title: 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.
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Grabarczyk
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Chappell
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
authors.forename: Steven
authors.initials: S
authors.lastname: Johnson
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;
authors.forename: Lukas S
authors.initials: LS
authors.lastname: Stelzl
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
authors.forename: Susan M
authors.identifier: http://orcid.org/0000-0001-9287-8053
authors.initials: SM
authors.lastname: Lea
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom; ben.berks@bioch.ox.ac.uk.
authors.forename: Ben C
authors.initials: BC
authors.lastname: Berks
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D010084:Oxidation-Reduction; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics; D013885:Thiosulfates / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D013885:Thiosulfates; D013455:Sulfur; D050862:Oxidoreductases Acting on Sulfur Group Donors; C413329:SoxZ protein, Paracoccus pantotrophus
keywords: Sox system; swinging arm; thiosulfate oxidation