Addition of PEtN to Thr285 however prevents POPE from accessing the active site. There is persistent interaction between the phosphate group of Thr285-bound PEtN and the active site zinc ion, replicating interactions observed in crystal structures (e.g. PDBPDB 5LRN ) that contain phosphorylated Thr285, and in the simulations described above. The ethanolamine moiety can then make more transient contacts with the "gating" residues His395 and/or His478. In consequence, modification of Thr285 prevents POPE from approaching the MCR-1 zinc center (Figure ), leading to a loss of consistency in orientation of bound POPE in the three replicate simulations. Similarly, addition of a second zinc equivalent to the Zn2 site precludes interaction of POPE with the Zn1 site or Thr285. In these simulations, the starting complex involves Zn2 coordination by the "gating" His395 NE2, Glu116 OE1 from the transmembrane domain, and a POPE phosphoryl oxygen atom. Zn2 remains bound for the full 500 ns duration of all three replicate simulations, but makes a variety of interactions as these proceed, most commonly losing contact with His395 and occupying a range of positions coordinated by combinations of POPE phosphoryl oxygens and Glu116. The situation is exacerbated by PEtN addition to Thr285, with Zn2 rapidly dissociating from its crystallographically defined binding site but remaining bound by combinations of the POPE phosphate group, Glu116 and solvent water molecules. Thus, in simulations of di-zinc MCR-1 in both free and PEtN-modified forms of Thr285 POPE remains bound via interactions of the acyl chains with the transmembrane domain, but is neither positioned nor oriented for interaction or reaction with the catalytic Zn1 center (Figure ).