Properties of the Thioredoxin Fold Superfamily Are Modulated by a Single Amino Acid Residue

Guoping Ren,Daniel Stephan,Zhaohui Xu,Ying Zheng,Danming Tang,Rosemary S Harrison,Mareike Kurz,Russell Jarrott,Stephen R Shouldice,Annie Hiniker,Jennifer L Martin,Begonña Heras,James C.A Bardwell

doi:10.1074/jbc.m809509200

Abstract

The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.

Highlights

Protein-disulfide isomerase [5], which catalyzes the oxidation and shuffling of disulfides in the endoplasmic reticulum of eukaryotic cells
Isoleucine is common in thioredoxins, threonine is common in the prokaryotic disulfide isomerases DsbC and DsbG, and valine is common in DsbA and glutaredoxins
Given this pattern of conservation, the close proximity in three-dimensional space of this residue to the active site and the existence of gain and loss of function mutations that affected this cisProminus1 residue in DsbG [23], we were intrigued by the possibility that the character of the cisPro-minus1 residue helps to determine the divergent properties of the various subfamilies of thioredoxin fold proteins

Summary

Sequence of the CXXC motif and cisPro loop

The Sequences of each protein family surrounding CXXC and cis-proline were compiled and used to generate sequence logos. To determine the conservation of the residues in the CXXC and cis-proline loop in these proteins, we analyzed the proteins from genomes that are as divergent as possible; we wanted to avoid comparing proteins that have been evolving over different evolutionary time frames. Thioredoxin and glutaredoxin, for instance, are present in eukaryotes, bacteria, and archaea, and have been evolving for at least 3.8 billion years [53], whereas DsbC and DsbG are restricted to proteobacteria and have probably been evolving for at least ϳ0.5 billion years [23, 53]. We restricted our comparison to genomes that contain an orthologue to DsbC. We obtained the sequence of the individual thioredoxin, DsbA, DsbG, and glutaredoxin orthologues present in these individual genomes using blast by searching with the E. coli homologue

EXPERIMENTAL PROCEDURES

Isomerase Activity Assay with

Redox potential mV

RESULTS

DISCUSSION