Manthei, K. A. | Search bioRxiv

bioRxiv2024

Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 [A], in what appears to be an inactive state. In this conformation, a predicted -helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg2+-dependent 3-5 DNA exonuclease. We show that Leu113 aids in coordinating the 3 end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/580553v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@ac964borg.highwire.dtl.DTLVardef@bf1f81org.highwire.dtl.DTLVardef@1ad4e89org.highwire.dtl.DTLVardef@ff0641_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2018

Molecular basis for activation of lecithin:cholesterol acyltransferase by a compound that increases HDL cholesterol

Lecithin:cholesterol acyltransferase (LCAT) and LCAT-activating small molecules are being investigated as treatments for coronary heart disease (CHD) and familial LCAT deficiency (FLD). Herein we report the crystal structure of LCAT bound to a potent activator and an acyl intermediate-like inhibitor, thereby revealing an active conformation of LCAT and that the activator is bound exclusively to its membrane-binding domain (MBD). Functional studies indicate that the compound does not modulate the affinity of LCAT for HDL, but instead stabilizes residues in the MBD and likely facilitates channeling of substrates into the active site. By demonstrating that these activators increase the activity of an FLD variant, we show that compounds targeting the MBD have therapeutic potential. In addition, our data better define the acyl binding site of LCAT and pave the way for rational design of LCAT agonists and improved biotherapeutics for augmenting or restoring reverse cholesterol transport in CHD and FLD patients.

biochemistry↗