Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme

Abstract

A 3D-printed modular droplet injector successfully delivered microcrystals of human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin with electrical stimulation in a serial crystallography experiment at 120 Hz repetition rate.

Structural dynamics and functional cooperativity of human NQO1 by ambient temperature serial crystallography and simulations

“…From this analysis, we have also found out that there are significant F I G U R E 8 Comparison of the packing in the two homodimers of hNQO1 from different structures. (a) Superposition of the two homodimers in the microcrystals of the free hNQO1 (orange) and hNQO1-NAD + /H (red), this study, and PDB 8C9J (light blue) (Doppler et al, 2023), to the two homodimers in the large crystals of PDB 8OK0 (purple) (Grieco et al, 2023). (b) Surface representation of the free hNQO1 structure (this study).…”

“…Protein expression and purification of hNQO1 (EC 1.6.99.2) were performed as previously described (Doppler et al, 2023). Briefly, Escherichia coli BL21 (DE3) cells containing the DNA sequence of hNQO1 were grown overnight in LB culture medium supplemented with 0.1 mg/mL ampicillin (LBA) at 37 C. This culture was diluted in 4 L of fresh LBA and grown at 37 C until the optical density at 600 nm reached a value between 0.6 and 0.8.…”

Structural dynamics and functional cooperativity of human NQO1 by ambient temperature serial crystallography and simulations

“…In that study, it was assumed that the interactions established by the FAD and the protein residues were equivalent in the two FAD binding sites, however, since our structure is a room temperature structure and based on previous results from our group (Doppler et al 2023), we decided to analyze the FAD sites from all chains in the ASU.…”

“…A further analysis of the homodimers interface with the PISA server (Krissinel and Henrick 2005) shows that the two homodimers in the room temperature structures of the free hNQO1 (PDB 8C9J (Doppler et al 2023); and this study) and the complex hNQO1-NAD + /H (this study), enclose a surface area that is similar in size (930 Å 2 on average), while the cryo-structure of hNQO1 in complex with PMSF (PDB 8OK0, (Grieco et al 2023)) buries a smaller surface area of 775 Å 2 . From this analysis, we have also found out that there are significant differences in the residues that participate in the interfaces between the two homodimers.…”

“…the active site of hNQO1 upon binding of the hydride donor NADH Since we have recently observed the existence of a high conformational heterogeneity of hNQO1 in the catalytic site with some of the residues being modeled in different conformations(Doppler et al 2023), the structural changes of the catalytic sites of both structures, the free hNQO1 and hNQO1-NAD + /H, were analyzed individually. Analysis of the hNQO1 protein with CASTp program(Tian et al 2018), shows that, overall, the catalytic pocket of hNQO1 buries a total area of 824.02 Å 2 (volume 694.64 Å 3 ) that is 21 found at the dimer interface and comprises the binding pocket for the FAD molecule and the NAD + /H molecule.…”