PubMed · 8424712
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B Barron. 1993. Corneal toxicity from acidic vancomycin solution.. https://doi.org/10.1001/archopht.1993.01090010020003
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The actin-ADP-ribosylating binary Clostridium botulinum C2 toxin consists of two individual proteins, the binding/translocation component C2II and the enzyme component C2I. To elicit its cytotoxic action, C2II binds to a receptor on the cell surface and mediates cell entry of C2I via receptor-mediated endocytosis. Here we report that binding of C2II to the surface of target cells requires cleavage of C2II by trypsin. Trypsin cleavage causes oligomerization of the activated C2II (C2IIa) to give SDS-stable heptameric structures, which exhibit a characteristic annular or horseshoe shape and form channels in lipid bilayer membranes. Cytosolic delivery of the enzyme component C2I is blocked by bafilomycin but not by brefeldin A or nocodazole, indicating uptake from an endosomal compartment and requirement of endosomal acidification for cell entry. In the presence of C2IIa and C2I, short term acidification of the extracellular medium (pH 5.4) allows C2I to enter the cytosol directly. Our data indicate that entry of C2 toxin into cells involves (i) activation of C2II by trypsin-cleavage, (ii) oligomerization of cleaved C2IIa to heptamers, (iii) binding of the C2IIa oligomers to the carbohydrate receptor on the cell surface and assembly with C2I, (iv) receptor-mediated endocytosis of both C2 components into endosomes, and finally (v) translocation and release of C2I into the cytosol after acidification of the endosomal compartment.
Protein stability in vitro can be influenced either by introduction of mutations or by changes in the chemical composition of the solvent. Recently, we have characterized the thermodynamic stability and the rate of folding of the engineered dimeric leucine zipper A(2), which has a strengthened hydrophobic core [Dürr, E., Jelesarov, I., and Bosshard, H. R. (1999) Biochemistry 38, 870-880]. Here we report on the energetic consequences of a cavity introduced by Leu/Ala substitution at the tightly packed dimeric interface and how addition of 30% glycerol affects the folding thermodynamics of A(2) and the cavity mutants. Folding could be described by a two-state transition from two unfolded monomers to a coiled coil dimer. Removal of six methylene groups by Leu/Ala substitutions destabilized the dimeric coiled coil by 25 kJ mol(-1) at pH 3.5 and 25 degrees C in aqueous buffer. Destabilization was purely entropic at around room temperature and became increasingly enthalpic at elevated temperatures. Mutations were accompanied by a decrease of the unfolding heat capacity by 0.5 kJ K(-1) mol(-1). Addition of 30% glycerol increased the free energy of folding of A(2) and the cavity mutants by 5-10 kJ mol(-1) and lowered the unfolding heat capacity by 25% for A(2) and by 50% for the Leu/Ala mutants. The origin of the stabilizing effect of glycerol varied with temperature. Stabilization of the parent leucine zipper A(2) was enthalpic with an unfavorable entropic component between 0 and 100 degrees C. In the case of cavity mutants, glycerol induced enthalpic stabilization below 50 degrees C and entropic stabilization above 50 degrees C. The effect of glycerol could not be accounted for solely by the enthalpy and entropy of transfer or protein surface from water to glycerol/water mixture. We propose that in the presence of glycerol the folded coiled coil dimer is better packed and displays less intramolecular fluctuations, leading to enhanced enthalpic interactions and to an increase of the entropy of folding. This work demonstrates that mutational and solvent effects on protein stability can be thermodynamically complex and that it may not be sufficient to only analyze changes of enthalpy and entropy at the unfolding temperature (T(m)) to understand the mechanisms of protein stabilization.
We have expanded our description of the energy landscape for folding of the SH3 domain of chicken alpha-spectrin by a detailed structural characterization of its denatured state ensemble (DSE). This DSE is significantly populated under mildly acidic conditions in equilibrium with the folded state. Evidence from heteronuclear nuclear magnetic resonance (NMR) experiments on (2)H, (15)N-labeled protein suggests the presence of conformers whose residual structure bears some resemblence to the structure of the folding transition state of this protein. NMR analysis in a mutant with an engineered, non-native alpha-helical tendency shows a significant amount of local non-native structure in the mutant, while the overall characteristics of the DSE are unchanged. Comparison with recent theoretical predictions of SH3 domain folding reactions reveals an interesting correlation with the predicted early events. Based on these results and recent data from other systems, we propose that the DSE of a protein will resemble the intermediate or transition state of its nearest rate-limiting step, as a consequence of simple energetic and kinetic principles.