High-resolution cryo-EM proteasome structures in drug development

Abstract

With the recent advances in biological structural electron microscopy (EM), protein structures can now be obtained by cryo-EM and single-particle analysis at resolutions that used to be achievable only by crystallographic or NMR methods. We have explored their application to study protein-ligand interactions using the human 20S proteasome, a well established target for cancer therapy that is also being investigated as a target for an increasing range of other medical conditions. The map of a ligand-bound human 20S proteasome served as a proof of principle that cryo-EM is emerging as a realistic approach for more general structural studies of protein-ligand interactions, with the potential benefits of extending such studies to complexes that are unfavourable to other methods and allowing structure determination under conditions that are closer to physiological, preserving ligand specificity towards closely related binding sites. Subsequently, the cryo-EM structure of the Plasmodium falciparum 20S proteasome, with a new prototype specific inhibitor bound, revealed the molecular basis for the ligand specificity towards the parasite complex, which provides a framework to guide the development of highly needed newgeneration antimalarials. Here, the cryo-EM analysis of the ligand-bound human and P. falciparum 20S proteasomes is reviewed, and a complete description of the methods used for structure determination is provided, including the strategy to overcome the bias orientation of the human 20S proteasome on electronmicroscope grids and details of the icr3d software used for three-dimensional reconstruction.

Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes

“…Quantifoil R1.2/1.3 electron microscope grids, with a 300 gold mesh, were coated with a thin layer of carbon freshly floated from mica, following the procedures we previously optimized for the preparation of grids with endogenous human proteasomes (da Fonseca and Morris, 2015, Morris and da Fonseca, 2017). We find that the continuous carbon layer favors an even particle distribution, while its electron scattering facilitates the assessment of the information in the recorded images, as judged by the recovery of Thon rings to high resolution in their power spectra, which also contributes to an accurate defocus estimation for the correction of the contrast transfer function associated effects.…”

Microfluidic protein isolation and sample preparation for high-resolution cryo-EM

“…Moreover, the similar subunits around the “pseudo-7-fold” axis can lead to misalignments of the particle projections. Together, these factors explain why existing cryo-EM maps are at the lower resolution of 3.5 Å (29, 32).…”

Microfluidic protein isolation and sample preparation for high-resolution cryo-EM

“…Moreover, the similar subunits around the 'pseudo-sevenfold' axis can lead to misalignments of the particle projections. Together, these factors explain why existing cryo-EM maps are at the lower resolution of 3.5 Å [23,24].…”