NMR structural and functional studies of the mithochondrial outer membrane protein VDAC by Thomas J. Malia.

Abstract

Apoptosis is a mechanism of programmed cell death required by multicellular organisms during development and for tissue maintenance. Bcl-2 family proteins are central regulators of apoptosis and many of their primary roles are carried out in the outer mitochondrial membrane. Anti-apoptotic Bcl-xL has been found previously to interact with VDAC, an outer mitochondrial membrane protein responsible for metabolite trafficking. Here, the NMR-based investigation of VDAC and its interaction with Bcl-xL in detergent micelles is described. As an integral membrane protein VDAC presented a challenge for producing a folded form amenable to solution NMR studies. Methods developed for expression and purification of human VDAC for structural studies by NMR spectroscopy were developed. Optimal sample conditions were explored in a screen of various detergents and buffers. Suitable NMR sample conditions for VDAC were identified and allowed further characterization of VDAC and its interactions by NMR and other methods. Obtained through various means, evidence of a concentration dependent self-association of VDAC is also presented. Chemical cross-linking, analytical size exclusion chromatography, and NMR spectroscopic studies strongly support an equilibrium model for LDAO-purified VDAC between monomer and trimer.

(cont.) Methods that were necessary to carry out NMR structural studies of VDAC and results from those studies are described. In addition to various methods previously developed for solution NMR spectroscopy of very large proteins, extensive labeling approaches and unconventional experimental NMR methods were necessary to obtain a high level of chemical shift assignment for micelle-bound VDAC. Chemical shift assignment has allowed the preliminary characterization of ATP and Bcl-xL binding to VDAC. Binding of Bcl-xL, a central regulator of apoptosis, to VDAC is demonstrated here. Using NMR spectroscopy, the VDAC-binding region of Bcl-xL has been mapped to a putative helical hairpin motif, which also mediates insertion into membranes. The stoichiometry of the VDAC/Bcl-xL complex is shown to be a heterotrimer of two VDAC monomers and one Bcl-xL. The demonstration of VDAC/Bcl-xL complex formation supports the concept that components of apoptosis and metabolism are integrally connected, and that interplay between the two processes is required for regulation of cell survival and cell death.