Stability and specificity of heterodimer formation for the coiled‐coil neck regions of the motor proteins Kif3A and Kif3B: the role of unstructured oppositely charged regions

Abstract: We investigated the folding, stability, and specificity of dimerization of the neck regions of the kinesin‐like proteins Kif3A (residues 356–416) and Kif3B (residues 351–411). We showed that the complementary charged regions found in the hinge regions (which directly follow the neck regions) of these proteins do not adopt any secondary structure in solution. We then explored the ability of the complementary charged regions to specify heterodimer formation for the neck region coiled‐coils found in Kif3A and Kif3B. Redox experiments demonstrated that oppositely charged regions specified the formation of a heterodimeric coiled‐coil. Denaturation studies with urea demonstrated that the negatively charged region of Kif3A dramatically destabilized its neck coiled‐coil (urea_1/2 value of 3.9 m compared with 6.7 m for the coiled‐coil alone). By comparison, the placement of a positively charged region C‐terminal to the neck coiled‐coil of Kif3B had little effect on stability (urea_1/2 value of 8.2 m compared with 8.8 m for the coiled‐coil alone). The pairing of complementary charged regions leads to specific heterodimer formation where the stability of the heterodimeric neck coiled‐coil with charged regions had similar stability (urea_1/2 value of 7.8 m ) to the most stable homodimer (Kif3B) with charged regions (urea_1/2 value of 8.0 m ) and dramatically more stable than the Kif3A homodimer with charged regions (urea_1/2, value of 3.9 m ). The heterodimeric coiled‐coil with charged extensions has essentially the same stability as the heterodimeric coiled‐coil on its own (urea_1/2 values of 7.8 and 8.1 m , respectively) suggesting that specificity of heterodimerization is driven by non‐specific attraction of the oppositely unstructured charged regions without affecting stability of the heterodimeric coiled‐coil.     

Oversulfated chondroitin sulfate interaction with heparin-binding proteins: New insights into adverse reactions from contaminated heparins

An oversulfated chondroitin sulfate (OSCS) was identified as a contaminant to pharmaceutical heparin and severe anaphylactoid reactions were ascribed to this contaminant. An examination of the biochemistry underlying both the anticoagulant activity and the toxic effects of oversulfated chondroitin sulfate was undertaken. This study demonstrates that the anticoagulant activity of this oversulfated chondroitin sulfate is primarily dependent on heparin cofactor II mediated inhibition of thrombin. Heparin and oversulfated chondroitin sulfate binding to coagulation, kinin-kallikrein and complement proteins were studied by surface plasmon resonance. While oversulfated chondroitin sulfate binds tightly to antithrombin III, unlike heparin, OSCS does not induce antithrombin III to undergo the conformational change required for its inactivation of thrombin and factor Xa. In contrast to heparin, oversulfated chondroitin sulfate tightly binds factor XIIa suggesting a biochemical mechanism for the factor XIIa-based enhancement of vasoactive bradykinin production.