Nicolas Mouz

Mutations in the Active Site of Penicillin-binding Protein PBP2x from Streptococcus pneumoniae ROLE IN THE SPECIFICITY FOR β-LACTAM ANTIBIOTICS

Penicillin-binding protein 2x (PBP2x) isolated from clinical β-lactam-resistant strains of Streptococcus pneumoniae (R-PBP2x) have a reduced affinity for β-lactam antibiotics. Their transpeptidase domain carries numerous substitutions compared with homologous sequences from β-lactam-sensitive streptococci (S-PBP2x). Comparison of R-PBP2x sequences suggested that the mutation Gln552 → Glu is important for resistance development. Mutants selected in the laboratory with cephalosporins frequently contain a mutation Thr550 → Ala. The high resolution structure of a complex between S-PBP2x* and cefuroxime revealed that Gln552 and Thr550, which belong to strand β3, are in direct contact with the cephalosporin. We have studied the effect of alterations at positions 552 and 550 in soluble S-PBP2x (S-PBP2x*) expressed in Escherichia coli. Mutation Q552E lowered the acylation efficiency for both penicillin G and cefotaxime when compared with S-PBP2x*. We propose that the introduction of a negative charge in strand β3 conflicts with the negative charge of the β-lactam. Mutation T550A lowered the acylation efficiency of the protein for cefotaxime but not for penicillin G. Thein vitro data presented here are in agreement with the distinct resistance profiles mediated by these mutations in vivo and underline their role as powerful resistance determinants.

Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti–HIV-1 drugs

Significance The envelope subunit gp41 is an attractive target for therapeutic intervention against HIV-1. Interfering with the interaction between the heptad-repeat regions of gp41 is a promising approach to inhibit HIV-1 fusion to the host cell membrane. Here, we present an alternative rational design and protein-engineering approach to produce highly stable single-chain proteins that accurately mimic the trimeric coiled-coil surface of the gp41 N-terminal heptad repeat. This approach has a strong potential for development to HIV-1 drugs, vaccines, or microbicides and could be extendable to the design of proteins interfering with other types of coiled-coil interactions. During HIV-1 fusion to the host cell membrane, the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) of the envelope subunit gp41 become transiently exposed and accessible to fusion inhibitors or Abs. In this process, the NHR region adopts a trimeric coiled-coil conformation that can be a target for therapeutic intervention. Here, we present an approach to rationally design single-chain protein constructs that mimic the NHR coiled-coil surface. The proteins were built by connecting with short loops two parallel NHR helices and an antiparallel one with the inverse sequence followed by engineering of stabilizing interactions. The constructs were expressed in Escherichia coli, purified with high yield, and folded as highly stable helical coiled coils. The crystal structure of one of the constructs confirmed the predicted fold and its ability to accurately mimic an exposed gp41 NHR surface. These single-chain proteins bound to synthetic CHR peptides with very high affinity, and furthermore, they showed broad inhibitory activity of HIV-1 fusion on various pseudoviruses and primary isolates.

Molecular and Physicochemical Factors Governing Solubility of the HIV gp41 Ectodomain.

The HIV gp41 ectodomain (e-gp41) is an attractive target for the development of vaccines and drugs against HIV because of its crucial role in viral fusion to the host cell. However, because of the high insolubility of e-gp41, most biophysical and structural analyses have relied on the production of truncated versions removing the loop region of gp41 or the utilization of nonphysiological solubilizing conditions. The loop region of gp41 is also known as principal immunodominant domain (PID) because of its high immunogenicity, and it is essential for gp41-mediated HIV fusion. In this study we identify the aggregation-prone regions of the amino acid sequence of the PID and engineer a highly soluble mutant that preserves the trimeric structure of the wild-type e-gp41 under physiological pH. Furthermore, using a reverse mutagenesis approach, we analyze the role of mutated amino acids upon the physicochemical factors that govern solubility of e-gp41. On this basis, we propose a molecular model for e-gp41 self-association, which can guide the production of soluble e-gp41 mutants for future biophysical analyses and biotechnological applications.

Liposomal formulation of Gp41 derivate with adjuvant MPLA: vaccine design, immunogenicity in animals and safety in humans

Background Gp41 of the HIV envelope, especially the MPER, contains highly conserved epitopes recognized by neutralizing monoclonal antibodies such as D5, 2F5 and 4E10. The correct presentation of the antigens is considered to be key for eliciting neutralizing immune response. The lipid bilayer of liposomes can mimic the virus surface and therefore provides the appropriate presentation of this membrane protein. In addition, liposomes can integrate the adjuvant monophosphoryl lipid A (MPLA).