Structural basis for specificity and promiscuity in a carrier protein/enzyme system from the sulfur cycle

Abstract

The bacterial Sox (sulfur oxidation) pathway is an important route for the oxidation of inorganic sulfur compounds. Intermediates in the Sox pathway are covalently attached to the heterodimeric carrier protein SoxYZ through conjugation to a cysteine on a protein swinging arm. We have investigated how the carrier protein shuttles intermediates between the enzymes of the Sox pathway using the interaction between SoxYZ and the enzyme SoxB as our model. The carrier protein and enzyme interact only weakly, but we have trapped their complex by using a "suicide enzyme" strategy in which an engineered cysteine in the SoxB active site forms a disulfide bond with the incoming carrier arm cysteine. The structure of this trapped complex, together with calorimetric data, identifies sites of protein-protein interaction both at the entrance to the enzyme active site tunnel and at a second, distal, site. We find that the enzyme distinguishes between the substrate and product forms of the carrier protein through differences in their interaction kinetics and deduce that this behavior arises from substrate-specific stabilization of a conformational change in the enzyme active site. Our analysis also suggests how the carrier arm-bound substrate group is able to outcompete the adjacent C-terminal carboxylate of the carrier arm for binding to the active site metal ions. We infer that similar principles underlie carrier protein interactions with other enzymes of the Sox pathway.swinging arm | thiosulfate oxidation | Sox system

Intermediates in the Sox sulfur oxidation pathway are bound to a sulfane conjugate of the carrier protein SoxYZ

“…SoxCD is related to sulfite oxidases [12]. Consistent with these inferred catalytic similarities SoxB has been shown to have thiosulfohydrolase activity using the small-molecule substrate analogue trithionate [7] while SoxCD is able to catalyse sulfite oxidation [13]. Nevertheless, it is important to realise that none of the proposed reactions of the Sox pathway has been directly demonstrated.…”

Computational investigation of the conformational dynamics in Tom20‐mitochondrial presequence tethered complexes

“…In the Tom20‐presequence complex, we found the common major and minor distributions of the conformational ensemble, independent of the choice of Ala or Tyr in the spacer region of the linker, but absence of a certain minor distribution due to the bulky side chain of the Tyr residue. In general, the cross‐linking via a disulfide‐bond is widely used as a biochemical technique to fix a certain conformation among multiple conformations of protein molecules or to stabilize the bound state of a ligand . The primary goal of cross‐linking is to trap one particular state/conformation for which preservation of dynamics in the cross‐linked complexes is generally not considered.…”

“…Disulfide‐bond formation has been successfully used for co‐crystallization of protein‐peptide complexes with low affinity, fixing a certain state of a biological complex among many conformations and studies on the binding properties of ligands . In these experiments, a disulfide‐bond was introduced to trap one particular state/conformation, without considering residual dynamics in the stabilized state/conformation.…”

Methodologies for “Wiring” Redox Proteins/Enzymes to Electrode Surfaces

“…In living systems, the exchange of electrons between soluble redox proteins is dependent on the two proteins “docking” so that the electron‐donor and electron‐acceptor redox centers are brought into close enough approach to facilitate rapid, direct electron transfer . These interactions are often mediated by areas of complementary polarity on the donor and acceptor proteins .…”

“…In living systems, the exchange of electrons between soluble redox proteins is dependent on the two proteins “docking” so that the electron‐donor and electron‐acceptor redox centers are brought into close enough approach to facilitate rapid, direct electron transfer . These interactions are often mediated by areas of complementary polarity on the donor and acceptor proteins . Thus, a simple model for understanding successful direct electroactive protein adsorption onto electrode surfaces is to envisage the electrode surface polarity complementing a region of oppositely charged residues on the protein surface that is proximal to the electron entry/exit redox center (Figure ) .…”