Interaction Domain on Thioredoxin for Pseudomonas aeruginosa 5′-Adenylylsulfate Reductase

Jung-Sung Chung,Valérie Noguera-Mazon,Jean-Marc Lancelin,Sung-Kun Kim,Masakazu Hirasawa,Maggy Hologne,Thomas Leustek,David B Knaff

doi:10.1074/jbc.m109.035634

Abstract

NMR spectroscopy has been used to map the interaction domain on Escherichia coli thioredoxin for the thioredoxin- dependent 5'-adenylylsulfate reductase from Pseudomonas aeruginosa (PaAPR). Seventeen thioredoxin amino acids, all clustered around Cys-32 (the more surface-exposed of the two active-site cysteines), have been located at the PaAPR binding site. The center of the binding domain is dominated by nonpolar amino acids, with a smaller number of charged and polar amino acids located on the periphery of the site. Twelve of the amino acids detected by NMR have non-polar, hydrophobic side chains, including one aromatic amino acid (Trp-31). Four of the thioredoxin amino acids at the PaAPR binding site have polar side chains (Lys-36, Asp-61, Gln-62 and Arg-73), with three of the four having charged side chains. Site-directed mutagenesis experiments have shown that replacement of Lys-36, Asp-61, and Arg-73 and of the absolutely conserved Trp-31 significantly decreases the V(max) for the PaAPR-catalyzed reduction of 5'-adenylylsulfate, with E. coli thioredoxin serving as the electron donor. The most dramatic effect was observed with the W31A variant, which showed no activity as a donor to PaAPR. Although the thiol of the active-site Cys-256 of PaAPR is the point of the initial nucleophilic attack by reduced thioredoxin, mutagenic replacement of Cys-256 by serine has no effect on thioredoxin binding to PaAPR.

Highlights

Oredoxin is capable of reducing almost all disulfide bonds in proteins
Given that the Kd for the non-covalent complex is in the range appropriate for studying protein/protein interactions by NMR spectroscopy [26], an attempt to map the site on thioredoxin involved in binding PaAPR using NMR seemed feasible
Thioredoxin from P. aeruginosa was not available, E. coli thioredoxin can serve as an efficient electron donor to the thioredoxin-dependent PaAPR, and previous work in our laboratory had already characterized a number of aspects of the interaction of this thioredoxin with PaAPR [17]

Summary

EXPERIMENTAL PROCEDURES

Wild-type PaAPR and its C256S variant were prepared and purified as described previously [17, 18]. Previous studies carried out in two of our laboratories these variants were confirmed by DNA sequencing in the Biodemonstrated that the APS reductase from Pseudomonas technology Core Facility at Texas Tech University) These aeruginosa (PaAPR), like PAPS reductase, could form a dis- mutated thioredoxin variants were expressed and purified ulfide-linked covalent complex with E. coli thioredoxin [17]. Variants of the E. coli and C. reinhardtii thioredoxins, in The gene encoding E. coli NADPH:thioredoxin oxidoreducwhich the resolving cysteine was eliminated by mutagenesis, tase (hereafter abbreviated NTR) was cloned into the pET28b were used to trap the disulfide-linked PaAPR-thioredoxin vector between the NdeI and BamHI restriction enzyme sites complexes These studies identified the C-terminal, cat- by PCR using 5Ј-ACGTCATATGGGCACGACCAAACA-. Where ⌬␦ is the chemical shift variation between free thioredoxin and thioredoxin bound to PaAPR

RESULTS

Relative Vmaxa

NA e

DISCUSSION