Pang, Yuan‐Ping

The mechanism by which the proapoptotic Bcl-2 family members Bax and Bak release cytochrome c from mitochondria is incompletely understood. In this paper, we show that activator BH3-only proteins bind tightly but transiently to the Bak hydrophobic BH3-binding groove to induce Bak oligomerization, liposome permeabilization, mitochondrial cytochrome c release, and cell death. Analysis by surface plasmon resonance indicated that the initial binding of BH3-only proteins to Bak occurred with similar kinetics with or without detergent or mitochondrial lipids, but these reagents increase the strength of the Bak–BH3-only protein interaction. Point mutations in Bak and reciprocal mutations in the BH3-only proteins not only confirmed the identity of the interacting residues at the Bak–BH3-only protein interface but also demonstrated specificity of complex formation in vitro and in a cellular context. These observations indicate that transient protein–protein interactions involving the Bak BH3-binding groove initiate Bak oligomerization and activation.

The mitochondrial enzyme, dihydrolipoamide dehydrogenase (DLD), is essential for energy metabolism across eukaryotes. Here, conditions known to destabilize the DLD homodimer enabled the mouse, pig, or human enzyme to function as a protease. A catalytic dyad (S456–E431) buried at the homodimer interface was identified. Serine protease inhibitors and an S456A or an E431A point mutation abolished the proteolytic activity, whereas other point mutations at the homodimer interface domain enhanced the proteolytic activity, causing partial or complete loss of DLD activity. In humans, mutations in the DLD homodimer interface have been linked to an atypical form of DLD deficiency. These findings reveal a previously unrecognized mechanism by which certain DLD mutations can simultaneously induce the loss of a primary metabolic activity and the gain of a moonlighting proteolytic activity. The latter could contribute to the metabolic derangement associated with DLD deficiency and represent a target for therapies of this condition.

Conformationally altered proteins and protein fragments derived from the extracellular matrix and hemostatic system may function as naturally occurring angiogenesis inhibitors. One example of such a protein is cleaved high molecular weight kininogen (HKa). HKa inhibits angiogenesis by inducing apoptosis of proliferating endothelial cells, effects mediated largely by HKa domain 5. However, the mechanisms underlying the antiangiogenic activity of HKa have not been characterized, and its binding site on proliferating endothelial cells has not been defined. Here, we report that the induction of endothelial cell apoptosis by HKa, as well as the antiangiogenic activity of HKa in the chick chorioallantoic membrane, was inhibited completely by antitropomyosin monoclonal antibody TM-311. TM-311 also blocked the high-affinity Zn 2+ -dependent binding of HKa to both purified tropomyosin and proliferating endothelial cells. Confocal microscopic analysis of endothelial cells stained with monoclonal antibody TM-311, as well as biotin labeling of cell surface proteins on intact endothelial cells, revealed that tropomyosin exposure was enhanced on the surface of proliferating cells. These studies demonstrate that the antiangiogenic effects of HKa depend on high-affinity binding to endothelial cell tropomyosin.

Atomic force microscope manipulations of single polysaccharide molecules have recently expanded conformational chemistry to include force-driven transitions between the chair and boat conformers of the pyranose ring structure. We now expand these observations to include chair inversion, a common phenomenon in the conformational chemistry of six-membered ring molecules. We demonstrate that by stretching single pectin molecules (1 → 4-linked α- d -galactouronic acid polymer), we could change the pyranose ring conformation from a chair to a boat and then to an inverted chair in a clearly resolved two-step conversion: 4 C 1 ⇄ boat ⇄ 1 C 4 . The two-step extension of the distance between the glycosidic oxygen atoms O 1 and O 4 determined by atomic force microscope manipulations is corroborated by ab initio calculations of the increase in length of the residue vector O 1 O 4 on chair inversion. We postulate that this conformational change results from the torque generated by the glycosidic bonds when a force is applied to the pectin molecule. Hence, the glycosidic bonds act as mechanical levers, driving the conformational transitions of the pyranose ring. When the glycosidic bonds are equatorial (e), the torque is zero, causing no conformational change. However, when the glycosidic bond is axial (a), torque is generated, causing a rotation around C—C bonds and a conformational change. This hypothesis readily predicts the number of transitions observed in pyranose monomers with 1a-4a linkages (two), 1a-4e (one), and 1e-4e (none). Our results demonstrate single-molecule mechanochemistry with the capability of resolving complex conformational transitions.