Limits...
Probing the mutational interplay between primary and promiscuous protein functions: a computational-experimental approach.

Garcia-Seisdedos H, Ibarra-Molero B, Sanchez-Ruiz JM - PLoS Comput. Biol. (2012)

Bottom Line: Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities.Overall, the results reported may help clarify the mechanisms of the evolution of new functions.From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes.

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias, Departamento de Quimica Fisica, Universidad de Granada, Granada, Spain.

ABSTRACT
Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes.

Show MeSH
Basic mechanism for disulfide oxidation catalyzed by a thioredoxin-fold domain.(Thioredoxin domain shown in blue). The mixed disulfide intermediate has been highlighted in grey. Note that, unlike the mechanism of disulfide reduction shown in Figure 2, resolution of the intermediate occurs through attack of a thiolate group of the substrate. Actually, attack of the thiolate from the catalyst (as in Figure 2) must be prevented since it would revert the substrate to the initial reduced state.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3375227&req=5

pcbi-1002558-g003: Basic mechanism for disulfide oxidation catalyzed by a thioredoxin-fold domain.(Thioredoxin domain shown in blue). The mixed disulfide intermediate has been highlighted in grey. Note that, unlike the mechanism of disulfide reduction shown in Figure 2, resolution of the intermediate occurs through attack of a thiolate group of the substrate. Actually, attack of the thiolate from the catalyst (as in Figure 2) must be prevented since it would revert the substrate to the initial reduced state.

Mentions: The processes of thioredoxin-domain catalyzed reduction, formation (oxidation) and reshuffling of disulfide bonds are all dependent on the active-site CXXC motif. Reduction (see Figure 2) starts with the reduced enzyme and involves the nucleophilic attack of the thiolate form of the amino-terminal cysteine on the disulfide bridge of the substrate [34], [35]. The mixed-disulfide thus formed is resolved by the nucleophilic attack of the carbonyl-terminal cysteine. Oxidation, on the other hand (see Figure 3), involves a nucleophilic attack of the substrate on the disulfide bridge of the oxidized enzyme and the mixed-disulfide intermediate is resolved by attack from the free cysteine (in the thiolate form) of the substrate [36]. It is relevant that the oxidation and reduction processes involve opposite chemical changes in the substrate (break-up and formation of disulfide bridges) as well as different mechanisms for the resolution of the mixed-disulfide intermediate. Furthermore, the two processes may be expected to be linked to different values of the redox potential (as suggested by the redox potentials of thioredoxin and PDI: see Figure 6 in Hatahet & Ruddock [36]) and, as it has been extensively discussed in the literature, they likely have different molecular requirements in terms of the conformational changes during catalysis, the stability of cysteine thiolates and the modulation of the pK values of the catalytic groups [33], [36]–[39]. Clearly, disulfide reduction and catalysis of oxidative folding (involving formation of disulfide bridges) may be expected to strongly trade-off.


Probing the mutational interplay between primary and promiscuous protein functions: a computational-experimental approach.

Garcia-Seisdedos H, Ibarra-Molero B, Sanchez-Ruiz JM - PLoS Comput. Biol. (2012)

Basic mechanism for disulfide oxidation catalyzed by a thioredoxin-fold domain.(Thioredoxin domain shown in blue). The mixed disulfide intermediate has been highlighted in grey. Note that, unlike the mechanism of disulfide reduction shown in Figure 2, resolution of the intermediate occurs through attack of a thiolate group of the substrate. Actually, attack of the thiolate from the catalyst (as in Figure 2) must be prevented since it would revert the substrate to the initial reduced state.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375227&req=5

pcbi-1002558-g003: Basic mechanism for disulfide oxidation catalyzed by a thioredoxin-fold domain.(Thioredoxin domain shown in blue). The mixed disulfide intermediate has been highlighted in grey. Note that, unlike the mechanism of disulfide reduction shown in Figure 2, resolution of the intermediate occurs through attack of a thiolate group of the substrate. Actually, attack of the thiolate from the catalyst (as in Figure 2) must be prevented since it would revert the substrate to the initial reduced state.
Mentions: The processes of thioredoxin-domain catalyzed reduction, formation (oxidation) and reshuffling of disulfide bonds are all dependent on the active-site CXXC motif. Reduction (see Figure 2) starts with the reduced enzyme and involves the nucleophilic attack of the thiolate form of the amino-terminal cysteine on the disulfide bridge of the substrate [34], [35]. The mixed-disulfide thus formed is resolved by the nucleophilic attack of the carbonyl-terminal cysteine. Oxidation, on the other hand (see Figure 3), involves a nucleophilic attack of the substrate on the disulfide bridge of the oxidized enzyme and the mixed-disulfide intermediate is resolved by attack from the free cysteine (in the thiolate form) of the substrate [36]. It is relevant that the oxidation and reduction processes involve opposite chemical changes in the substrate (break-up and formation of disulfide bridges) as well as different mechanisms for the resolution of the mixed-disulfide intermediate. Furthermore, the two processes may be expected to be linked to different values of the redox potential (as suggested by the redox potentials of thioredoxin and PDI: see Figure 6 in Hatahet & Ruddock [36]) and, as it has been extensively discussed in the literature, they likely have different molecular requirements in terms of the conformational changes during catalysis, the stability of cysteine thiolates and the modulation of the pK values of the catalytic groups [33], [36]–[39]. Clearly, disulfide reduction and catalysis of oxidative folding (involving formation of disulfide bridges) may be expected to strongly trade-off.

Bottom Line: Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities.Overall, the results reported may help clarify the mechanisms of the evolution of new functions.From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes.

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias, Departamento de Quimica Fisica, Universidad de Granada, Granada, Spain.

ABSTRACT
Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes.

Show MeSH