Trp101-Mediated Cold Adaptation in Sphingomonas sp. Thioredoxin: Increased α4-Helix Rigidity with Preserved Overall Flexibility

TL;DR

This study explores how a cold-adapted thioredoxin from Sphingomonas sp. maintains flexibility and activity at low temperatures through specific structural features.

Contribution

The paper identifies Trp101 as a key residue that increases α4-helix rigidity while preserving overall flexibility in cold-adapted thioredoxin.

Findings

Trp101 in Sphingomonas sp. Trx enhances α4-helix rigidity through hydrophobic interactions.

Mutations at Trp101 caused significant structural destabilization and reduced enzyme activity.

Cold-adapted thioredoxin uses distinct stabilization mechanisms compared to Escherichia coli Trx.

Abstract

Thioredoxin (Trx)
is a small, conserved redox protein
composed
of five β-strands and four α-helices, and it reduces disulfide
bonds and helps maintain cellular redox homeostasis. During evolution
from the Trx of the last bacterial common ancestor (LBCA Trx) to Escherichia coli Trx (EcTrx), the α3 helix
became more flexible, while the α4 helix gained rigidity. To
investigate the role of α4-helix rigidity in cold adaptation,
we analyzed Sphingomonas sp. Trx (SpTrx), a cold-adapted
ortholog, focusing on α2−α4 salt bridges and α4−β5
hydrophobic interactions. We constructed single and double mutants,
targeting Glu43, Glu47, and Trp101, and assessed structural stability
using thermal shift assays, chemical denaturation, fluorescence spectroscopy,
and circular dichroism. Catalytic activity was evaluated using insulin
reduction and DTNB-based kinetic assays. Salt bridge mutations (E43A,
E47A, and E43A/E47A) modestly decreased both stability and enzyme
activity. In contrast, hydrophobic interface mutations (W101A and
W101F) caused more substantial destabilization, with W101A inducing
the most pronounced structural disruption. The E47A/W101F double mutant
exhibited poor expression and the lowest stability and activity. In
a comparative study with EcTrx, salt bridge mutations had a greater
impact on thermal stability than in SpTrx. While F102A significantly
reduced stability and increased flexibility, F102W (the SpTrx-equivalent
substitution) increased both stability and rigidity. These findings
demonstrate that in SpTrx, Trp101 enhances α4-helix rigidity
through hydrophobic packing while preserving overall flexibility.
This study highlights the evolutionary divergence of stabilization
mechanisms in Trx orthologs and provides insight into how cold-adapted
enzymes maintain activity at low temperatures.

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