Probing the Mutational Interplay between Primary and Promiscuous Protein Functions: A Computational-Experimental Approach

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

How Thioredoxin Dissociates Its Mixed Disulfide

The dissociation mechanism of the thioredoxin (Trx) mixed disulfide complexes is unknown and has been debated for more than twenty years. Specifically, opposing arguments for the activation of the nucleophilic cysteine as a thiolate during the dissociation of the complex have been put forward. As a key model, the complex between Trx and its endogenous substrate, arsenate reductase (ArsC), was used. In this structure, a Cys29Trx-Cys89ArsC intermediate disulfide is formed by the nucleophilic attack of Cys29Trx on the exposed Cys82ArsC-Cys89ArsC in oxidized ArsC. With theoretical reactivity analysis, molecular dynamics simulations, and biochemical complex formation experiments with Cys-mutants, Trx mixed disulfide dissociation was studied. We observed that the conformational changes around the intermediate disulfide bring Cys32Trx in contact with Cys29Trx. Cys32Trx is activated for its nucleophilic attack by hydrogen bonds, and Cys32Trx is found to be more reactive than Cys82ArsC. Additionally, Cys32Trx directs its nucleophilic attack on the more susceptible Cys29Trx and not on Cys89ArsC. This multidisciplinary approach provides fresh insights into a universal thiol/disulfide exchange reaction mechanism that results in reduced substrate and oxidized Trx

Redox-active cysteines of a membrane electron transporter DsbD show dual compartment accessibility

The membrane-embedded domain of the unusual electron transporter DsbD (DsbDβ) uses two redox-active cysteines to catalyze electron transfer between thioredoxin-fold polypeptides on opposite sides of the bacterial cytoplasmic membrane. How the electrons are transferred across the membrane is unknown. Here, we show that DsbDβ displays an inherent functional and structural symmetry: first, the two cysteines of DsbDβ can be alkylated from both the cytoplasm and the periplasm. Second, when the two cysteines are disulfide-bonded, cysteine scanning shows that the C-terminal halves of the cysteine-containing transmembrane segments 1 and 4 are exposed to the aqueous environment while the N-terminal halves are not. Third, proline residues located pseudo-symmetrically around the two cysteines are required for redox activity and accessibility of the cysteines. Fourth, mixed disulfide complexes, apparent intermediates in the electron transfer process, are detected between DsbDβ and thioredoxin molecules on each side of the membrane. We propose a model where the two redox-active cysteines are located at the center of the membrane, accessible on both sides of the membrane to the thioredoxin proteins