*3.1. Protein Expression and Purification*

The codon-optimized full-length and Δ3-22 human CYP3A4 were produced as reported previously [29] and used for the spectral and structural studies, respectively.

#### *3.2. Spectral Binding Titrations*

Ligand binding to CYP3A4 was monitored in a Cary 300 spectrophotometer at ambient temperatures in 0.1 M phosphate buffer, pH 7.4, containing 20% glycerol and 1 mM dithiothreitol. The investigated compounds were dissolved in dimethyl sulfoxide (DMSO) to 0.2–20 mM concentration and added to a 1.5–2 μM protein solution in small aliquots, with the final solvent concentration <2%. After the addition of each aliquot, the reaction mixture was permitted to stand until no further absorbance changes could be detected, usually less than 20 min. At the end of titrations, the spectra of the ferrous CO-adduct were recorded to ensure that there was no CYP3A4 inactivation during lengthy measurements. Difference spectra were recorded in a separate experiment, where glycerol was omitted and equal amounts of DMSO were added to the reference cuvette to correct for the solvent-induced spectral perturbations. The spectral dissociation constant (Ks) was derived from the hyperbolic, sigmoidal or quadratic fits to the titration curves using IgorPro (WaveMetrics, Inc., Portland, OR, USA). To assess the ligands' dissociability, CYP3A4 was saturated with the substrate and then titrated with ritonavir (0.2–5 mM solution in DMSO) to determine how occupation of the active site affects its dissociation constant (Ks RIT). Each titration experiment was repeated three times. The average Ks values and standard errors are given in Table 1. The high-spin content was estimated based on the absorbance spectra of ligand-free (100% low-spin) and azamulin-bound CYP3A4 (100% high-spin conversion).

#### *3.3. Determination of the X-ray Structures*

Δ3-22 CYP3A4 was co-crystallized with the investigated compounds at room temperature using a sitting drop vapor diffusion method. The protein (50–70 mg/mL or 0.9–1.25 mM in 100 mM potassium phosphate buffer, pH 7.4, 20% glycerol and 2 mM dithiothreitol) was incubated with a 5-fold ligand excess and centrifuged to remove the precipitate (<5% protein loss). The supernatant (0.4–0.5 μL) was mixed with 0.4–0.6 μL of the crystallization solution containing 6–10% polyethylene glycol 3350 and either 70 mM malate, 50 mM succinate, or 80 mM ammonium citrate tribasic, pH 7.0, for mibefradil-, azamulin- and DHB-bound CYP3A4, respectively. After harvesting, crystals were cryoprotected with Paratone-N and frozen in liquid nitrogen. The X-ray diffraction data were collected at the Stanford Synchrotron Radiation Lightsource beamline 7-1 and the Advanced Light Source beamline 5.0.2. The high-resolution cutoffs were chosen based on the CC1/2 value [30]. Crystal structures were solved by molecular replacement with PHASER [31], using the 5VCC structure as a search model. The ligands were built with eLBOW [32] and manually fit into the density with COOT [33]. The initial models were rebuilt and refined with COOT and PHENIX [32]. The polder and simulated annealing omit electron density maps were calculated with PHENIX. Data collection and refinement statistics are summarized in Table S1. The atomic coordinates and structure factors for mibefradil-, azamulin- and DHB-bound CYP3A4 were deposited to the Protein Data Bank with the ID codes 6OO9, 6OOA and 6OOB, respectively.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/17/ 4245/s1 and include five figures, showing spectral changes observed upon displacement of CYP3A4-bound bromocryptine, midazolam, bergamottin and DHB by ritonavir, simulated annealing omit electron density maps, superposition of the ligand-free and mibefradil-, DHB- and azamulin-bound structures of CYP3A4, and a table with the X-ray data collection and refinement statistics.

**Funding:** This work was funded by the National Institutes of Health grant ES025767.

*Int. J. Mol. Sci.* **2019**, *20*, 4245

**Acknowledgments:** This study involves research carried out at the Stanford Synchrotron Radiation Lightsource and the Advanced Light Source. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

**Conflicts of Interest:** The author declares no competing financial interest.
