by improvement of the stability of individual alpha-helices in dimeric as well as monomeric forms of the protein.

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.

Spectroscopic investigation of structure in octarellin (a de novo protein designed to adopt the alpha/beta-barrel packing).

We present here a spectroscopic structural characterization of octarellin, a recently reported de novo protein modelled on alpha/beta-barrel proteins [K. Goraj, A. Renard and J.A. Martial (1990) Protein Engng, 3, 259-266]. Infrared and Raman spectra analyses of octarellin's secondary structure reveal the expected percentage of alpha-helices (30%) and a higher beta-sheet content (40%) than predicted from the design. When the Raman spectra obtained with octarellin and native triosephosphate isomerase (a natural alpha/beta-barrel) are compared, similar percentages of secondary structures are found. Thermal denaturation of octarellin monitored by CD confirms that its secondary structures are quite stable, whereas its native-like tertiary fold is not. Tyrosine residues, predicted to be partially hidden from solvent, are actually exposed as revealed by Raman and UV absorption spectra. We conclude that the attempted alpha/beta-barrel conformation in octarellin may be loosely packed. The criteria used to design octarellin are discussed and improvements suggested.

Modular mutagenesis of a TIM-barrel enzyme: the crystal structure of a chimeric E. coli TIM having the eighth beta alpha-unit replaced by the equivalent unit of chicken TIM.

The crystal structure of a hybrid Escherichia coli triosephosphate isomerase (TIM) has been determined at 2.8 A resolution. The hybrid TIM (ETIM8CHI) was constructed by replacing the eighth beta alpha-unit of E. coli TIM with the equivalent unit of chicken TIM. This replacement involves 10 sequence changes. One of the changes concerns the mutation of a buried alanine (Ala232 in strand 8) into a phenylalanine. The ETIM8CHI structure shows that the A232F sequence change can be incorporated by a side-chain rotation of Phe224 (in helix 7). No cavities or strained dihedrals are observed in ETIM8CHI in the region near position 232, which is in agreement with the observation that ETIM8CHI and E.coli TIM have similar stabilities. The largest CA (C-alpha atom) movements, approximately 3 A, are seen for the C-terminal end of helix 8 (associated with the outward rotation of Phe224) and for the residues in the loop after helix 1 (associated with sequence changes in helix 8). From the structure it is not clear why the kcat of ETIM8CHI is 10 times lower than in wild type E.coli TIM.

Structure of triosephosphate isomerase from Escherichia coli determined at 2.6 A resolution.

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 beta-barrel.

Replacing the (beta alpha)-unit 8 of E.coli TIM with its chicken homologue leads to a stable and active hybrid enzyme.

In order to investigate how structural modifications interfere with protein stability, we modified a (beta alpha)-unit in E.coli triosephosphate isomerase (TIM), a typical (beta alpha)-barrel protein, assuming that the pseudosymmetrical beta-barrel can be divided into eight successive loop/beta-strand/loop/alpha-helix motifs. We replaced the eighth (beta alpha)-unit of E.coli TIM with the corresponding chicken (beta alpha)-unit. The substitution, involving the replacement of 10 of the 23 residues of this (beta alpha)-unit, was evaluated first by modelling, then experimentally. Modelling by homology suggests how the amino acid replacements might be accommodated in the hybrid E.coli/chicken TIM (ETIM8CHI). Both natural and hybrid recombinant TIMs, overproduced in E.coli, were purified to homogeneity and characterized as to their stability and kinetics. Our kinetic studies show that the modification performed here leads to an active enzyme. The stability studies indicate that the stability of ETIM8CHI is comparable to that of the wild type TIM.