Amino acid function relates to its embedded protein microenvironment: A study on disulfide‐bridged cystine

Abstract

In our previous study, we have shown that the microenvironments around conserved amino acids are also conserved in protein families (Bandyopadhyay and Mehler, Proteins 2008; 72:646-659). In this study, we have hypothesized that amino acids perform similar functions when embedded in a certain type of protein microenvironment. We have tested this hypothesis on the microenvironments around disulfide-bridged cysteines from high-resolution protein crystal structures. Although such cystines mainly play structural role in proteins, in certain enzymes they participate in catalysis and redox reactions. We have performed and report a functional annotation of enzymatically active cystines to their respective microenvironments. Three protein microenvironment clusters were identified: (i) buried-hydrophobic, (ii) exposed-hydrophilic, and (iii) buried-hydrophilic. The buried-hydrophobic cluster encompasses a small group of 22 redox-active cystines, mostly in alpha-helical conformations in a -C-x-x-C- motif from the Oxido-reductase enzyme class. All these cystines have high strain energy and near identical microenvironments. Most of the active cystines in hydrolase enzyme class belong to buried hydrophilic microenvironment cluster. In total there are 34 half-cystines detected in buried hydrophilic cluster from hydrolases, as a part of enzyme active site. Even within the buried hydrophilic cluster, there is clear separation of active half-cystines between surface exposed part of the protein and protein interior. Half-cystines toward the surface exposed region are higher in number compared to those in protein interior. Apart from cystines at the active sites of the enzymes, many more half-cystines were detected in buried hydrophilic cluster those are part of the microenvironment of enzyme active sites. However, no active half-cystines were detected in extremely hydrophilic microenvironment cluster, that is, exposed hydrophilic cluster, indicating that total exposure of cystine toward the solvent is not favored for enzymatic reactions. Although half-cystines in exposed-hydrophilic clusters occasionally stabilize enzyme active sites, as a part of their microenvironments. Analysis performed in this work revealed that cystines as a part of active sites in specific enzyme families or folds share very similar protein microenvironment regions, despite of their dissimilarity in protein sequences and position specific sequence conservations. Proteins 2016; 84:1576-1589. © 2016 Wiley Periodicals, Inc.

Characterization of cysteine thiol modifications based on protein microenvironments and local secondary structures

“…Both the cysteines from CxxC motif have exhibited helical secondary structure (Figure A). This observation was in contrast to the half‐cystines in CxxC motifs of disulfides, where 1 cysteine belonged to helix and the other 1 belongs to turn . Total 30 out of 38 cysteines in intermediate cluster belongs to helical conformation (Figure B).…”

“…Protein microenvironment played a crucial role in modulating the structure and catalytic activity of disulfide‐bridged cysteine . In this work, we have demonstrated the functions (chemical modifications) of cysteine thiol (reduced form of disulfide‐bridged cystine) side chains embedded in different protein microenvironments and secondary structures.…”

“…Population of cysteine thiol group gradually decreased from buried‐hydrophobic microenvironment cluster to exposed‐hydrophilic micro‐environment cluster. Cystine disulfide, the oxidized form of thiol, was also distributed in 3 different microenvironment clusters . However, the frequencies of populations differ among these 2 redox forms.…”

“…Frequency of population was defined here as number of cysteines (or cystines) in respective cluster divided by total number of cysteines (or cystines). According to the protein structure based hydrophobicity scale developed earlier, disulfide containing cystine exhibited maximum hydrophobicity, hence minimum frequency of population was observed in exposed hydrophilic cluster (0.015) [based on the data from table of reference]. On the other hand, the frequency of population for cysteine thiol group in exposed hydrophilic cluster was 0.03, twice compared to the value in disulfide group.…”

“…We have earlier developed protein microenvironment quantitative descriptor that is the summation of hydrophobic contribution from all the protein fragments and solvent molecules present within the first contact shell of a particular amino acid (or its functional group) . Recently, we have demonstrated that protein microenvironments around ‐S‐S‐ linkage of cystines are conserved, those are part of the active sites in specific enzyme classes, irrespective of their very low sequence similarity . However, the study was confined to enzymatic cystines only, no references were made with respect to the non‐enzymatic cystines.…”