Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery

Abstract

Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ ∼200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.

Figure 1. Cryo-EM Analysis of LDHB Bound to Inhibitor GSK2837808A

(A) Cryo-EM density map of complex showing density for GSK2837808A (red) and selected residues in the binding pocket. (B) Ribbon diagram of refined structure of LDHB showing the location of the bound inhibitor (marked by arrows) on the periphery of the tetramer. (C) Structure of GSK2837808A. (D and E) Visualization of side-chains in the cryo-EM density map in an α-helical segment (D) and loop region (E) of the polypeptide. See also Figure S1.

Figure 2. Cryo-EM Analysis of IDH1 in the Absence and Presence of the Inhibitor ML309

(A) Cryo-EM map of the apo-IDH1 (isocitrate dehydrogenase) dimer, colored by subunit. (B and C) Selected regions of the IDH1 map demonstrating density for side-chains in a α-helical region (B) and a β sheet region (C). (D) Cryo-EM map of IDH1 in complex with ML309 showing density (red) for the inhibitor (inset) close to the dimer interface. (E) Superposition of the apo-(gray) and ML309-bound (yellow/blue) IDH1 structures shows the outward movement of the subunits with ML309 binding (density shown in red). The black arrows indicate the direction of the changes in tertiary structure while the yellow and blue arrows show the overall movements at the level of quaternary structure. See also Figures S2 and S3, and Movie S1.

Figure 3. Cryo-EM Analysis of GDH

(A and B) Projection views of two 3D classes from cryo-EM analysis of GDH (glutamate dehydrogenase). In both classes, there is well-defined density at the core, but it is weakly defined at the peripheral nucleotide binding domain (NBD). The two classes display similar structures in the interior but differ slightly in the peripheral NBD. The two classes are likely to be subsets of a continuum of states with varying orientations for the outer domain relative to the core. (C) Ribbon diagrams of the open and closed structures (Borgnia et al., 2016) demonstrating the more extensive NBD movement associated with substrate binding and catalytic cleft closure. (D) A selected region of the cryo-EM map of the GDH structure, highlighting high-resolution features such as “holes” in the aromatic rings of Tyr³⁸², Phe³⁸³, and Trp³⁸⁵, water molecules (shaded yellow), and well-resolved densities for carbonyl bonds. See also Figures 4 and S4 and Movie S2.

Figure 4. Density Representations for a Representative Example of Each of the 20 Amino Acid Types from the 1.8 Å Resolution Cryo-EM Structure of apo-GDH

Features such as holes in aromatic rings, as well as the “zigzag” structure of extended sidechains such as Arg and Lys, are visible in the density maps. See also Figure S5 and Movie S3.

Figure 5. Relative Sizes of Small Metabolic Complexes Compared to Other Structures Solved by Cryo-EM and Variations in Flexibility across Different Regions

(A) Relative sizes of GDH, LDHB, and IDH1 in comparison to a representative set of other structures that have been solved by cryo-EM to near-atomic resolution including icosahedral viruses, ribosomes, and protein complexes such as β-galactosidase and the AAA ATPase p97. (B–D) Ribbon diagram of hexameric GDH (B), tetrameric LDHB (C), and dimeric IDH1 (D), colored to show B-factor variation from blue (lowest) to red (highest) B factors, respectively. See also Movie S3.