Micrococcin Biosynthesis


Thiopeptides, such as micrococcin P1 (MP1), are ribosomally synthesized and post-translationally modified peptides (RiPPs).


MerA C-terminal Tail (CTT) Flexibility

MerA mercury pathway MerA C-terminal tail
Pathway for Hg2+ acquisition and reduction by MerA (left), and the ionic triad modulating the C-terminal tail rates of Hg2+ attainment and tail equilibrium (right). The yellow spheres represent relavant cysteine residues.

Mercuric ion reductase (MerA) is an oxidoreductase part of the Hg2+-inducible mer operon. The role of MerA is to acquire cytosolic Hg2+ bound to cysteine residues in proteins or thiol containing metabolites (e.g., glutathione). Structurally, MerA is a homodimer with each monomer containing a 50 kDa catalytic core and a 7 kDa metallochaperone-like N-terminal domain (NmerA) connected by a 30-residue linker. NmerA facilitates Hg2+ attainment from the cytosol, especially proteins. It docks to the side of the catalytic core (red domain in image) where the flexible C-terminal Tail (CTT) protrudes from the dimer cleft to obtain Hg2+ from NmerA and delivers it to the inner cysteines. The inner cysteines participate in an FAD-mediated NADPH reduction of Hg2+ to the volitle and substaintially less nucleophilic Hg0.


Crystal structures of MerA indicate high B-factors in the CTT atoms, which suggests flexibility in the CTT. Given this flexibility, it was reasoned that an anchor must exist to hold the tail in the dimer cleft, avoiding the entorpically and sterically more favorable state of the tail free in solution. Steady-state and pre-equilibrium kinetics of of an alanine mutant of a conserved Lys suggested that this residue assists in favoring the bound state of the CTT in the dimer cleft. Since there exists a large network of uncharacterized electrostatic residues in the dimer cleft, we computationally investigated their role in the CTT dynamics with the Rosetta protocool FloppyTail. This revealed a series of residues that play a role in modulating the CTT dynamics and kinetics of Hg2+ acquisition. We are validating the computational work with with pre-equilibrium kinetics and NMR spectroscopy.





Hg2+ Proteomics