Chemokines form a family of signaling proteins mainly responsible for directing the traffic of leukocytes, where their biological activity can be modulated by their oligomerization state. We characterize the dynamics and thermodynamic stability of monomer and homodimer structures of CXCL7, one of the most abundant platelet chemokines, using experimental methods that include circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, and computational methods that include the anisotropic network model (ANM), molecular dynamics (MD) simulations and the distance constraint model (DCM). A consistent picture emerges for the effects of dimerization and Cys5-Cys31 and Cys7-Cys47 disulfide bonds formation. The presence of disulfide bonds is not critical for maintaining structural stability in the monomer or dimer, but the monomer is destabilized more than the dimer upon removal of disulfide bonds. Disulfide bonds play a key role in shaping the characteristics of native state dynamics. The combined analysis shows that upon dimerization flexibly correlated motions are induced between the 30s and 50s loop within each monomer and across the dimer interface. Interestingly, the greatest gain in flexibility upon dimerization occurs when both disulfide bonds are present, and the homodimer is least stable relative to its two monomers. These results suggest that the highly conserved disulfide bonds in chemokines facilitate a structural mechanism that is tuned to optimally distinguish functional characteristics between monomer and dimer.
RiVax is a candidate ricin toxin subunit vaccine antigen that has proven to be safe in human Phase I clinical trials. In this study we introduced double and triple cavity-filling point mutations into the RiVax antigen with the expectation that stability enhancing modifications would have a beneficial effect on overall immunogenicity of the recombinant proteins. We demonstrate that two RiVax triple mutant derivatives, RB (V81L/C171L/V204I) and RC (V81I/C171L/V204I), when adsorbed to aluminum salts adjuvant and tested in a mouse prime-boost-boost regimen were 5 to 10-fold more effective than RiVax at eliciting toxin-neutralizing serum IgG antibody (TNA) titers. Increased TNA values and seroconversion rates were evident at different antigen dosages and within seven days following the first booster. Quantitative stability/flexibility relationships (QSFR) analysis revealed that the RB and RC mutations affect rigidification of regions spanning residues 98 to 103, which constitutes a known immunodominant neutralizing B cell epitope. A more detailed understanding of the immunogenic nature of RB and RC may provide insight into the fundamental relationship between local protein stability and antibody reactivity.
DNA transposons can be employed for stable gene transfer in vertebrates. The Sleeping Beauty (SB) DNA transposon has been recently adapted for human application and is being evaluated in clinical trials, however its molecular mechanism is not clear. SB transposition is catalyzed by the transposase enzyme, which is a multi-domain protein containing the catalytic and the DNA-binding domains. The DNA-binding domain of the SB transposase contains two structurally independent subdomains, PAI and RED. Recently, the structures of the catalytic domain and the PAI subdomain have been determined, however no structural information on the RED subdomain and its interactions with DNA has been available. Here, we used NMR spectroscopy to determine the solution structure of the RED subdomain and characterize its interactions with the transposon DNA.
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