As an author cited in this publication for the HIV-1 PR substrate-groove model (S-groove, Laco GS, Biochimie, 2015, 118, 90-103, https://doi.org/10.1016/j...) and acknowledged contributor of the model for the HIV-1 PR with bound 24-mer substrate (S1 Fig.) I was interested in the author’s results on Ty1 PR, and also because the Ty1 PR was reported in this publication to be resistant to eight HIV-1 PR active site inhibitors making the Ty1 PR a multidrug resistant protease. And while not noted in the Fig. 7A protease alignment, the Ty1 PR residue T111 aligned with HIV-1 PR residue A71, a key S-groove residue that when mutated to T/V/I/L contributed to multidrug resistance in HIV-1 PR as well as in other retroviral proteases including HIV-2, SIV, EIAV, and FIV (Laco GS, J. Mol. Biochem., 2017, 6, 45-63, http://jmolbiochem.com/in...). The Ty1 PR also contains other potential S-groove resistance residues based on the alignment in Fig. 7A with HIV-1 PR, see above cited publications and the Stanford University HIV Drug Resistance Data Base (https://hivdb.stanford.ed...).
While the HIV-1 PR S-groove studies used a 24aa cleavage site, the authors here used only a 20aa cleavage site. All the same, selected results from Table 1C support the Ty1 PR having S-groove that increased binding affinity for 20aa cleavage sites, versus 10aa cleavage sites: PR/IN cleavage site (2.4-fold lower Km for 20aa cleavage site versus 10aa site); and Gag/PR cleavage site (2-fold lower Km for 20aa cleavage site versus 10aa site). Though when the PR/IN_20aa_wt cleavage site P10-P6/P6’-P10’ residues (predicted to bind in the S-grooves) were mutated to Gly the PR/IN_20aa_mut cleavage site had almost identical Km and kcat values as the wild-type PR/IN_20aa_wt site (Table 1C). A potential problem with mutating 5 cleavage site residues in a row to Gly is that the peptide backbone becomes exposed and flexible allowing the amide nitrogens and carbonyl oxygens to make electrostatic interactions with potential Ty1 PR S-groove residues, this may in part explain the results for the PR/IN_20aa_mut cleavage site. In Fig. 5B the significant increase in cleavage of the PR/IN_20aa_wt site, versus the PR/IN_10aa_wt site, was evident especially for the N-terminal cleavage product (see “N”, lower stained gel, 16 h lane). Unfortunately, a similar gel comparison was not made for the IN/RT and Gag/PR cleavage sites (only 20aa wt results shown).
However, there is potential issue with the assay used by the authors to measure Ty1 PR cleavage of 10aa and 20aa cleavage sites inserted into the middle of the non-native His6 MBP-mTurquoise2 recombinant fluorescent protein (Fig. 5A/C). In the case of the HIV-1 PR, it was shown in structure-based modeling studies that the HIV-1 PR makes a number of direct contacts with residues in a MA/CA polyprotein “outside” of the 24aa cleavage site, and those interactions significantly increased the overall binding and interaction energy score (see Fig. 7 and Results, Laco GS, Biochimie, 2015, 118, 90-103). If the Ty1 PR makes similar contacts with native Gag/Pol residues outside of the 10aa/20aa cleavage sites, it would then make non-native interactions (positive/negative) with the His6 MBP-mTurquoise2 protein on either side of the inserted cleavage sites. As a consequence, the Ty1 PR affinity for the inserted cleavage sites would be affected by both the length and secondary structure of the cleavage sites, since those factors would affect the proximity and orientation, respectively, of the His6 MBP-mTurquoise2 protein on either side of the inserted cleavage site. A cleavage assay that eliminated those potential factors may yield different results for the cleavage sites tested in Table 1C and D.
Given the above and the remarkable similarity of the Ty1 and HIV-1 PRs secondary and tertiary structure (Fig. 7B), including the vertically aligned anti-parallel beta-sheet and alpha helix which form the S-groove in HIV-1 PR (see left half of HIV-1 PR model in Fig. 7B for front facing S-groove), I think the Ty1 PR may have S-grooves that bind substrate residues outside of P5-P5’. Hopefully further studies will either confirm, or more convincingly disprove, that hypothesis.