Referee 2's review:

The manuscript 'Inhibition of MAPKK activation by Yesinia YopJ requires binding to its substrate' by Hao et al. explores the molecular details of the interaction between the bacterial toxin YopJ and its substrate MEKs. The manuscript reports the identification of mutations on MEKs that render them refractory to inhibition by the acetyl transferase activity of YopJ. Analysis of these mutant MEKs identifies a mutation that renders MEK constitutively active. This analysis also suggests a putative binding site for YopJ on MEKs. The study is well planned and executed and reports results that will be of interest to those working in the field of signal transduction. The results will help our understanding of the MEKs as well as of the process by which the bacterial toxin YopJ inactivates MEK. I recommend that the manuscript be accepted in principle for publication in PLOS One.

I have only two comments that need to be addressed:
(a) It is repeatedly mentioned in the manuscript that YopJ does not interact with and thus does not block the activation of IKKalpha (based on Reference 5). However, it has been shown in Reference 7 that IKKalpha is indeed acetylated by YopJ - in fact the site(s) of acetylation on IKKalpha have been identified and reported. Granted that Reference 5 shows a lack of interaction of YopJ with IKKalpha - however, absence of proof of interaction cannot be taken to be proof of absence of interaction. The positive identification of sites of acetylation on IKKalpha is proof that
YopJ can interact with IKKalpha in mammalian cells. The authors of the current manuscript should make note of this observation in the text and then develop the logic of their experimental approach in this context. Ignoring the observation is not proper. This is an important point because the assumed inability of YopJ to acetylate IKKalpha is taken to be the basis for designing the I531F mutation in Pbs2.

(b) The text on Page 7 states that the mutation P1 maps to a residue on a loop between beta strands 4 and 5. However, that does not appear to be the case in the depiction of Figure 1D.

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N.B. These are the general comments made by the reviewer when reviewing this paper in light of which the manuscript was revised. Specific points addressed during revision of the paper are not shown.