Microcrystallization and room-temperature serial crystallography structure of human cytochrome P450 3A4

Abstract

The cytochrome P450 family of enzymes are key players in the metabolism of foreign substances in the body, including pharmaceutical compounds, and therefore important to take into consideration during drug development. The main human isoform is CYP3A4, a highly flexible protein that can act on a diverse set of substances and that is inhibited by compounds varying greatly in size. To accompany the different ligands, substantial conformational changes occur that transform the active-site binding pocket between a collapsed form and various open states. A large body of biophysical data including high-resolution structures are available but there is still a lack of understanding of the dynamic properties of CYP3A4. Here, we present the first room-temperature structure of CYP3A4 solved by serial crystallography. The structure is overall very similar to structures solved at cryo-temperature of the un-bound form of the enzyme including the conformation of the active-site lid. We observe that loops are better defined at room-temperature despite the lower resolution of this structure. Based on an internal distance matrix analysis of a large set of CYP3A4 structures, we conclude that the crystal form rather than temperature is determining for how the structures cluster. Finally, a workflow for generating microcrystals suitable for fixed-target serial crystallography data collection is described. This work lays the foundation for future studies of ligand-induced dynamics and structural transitions during the catalytic reaction of CYP3A4.