The lability of the Zn2 site is a consistent feature of the simulations described above and, notwithstanding the potential for the additional phosphoryl groups to act as zinc ligands, is not substantially reduced by PEtN addition to Thr285 or by the presence of POPE. However, both the importance to activity of the conserved potential Zn2 ligands Glu116, His395 and His478 15 , and our previous molecular simulations suggesting that a second zinc equivalent can facilitate PEtN transfer to the lipid A acceptor , indicate a potential role for a candidate Zn2 site. Accordingly, as a further investigation of the architecture and stability of a potential Zn2 site, simulations of di-zinc MCR-1 were repeated with coordinating residues to both Zn1 and Zn2 sites restrained throughout minimization, heating, equilibration and production MD to the crystallographic distances and angles measured in the crystal structure of the catalytic domain in the di-zinc form (PDB 5LRM ). In simulations in the absence of POPE, a stable Zn2 site, involving coordination by Glu116 as well as His395 and His478, is observed (Figure ). On modification by PEtN addition the Thr285-bound phosphoryl group then "bridges" the two zinc ions with each coordinated by an additional oxygen atom (Figure ). These arrangements were stable and consistent across the two sets of replicate simulations. When POPE is present Zn2 coordination can involve both POPE and Glu116 (Figure ) but varies between replicates, and is stabilized by PEtN addition to Thr285 to generate an arrangement whereby the Thr285 backbone rotates to enable coordination of Zn2. In consequence the bridging interaction of the two Zn ions by the PEtN phosphoryl group is lost, and Zn2 is coordinated by the phosphoryl groups of both PEtN and POPE (Figure ). This stable architecture was observed in all three replicate simulations.