Our earlier cryo-electron microscopic studies provided detailed insights into the three-dimensional (3D) organization of icosahedral PDH complexes. Our studies established the basic architecture that enables substrate channeling in this complex. The critical role of PDH at a central branch point in cellular metabolism is reflected by the multiple layers of regulation, often combined in a highly cell-specific manner, that control the precise flux of carbon between pyruvate and acetyl-CoA in response to cellular bioenergetic and biosynthetic requirements. Over the last year, we have initiated a series of experiments aimed at studies of the structure and dynamics of enzymes that are directly connected to PDH in the larger context of mitochondrial metabolism. One such enzyme of considerable interest is glutamate dehydrogenase, a hexameric inner mitochondrial enzyme that is found in all organisms and catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate using either NADP(H) or NAD(H). In contrast to GDH from bacteria, mammalian GDH demonstrates negative cooperativity with respect to coenzyme, activation by ADP, and inhibition by GTP. The enzyme plays a pivotal role by acting as a redox sensor in the catabolic and biosynthetic pathways. Depending on the energetic demands of the cell, GDH is allosterically regulated. Using cryo-EM analyses, we show that in contrast to prior EM findings, GDH does not associate as elongated polymeric chains and the addition of ADP did not promote such phenomena. Instead, our results indicate that the enzyme exists as discrete and well-defined hexamers. Furthermore, the addition of GTP to the apo form of the enzyme did not cause the conformation of the enzyme to shift from an open to closed state. Another enzyme we are studying in a similar vein is lactate dehydrogenase, a critical protein in the metabolism of lactate, a metabolite that is produced in large quantities by cancer cells. We are using cryo-EM to systematically map the conformational changes in these enzymes by the binding of small molecules, including nucleotides, divalent ions, and other common cellular molecules, which enable rapid modulation of enzyme activity. The prospect that cryo-EM can be used as a tool to decipher conformational changes in these complexes is very appealing, especially if the structural information is sufficient to obtain atomic resolution models, because of the potential that they can serve a critical role in the process of drug development. In joint work with the Subramaniam lab, we have now shown that the solution structure of a small 465 kDa enzyme beta-galactosidase can be solved at 3.2 Angstrom resolution using single particle cryo-EM. Densities for most side-chains, including those of residues in the active site, and a catalytic Mg2+ ion can be discerned in the map obtained by cryo-EM. The atomic model derived from our cryo-EM analysis closely matches the 1.7 Angstrom crystal structure with a global RMSD of 0.66 Angstrom. There are significant local differences throughout the protein, with clear evidence for conformational changes resulting from contact zones in the crystal lattice. Detailed comparison of the atomic model derived by cryo-EM with several X-ray structures shows small, but significant differences. For example, the RMSD of all C-alpha atoms relative to the 1.7 Angstrom resolution X-ray structure (1DP0) is only 0.66 Angstrom, but it is worth noting that individual C-alpha atoms differ by as much as 4.6 Angstrom. As expected, the best agreement is in the more central regions, and the greatest differences are in the periphery of the tetramer. Interestingly, some of the largest deviations occur precisely at zones of crystal contacts. Comparison of the crystal structure with the structure of the protein in solution in the absence of lattice contacts shows a measurable shift of the C-alpha trace and an alteration of the local side-chain conformations at these zones, although significant deviations are also observed elsewhere in the protein. These differences show that contact with neighboring molecules in the crystal results both in stabilization and local perturbation of the protein structure. Overall, this comparison confirms the potential of cryo-EM maps for de novo protein structure determination, especially in instances in which deletions, mutations and crystal contacts can result in differences in conformation between the solution structure and the crystallized form. The advances in cryo-EM that allow determination of structures of small protein complexes and membrane proteins at near-atomic resolution mark a critical shift in structural biology. Even when a number of crystal structures are already available, the determination of an atomic resolution model that captures the conformation and active site geometry in solution can be very important for detailed quantitative analysis of the reaction mechanism.
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