UbiD domain dynamics underpins aromatic decarboxylation

The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic compounds based on reversible 1,3-dipolar cycloaddition between substrate and prFMN occurs within the confines of the active site. In contrast, during aromatic acid decarboxylation, substantial rearrangement of the substrate aromatic moiety associated with covalent catalysis presents a molecular dynamic challenge. Here we determine the crystal structures of the multi-subunit vanillic acid decarboxylase VdcCD. We demonstrate that the small VdcD subunit acts as an allosteric activator of the UbiD-like VdcC. Comparison of distinct VdcCD structures reveals domain motion of the prFMN-binding domain directly affects active site architecture. Docking of substrate and prFMN-adduct species reveals active site reorganisation coupled to domain motion supports rearrangement of the substrate aromatic moiety. Together with kinetic solvent viscosity effects, this establishes prFMN covalent catalysis of aromatic (de)carboxylation is afforded by UbiD dynamics.

Large Multidomain Protein NMR: HIV-1 Reverse Transcriptase Precursor in Solution

NMR studies of large proteins, over 100 kDa, in solution are technically challenging and, therefore, of considerable interest in the biophysics field. The challenge arises because the molecular tumbling of a protein in solution considerably slows as molecular mass increases, reducing the ability to detect resonances. In fact, the typical 1H-13C or 1H-15N correlation spectrum of a large protein, using a 13C- or 15N-uniformly labeled protein, shows severe line-broadening and signal overlap. Selective isotope labeling of methyl groups is a useful strategy to reduce these issues, however, the reduction in the number of signals that goes hand-in-hand with such a strategy is, in turn, disadvantageous for characterizing the overall features of the protein. When domain motion exists in large proteins, the domain motion differently affects backbone amide signals and methyl groups. Thus, the use of multiple NMR probes, such as 1H, 19F, 13C, and 15N, is ideal to gain overall structural or dynamical information for large proteins. We discuss the utility of observing different NMR nuclei when characterizing a large protein, namely, the 66 kDa multi-domain HIV-1 reverse transcriptase that forms a homodimer in solution. Importantly, we present a biophysical approach, complemented by biochemical assays, to understand not only the homodimer, p66/p66, but also the conformational changes that contribute to its maturation to a heterodimer, p66/p51, upon HIV-1 protease cleavage.