Crystal Structure of Candida antarctica Lipase B with a Putative Pro-Peptide Region

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript "Crystal structure of candida antarctica lipase B with the putative pro-peptide region" by Sohail et al describes the structure of CalB with density for the amino acids 19-25 visible. The structure shown provides some support for the lack of classical pro-peptide function in this region (lack of refolding/stabilisation of an auto-inhibited form). The manuscript is well written and should be published with minor revisions.

 

1. The potential for impact of the crystallisation conditions on structure should be more fully indicated. It is not impossible that the indicated crystallisation conditions may have caused the N-terminal residues to be re-arranged in the crystal lattice. A demonstration of the full-length protein (including 19-25) activity should be included for the sample that was used for crystallisation (along with an accompanying SDS showing no change in primary sequence). 
2. Some concentrations are incompletely described (eg. ln 83 : is the 0.8% w/v or v/v?). The authors should check the manuscript and fix these omissions.
3. The IMAC buffers are not described. These should be shown.
4. If the concentration measurement was performed using UV/Vis the extinction coefficient used should be shown.
5. Table 3 - the completeness in the high-resolution shell is 3% (280 reflections). While I do understand there may be differing opinions on where to cut this data (and the challenges of creating another 1.1A diffracting sample?). I think this is far too low to be meaningful. The authors should report the data collection statistics to 1.45 in line with their refinement statistic report.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Key note:

This work is confusing to me. The introduction poses two questions the authors want to answer, but in the same introduction, they write that the answer to these questions was found in the previous article (Van Tassel et al., 2020). Therefore, it turns out that this work is completely pointless.

 

I quote:

«This suggests that the “pro-peptide” does not have  a significant function in folding to the native state and hence implies if it is a pro-peptide it may maintain the enzyme in an inactive state. However, we have reported high-level production of CalB that includes the N-terminal putative pro-peptide along with the mature protein (Ala19 – Pro 342), with the enzyme having higher activity than commercially sourced CalB (Van Tassel et al., 2020)....

... This raises the questions as to whether this segment of seven acid acids is a pro-peptide region. If so, what the function of this segment might be and if it interacts with the lid domain or the active site in any way to modulate activity?»

 

But to understand that seven residues from the N-terminus to the active site are insufficient, can be calculated from previous structures; a crystal structure is not required. Furthermore, the fact that the enzyme is not inhibited is clear from previous work showing its activity. So, the authors were simply lucky that the crystal contacts captured these seven residues in chain A, but they could have obtained a structure similar to chain B, and this would not have changed anything. This tail is clearly mobile in solution, and its structure in chain A is not its natural state.

The authors need to give a more clear answer as to why this work is needed, other than the fact that it was simply done.

 

 

Minor comments:

Line 18   candida antarctica change to Candida antarctica

 

Table 3

If Rwork in % it must be 13.59 (19.45)

Rfree the same

 

Line 142  “space group P21” change to  “space group P2₁ “

 

Fig. 2 Please add to panel b crystal contacts

and describe them in the text, so in fact it was the crystal contacts that allowed us to fix this area in chain A, in chain B these contacts are different, therefore 7 residues of chain B are not visible.

 

Figure 3: There is no description for the black-colored end of the protein. Is it likely the N-terminus? Please add the end labels to the figure and the loop numbering in the figure description.

 

In Table 4, please add a column with the structure resolution, cell parameters, and symmetry group. This will clarify whether the crystal packing has changed due to the extension of the N-terminus.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

the authors answered to all my questions