Gyu Hyun Nam

Engineering N-glycosylation mutations in IL-12 enhances sustained cytotoxic T lymphocyte responses for DNA immunization

Interleukin-12 (IL-12), consisting of p40 and p35 subunits, produces both p70 heterodimer and free p40. p70 is essential for the induction of T-helper 1 (Th1) and cytotoxic T-cell (CTL) immunity, whereas p40 inhibits p70-mediated function. Here, we found that mutations introduced into N-glycosylation sites (N220 of murine p40 and N222 of human p40) reduced secretion of p40 but not p70. Co-immunization of N220 mutant mIL-12 gene with hepatitis C virus (HCV) E2 DNA significantly enhanced long-term E2-specific CD8+ T-cell response and protection against tumor challenge compared with that of wild type. Our results indicate that the ratio of p70 to p40 is important for generating sustained long-term cell-mediated immunity. Thus, the mutant IL-12 could be utilized for the development of DNA vaccines as an adjuvant for the generation of long-term memory T-cell responses.

Roles of dimerization in folding and stability of ketosteroid isomerase from Pseudomonas putida biotype B

Equilibrium and kinetic analyses have been performed to elucidate the roles of dimerization in folding and stability of KSI from Pseudomonas putida biotype B. Folding was reversible in secondary and tertiary structures as well as in activity. Equilibrium unfolding transition, as monitored by fluorescence and ellipticity measurements, could be modeled by a two‐state mechanism without thermodynamically stable intermediates. Consistent with the two‐state model, one dimensional (1D) NMR spectra and gel‐filtration chromatography analysis did not show any evidence for a folded monomeric intermediate. Interestingly enough, Cys 81 located at the dimeric interface was modified by DTNB before unfolding. This inconsistent result might be explained by increased dynamic motion of the interface residues in the presence of urea to expose Cys 81 more frequently without the dimer dissociation. The refolding process, as monitored by fluorescence change, could best be described by five kinetic phases, in which the second phase was a bimolecular step. Because <30% of the total fluorescence change occurred during the first step, most of the native tertiary structure may be driven to form by the bimolecular step. During the refolding process, negative ellipticity at 225 nm increased very fast within 80 msec to account for >80% of the total amplitude. This result suggests that the protein folds into a monomer containing most of the α‐helical structures before dimerization. Monitoring the enzyme activity during the refolding process could estimate the activity of the monomer that is not fully active. Together, these results stress the importance of dimerization in the formation and maintenance of the functional native tertiary structure.

The conserved cis-Pro39 residue plays a crucial role in the proper positioning of the catalytic base Asp38 in ketosteroid isomerase from Comamonas testosteroni.

KSI (ketosteroid isomerase) from Comamonas testosteroni is a homodimeric enzyme that catalyses the allylic isomerization of Delta5-3-ketosteroids to their conjugated Delta4-isomers at a reaction rate equivalent to the diffusion-controlled limit. Based on the structural analysis of KSI at a high resolution, the conserved cis-Pro39 residue was proposed to be involved in the proper positioning of Asp38, a critical catalytic residue, since the residue was found not only to be structurally associated with Asp38, but also to confer a structural rigidity on the local active-site geometry consisting of Asp38, Pro39, Val40, Gly41 and Ser42 at the flexible loop between b-strands B1 and B2. In order to investigate the structural role of the conserved cis-Pro39 residue near the active site of KSI, Pro39 was replaced with alanine or glycine. The free energy of activation for the P39A and P39G mutants increased by 10.5 and 16.7 kJ/mol (2.5 and 4.0 kcal/mol) respectively, while DG(U)H2O (the free-energy change for unfolding in the absence of urea at 25.00+/-0.02 degrees C) decreased by 31.0 and 35.6 kJ/mol (7.4 and 8.5 kcal/mol) respectively, compared with the wild-type enzyme. The crystal structure of the P39A mutant in complex with d-equilenin [d-1,3,5(10),6,8-estrapentaen-3-ol-17-one], a reaction intermediate analogue, determined at 2.3 A (0.23 nm) resolution revealed that the P39A mutation significantly disrupted the proper orientations of both d-equilenin and Asp38, as well as the local active-site geometry near Asp38, which resulted in substantial decreases in the activity and stability of KSI. Upon binding 1-anilinonaphthalene-8-sulphonic acid, the fluorescence intensities of the P39A and P39G mutants were increased drastically, with maximum wavelengths blue-shifted upon binding, indicating that the mutations might alter the hydrophobic active site of KSI. Taken together, our results demonstrate that the conserved cis-Pro39 residue plays a crucial role in the proper positioning of the critical catalytic base Asp38 and in the structural integrity of the active site in KSI.

Small exterior hydrophobic cluster contributes to conformational stability and steroid binding in ketosteroid isomerase from Pseudomonas putida biotype B

A structural motif called the small exterior hydrophobic cluster (SEHC) has been proposed to explain the stabilizing effect mediated by solvent‐exposed hydrophobic residues; however, little is known about its biological roles. Unusually, in Δ5‐3‐ketosteroid isomerase from Pseudomonas putida biotype B (KSI‐PI) Trp92 is exposed to solvent on the protein surface, forming a SEHC with the side‐chains of Leu125 and Val127. In order to identify the role of the SEHC in KSI‐PI, mutants of those amino acids associated with the SEHC were prepared. The W92A, L125A/V127A, and W92A/L125A/V127A mutations largely decreased the conformational stability, while the L125F/V127F mutation slightly increased the stability, indicating that hydrophobic packing by the SEHC is important in maintaining stability. The crystal structure of W92A revealed that the decreased stability caused by the removal of the bulky side‐chain of Trp92 could be attributed to the destabilization of the surface hydrophobic layer consisting of a solvent‐exposed β‐sheet. Consistent with the structural data, the binding affinities for three different steroids showed that the surface hydrophobic layer stabilized by SEHC is required for KSI‐PI to efficiently recognize hydrophobic steroids. Unfolding kinetics based on analysis of the ΦU value also indicated that the SEHC in the native state was resistant to the unfolding process, despite its solvent‐exposed site. Taken together, our results demonstrate that the SEHC plays a key role in the structural integrity that is needed for KSI‐PI to stabilize the hydrophobic surface conformation and thereby contributes both to the overall conformational stability and to the binding of hydrophobic steroids in water solution.