As our starting point, we performed EVB simulations of Chimeras 2 and 3 for comparison to our prior simulations of wild-type PTP1B and YopH . As in prior work , we focused here on modelling just the second hydrolysis step (Figure ), as this is rate-limiting . Important to note is that due to the lack of ligand-bound crystal structures of either chimera with the WPD-loop in its catalytically active closed conformation, simulations were initiated from the wild-type YopH crystal structure (PDB ID: 2I42 ), with the relevant WPD-loop substitutions for each chimera modelled in manually to mimic a hypothetical closed structure of each chimera, as described in the Methods section. This thus represents an "idealized" conformation of the active site and WPD-loop which is not necessarily sampled in reality; however, starting from this idealized conformation is useful in that it allows us to distinguish between chemical effects caused by the loop-substitutions (that would manifest even in an idealized loop-closed conformation) and those caused by loop dynamics (including an inability to sample the narrowly defined closed conformation). Specifically, we know that Chimera 2 and Chimera 3 both sample wide-open conformations, at least in their unliganded forms, and that Chimera 3 retains activity (albeit diminished compared to wild-type), while Chimera 2 is inactive . Our EVB simulations aim to address whether there are differences at the level of the active sites of the closed conformation of each chimera that led to the loss of activity in these constructs, or whether the loss of activity is more likely to be a result of the inability of the WPD-loop to sample a catalytically-optimal closed conformation, in particular in Chimera 2, which is not catalytically active. We note that our prior work has suggested a strong link between WPD-loop sequence and the conformational dynamics of the WPD-loop in chimeras with some or all of the YopH WPDloop grafted onto the PTP1B scaffold. Furthermore, the PTP1B chimeras shifted to a closed conformation of the WPD-loop, in contrast to the YopH chimeras studied here.