Review for " Use of Computational and Structural Bioinformatics Strategies in Controlling
Fusarium Wilt in Cotton" Authors Reply in RED
S. Prabhakaran et al 2009.
Despite a few minor errors in English the manuscript is well presented and in a logical clear
order. However a few points need to be expanded upon or clarified before the manuscript is
suitable for publication
Specific Points
p3, line 18 it is stated as fact, without reference that "As a first step in molecular – plant
pathogen interaction, the pathogenecity proteins interact with host proteins and make
modifications in plant proteome." Has this been proven by the authors? Or is there literature
evidence that plant proteins involved in chemical defence are up/down-regulated at the RNA
level?
Reference for this statement has been included in the article.
In the same paragraph - "...there are very minimum reports on the sequence of interaction and
its importance..."
Is this meant to be about the specific amino acids sequences in the proteins that are important
in protein-protein recognition?
The line has been changed now to exactly mean " the order of interaction
i.e., which proteins interact first"
In the general discussion section the authors state that the homology models were assessed
using WhatIf and RAMPAGE to validate the structure but make no mention if the 10% of
residues that fall into dis-allowed areas of Ramachandran space fall in the regions assumed to
be important for binding. This would have broad implications for their docking results if the
crucial regions are not clearly defined in the models used. Similarly a brief statement about the
cutoffs used in the steepest descents and gradients minimisation protocols should be included.
The number of significant figures used for the final energies (up to 5!) is excessive given the
natural error in these calculations. Whole numbers would suffice here.
None of the residues were fall into dis-allowed regions in this analysis. The
default parameters were used and it has also been now included in the
manuscript.
With regards to the modelling studies too, it is not apparent that the authors have modelled the
structures in their physiologically relevent protonation states. For example, terbinafine,
amphotericin B, pentamidine, and probably all the 1,2,4-triazoles are protonated at phsiological
pH, something not apparent in the raw structures downloaded from PubChem. It is stated that
Gasteiger charges were added, but was this to the neutral or protonated species?
To the neutral species
The protein-protein docking calculations were performed treating both partners as rigid bodies,
and conclusions were drawn based on the initial docking results. However in the field this is
clearly not a considered a sufficiently accurate protocol to use. As fully flexible docking of
proteins is not generally feasible using Autodock in comparison to some other docking packages,
at the very least the final docked complexes should be subjected to several rounds of molecular
dynamics/minimisation to allow bad contacts to be relieved, and lower energy loop
conformations to be obtained. Only then can a detailed analysis of the specific interacting
residues be performed. In particular the relative scoring of the small molecules compared to the
proteins would be expected to be significantly lower when the protein-protein structure
complexes are minimised.
This fact is also accepted and it is included in the concluding remarks.