第二篇文章被接收了,还是EMBO,review 非常的postive,可能是老板的friends的,晒晒他们的回复
Referee #1 (Remarks to the Author):
Authors determined the dimeric crystal structure of the second PDZ
domain of ZO-1, which plays a important role in intracellular junctions.
The PDZ-ligand complex crystal structures are novel and interesting.
To further support the importance of this structure, the following
biochemical experiments are required.
Major points:
1. In order to know the physiological importance of the charge-charge
interaction of novel dimeric interface, the Gap junction formation should
be examined with Lys209 mutant and Glu238 mutant, respectively.
2. Authors could show that the GGGA-inserted mutant and the wild type ZO-1
PDZ domain are similar in the structure by another technique, such as
circular dichroism. The content of helix and β sheet should be
similar between the two PDZ protein in the analysis.
3. In the dimeric PDZ domain of ZO-1, the two ligands bind to the
canonical binding sites. However, in the Abstract, authors write that
'...the domain-swapped dimerization of ZO-1 PDZ2 generates an
interface...binding site for connexin43 (Cx43) in addition to the
well-separated canonical carboxyl tail binding pocket in each PDZ unit.'
This sentence may mislead the readers to the existence of two different
(independent) binding sites.
In addition, P11, 2nd paragraph, 'second ligand binding pocket located in
the interface...' should be explained carefully.
Referee #2 (Remarks to the Author):
In the present manuscript, Chen et al. investigate the structure and
function of the second PDZ domain of Zonula occludens-1 (ZO-1) and its
interaction with connexin 43 (Cx43) using a variety of structural
biochemical and biophysical techniques and functional analysis. ZO-1 is a
scaffolding protein involved in formation and maintenance of various types
of cellular junctions. Of particular interest is the interaction of the
second PDZ domain of ZO-1 (ZO-1 PDZ2) with connexins, which is important
for the formation and regulation of gap junctions.
The authors used a range of biophysical techniques (including X-ray
crystallography, NMR, fluorescence spectroscopy and analytical
ultracentrifugation) and various biochemical approaches to characterize an
important PDZ-ligand peptide interaction. They determined the crystal
structure of ZO-1 PDZ2 in complex with a Cx43 peptide. Cx43 is the most
abundant connexin in mammals. They observed a domain-swapped dimer formed
between two ZO-1 PDZ2 molecules and showed by NMR that the domain-swapped
dimer exists in solution and is kinetically exceedingly stable. This dimer
provides a canonical and a second, non-canonical binding site for the Cx43
peptide. The authors used mutational analysis to demonstrate the
importance of dimerization for Cx43 binding (they managed to make a
dysfunctional monomeric ZO-1 PDZ2 variant by rational design) and the
importance of a charge-charge interaction network at the second binding
site. By this mutational analysis in combination with conservation pa
ttern
and the structure, they were able to explain the high specificity of the
interaction. They rationalized the domain-swapping from the structure and
used their findings to predict other PDZ domains that may form
domain-swapped dimers. They experimentally showed that dimerization indeed
takes place in at least one of the predicted candidates (the DLG5 PDZ2).
It is also shown in this paper (by using phosphate-mimicking mutations and
by monitoring the binding of a phosphorylated ligand) that phosphorylation
of two serine residues of Cx43, which are neighboring the bound peptide
region, significantly weakens the binding to ZO-1 PDZ2. Finally, the
authors show, using a cell biological assay based on GFP fusion proteins,
that the strength of the specific interaction between Cx43 and ZO-1 PDZ2
is required for the formation of gap junctions in vivo. Taken together,
the work also suggests (but does not prove) that this interaction could be
regulated by phosphorylation of Ser372 and Ser
373
of Cx43.
This paper represents an important contribution to our understanding of
the PDZ domain-ligand recognition code. In addition, the authors
demonstrate how an important PDZ-ligand interaction is regulated by
phosphorylation and rationalize the interaction by structural analysis.
They demonstrate the importance of this interaction and perhaps its
regulation by phosphorylation for gap junction formation in vivo. Thus,
the results reported here are relevant both because of fundamental as well
as specific aspects regarding a cellular recognition event.
The work is technically sound and the synergism from a combination of
analytical techniques makes it particularly appealing. The paper is very
well written and should address a wide audience. Importantly, the authors
did not stop with the in-depth structural and in vitro functional analysis
of the protein complex but they went on to link their findings to in vivo
activity using cell biological assays. All the results are combined in the
end in a functional model.
This referee has virtually no points to raise. Only one very minor issue:
Figure 3B appears to be a cross-eyed stereo view, while the relaxed view
is the appropriate display in journals (as used in Fig. 1B for example).
Thus, the panels of Fig. 3B should be exchanged.
Referee #3 (Remarks to the Author):
The manuscript by Chen et al. describes an elegant structural study of the
interaction of the domain swapped ZO-1 PDZ2 domain with connexin43 (Cx43).
The authors conducted NMR and X-ray diffraction studies that revealed the
mode of the interactions of a 9-residue long peptide representing the
carboxyl tail of Cx43, with the ZO-1 PDZ2 domain dimer. The crystal
structure was solved at 2.4 Å resolution and the quality of the
atomic model, as judged by the statistics and the electron density map is
satisfactory.
The authors have shown by a creative experiment that generated a monomer
of ZO-1 PDZ2, that dimerization is essential for the specificity of the
interaction with Cx43. They then investigated the structural role of the
two serine residues located upstream, and showed that the phosphorylation
of these residues negatively regulates the interaction with ZO-1 PDZ2.
Finally, the authors conducted functional studies using HeLa cells, in
which they were able to monitor the function of Cx43, because these cells
do not have the endogenous protein. Taken together, the experimental data
provided the basis for a model of ZO-1 mediated Cx43 GJ complex assembly.
The manuscript is professionally and clearly written, all the experiments
were ably conducted, and resulted in a multidisciplinary, comprehensive
study clearly worthy of publication. I recommend this paper for
publication.
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四个好沉重的字,但岁月不饶人,27岁了也该到好好考虑考虑的时候了
日子一天一天的过,人一天天的老,千篇一律的工作式研究无情地打击着曾经的血气方刚和敢作敢为,
残酷的选择题又一次摆在面前
是低着头毫无杂念默默地沿着既定的路线前行呢?
还是勇敢地抬起头去找最适合自己前行的路线呢?
前者几乎毫无风险,但是自己未来的角色太窄,太清晰了
后者风险很大,不确定因素很多,也许会飞黄腾达,也许会得不偿失
sigh,成长的烦恼吧
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