The disruption of protein hydroxylation is highly associated with several serious diseases and consequently the identification of protein hydroxylation sites has attracted significant attention recently. Here, we report the development of an improved method, called HydPred, to identify protein hydroxylation sites (hydroxyproline and hydroxylysine) based on the synthetic minority over-sampling technique (SMOTE), the random forest (RF) algorithm and four blocks of newly composed features that are derived from the protein primary sequence. The HydPred method achieved the best prediction performance reported until now with Matthew's correlation coefficient values of 0.770 and 0.857 for hydroxyproline and hydroxylysine, respectively, according to jack-knife cross-validation. This represents an improvement of 8% for hydroxyproline and 19% for hydroxylysine compared to the best results of available predictors. The prediction performance of HydPred for the external validation of hydroxyproline and hydroxylysine was also improved compared with other published methods. We subsequently applied HydPred to study the association of disruption of hydroxylation sites with human inherited disease. The analyses suggested that the loss of hydroxylation sites is more likely to cause disease instead of the gain of hydroxylation sites and 52 different human inherited diseases were found to be highly associated with the loss of hydroxylation sites. Therefore, HydPred represents a new strategy to discover the molecular basis of pathogenesis associated with abnormal hydroxylation. HydPred is now available online as a user-friendly web server at http://lishuyan.lzu.edu.cn/hydpred/.

Phthalate esters (PAEs) are a ubiquitous kind of environmental endocrine that disrupt chemicals, causing environmental and health issues. EstJ6 is an effective phthalate-degrading hydrolase, and its mutant with a combination of three non-conservative distal mutations has an improved activity against PAEs with unknown molecular mechanisms. Herein, we attempt to fill the significant gap between distal mutations and the activity of this enzyme using computational approaches. We found that mutations resulted in a redistribution of the enzyme’s preexisting conformational states and dynamic changes of key functional regions, especially the lid over the active site. The outward motion of the lid upon the mutations made it easier for substrates or products to enter or exit. Additionally, a stronger substrate binding affinity and conformational rearrangements of catalytic reaction-associated residues in the mutant, accompanied by the strengthened communication within the protein, could synergistically contribute to the elevated catalytic efficiency. Finally, an attempt was made to improve the thermostability of EstJ6 upon introducing a distal disulfide bond between residues A23 and A29, and the simulation results were as expected. Together, our work explored the allosteric effects caused by distal mutations, which could provide insights into the rational design of esterases for industrial applications in the future.

The parathyroid hormone type 1 receptor (PTH1R) acts as a canonical class B G protein-coupled receptor, regulating crucial functions including calcium homeostasis and bone formation. The identification and development of PTH1R non-peptide allosteric modulators have obtained widespread attention. It has been found that a negative allosteric modulator (NAM) could inhibit the activation of PTH1R, but the implied mechanism remains unclear. Herein, extensive molecular dynamics simulations together with multiple analytical approaches are utilized to unravel the mechanism of PTH1R allosteric inhibition. The results suggest that the binding of NAM destabilizes the structure of the PTH1R–PTH–spep/qpep (the C terminus of Gs/Gq proteins) complexes. Moreover, the presence of NAM weakens the binding of PTH/peps (spep and qpep) and PTH1R. The intra- and inter-molecular couplings are also weakened in PTH1R upon NAM binding. Interestingly, compared with our previous study of the positive allosteric effects induced by extracellular Ca²⁺, the enhanced correlation between the PTH and G-protein binding sites is significantly reduced by the replacement of this negative allosteric regulator. Our findings might contribute to the development of new therapeutic agents for diseases caused by the abnormal activation of PTH1R.