Disorder and cysteines in proteins: A design for orchestration of conformational see-saw and modulatory functions

Abstract

Being responsible for more than 90% of cellular functions, protein molecules are workhorses in all the life forms. In order to cater for such a high demand, proteins have evolved to adopt diverse structures that allow them to perform myriad of functions. Beginning with the genetically directed amino acid sequence, the classical understanding of protein function involves adoption of hierarchically complex yet ordered structures. However, advances made over the last two decades have revealed that inasmuch as 50% of eukaryotic proteome exists as partially or fully disordered structures. Significance of such intrinsically disordered proteins (IDPs) is further realized from their ability to exhibit multifunctionality, a feature attributable to their conformational plasticity. Among the coded amino acids, cysteines are considered to be "order-promoting" due to their ability to form inter- or intramolecular disulfide bonds, which confer robust thermal stability to the protein structure in oxidizing conditions. The co-existence of order-promoting cysteines with disorder-promoting sequences seems counter-intuitive yet many proteins have evolved to contain such sequences. In this chapter, we review some of the known cysteine-containing protein domains categorized based on the number of cysteines they possess. We show that many protein domains contain disordered sequences interspersed with cysteines. We show that a positive correlation exists between the degree of cysteines and disorder within the sequences that flank them. Furthermore, based on the computational platform, IUPred2A, we show that cysteine-rich sequences display significant disorder in the reduced but not the oxidized form, increasing the potential for such sequences to function in a redox-sensitive manner. Overall, this chapter provides insights into an exquisite evolutionary design wherein disordered sequences with interspersed cysteines enable potential modulatory protein functions under stress and environmental conditions, which thus far remained largely inconspicuous.

Keywords: Cysteine-rich sequences; Cysteines; Disulfide bonds; Intrinsically disordered proteins; Intrinsically disordered regions; Metal binding; Modulatory functions; Redox sensitivity.

Fig. 1

Positive correlation between the percentage of cysteines in the protein and their corresponding redox sensitivity based on IUPred2A predictions.

Fig. 2

Correlation between the structure, disorder propensities and redox sensitivity in proteins with cysteine content: <5%. (A–E) PDB-derived structures of the cysteine-containing domains of the respective proteins are displayed in the left panels and the prediction of structural disorder within the proteins performed using IUPred2A computational platform are the right panels. The positions of cysteines in the domains are annotated and their corresponding location in the primary sequence are marked on the right panels. Shown is the disorder score for oxidized forms of the protein (red) with disulfide bonds and reduced forms (dark purple) where cysteine residues in the sequence are swapped with serine. A higher score (0–1) indicates disorder. All five proteins; amyloid precursor protein (A), AEBP2 (B), eotaxin-2 (C), complement decay accelerating factor (D) and phenoloxidase-activating factor-2 (E), have a cysteine content of less than 5%. The positions of cysteine residues within the sequences are indicated with a yellow vertical line while the position of the individual cysteine-rich domains is pointed using the green line. Redox sensitivity (colored light purple) of proteins is marked by regions where the oxidized and reduced forms display significant difference in structural disorder.

Fig. 3

Correlation between the structure, disorder propensities and redox sensitivity in proteins with cysteine content: 5%–10%. (A–E) β defensin (A), phospholipase A2 (B), plasminogen (C) and lysozyme (D) have a cysteine content in the range of 5%–10%. β defensin (A) shows a lack of predicted redox sensitivity across its sequence due to relatively mild differences in disorder propensities of its redox forms while overall, more cysteine residues within these proteins impart greater redox sensitivity.

Fig. 4

Correlation between the structure, disorder propensities and redox sensitivity in proteins with cysteine content: 10%–20%. (A–E) Cysteine residues account for 10%–20% of the sequence in α cobratoxin (A), α bungarotoxin (B), follistatin (C), epidermal growth factor (D) and vanabin-2 (E). Significant differences are observed between the disorder scores of the oxidized and reduced forms of these proteins highlighting a clear trend of increased redox sensitivity with increased cysteine content along with an increase in the loops and coils within the structure as evidenced in the left panels.

Fig. 5

Correlation between the structure, disorder propensities and redox sensitivity in proteins with cysteine content: > 20%. (A–C) GRN-3 (A), GRN-4 and MT-2 (C) have sequences significantly enriched in cysteine residues accounting for more than 20% of the total. These proteins also show stark contrast for disorder propensity among their redox states, with the entire length of the sequence being redox sensitive along with a structure dominated by loops and coils.