The -Cys-X1-X2-Cys- Motif
of Reduced Glutaredoxins
Adopts a Consensus Structure That Explains the Low p<i>K</i><sub>a</sub> of Its Catalytic Cysteine
- Publication date
- Publisher
Abstract
The -Cys-X1-X2-Cys- active site motif is central to the
function
of enzymes of the thioredoxin superfamily, including glutaredoxins.
Their chemistry depends on the lowered p<i>K</i><sub>a</sub> of the N-terminal thiolate cysteine of the -Cys-X1-X2-Cys- sequence;
therefore its structure, dynamics, and electrostatics matter. Much
information about the glutaredoxin structures was obtained by nuclear
magnetic resonance (NMR), yet these various NMR structures produced
heterogeneous and discordant views of the -Cys-X1-X2-Cys- motifs.
This study addresses these inconsistencies by a computational and
experimental investigation of three diverse reduced -Cys-X1-X2-Cys-
motifs, from human glutaredoxin 1 (hGrx1), <i>Escherichia coli</i> glutaredoxin 2 (EcGrx2), and T4 virus glutaredoxin (T4Grx). The
NMR models do not account for the low p<i>K</i><sub>a</sub> of the N-terminal cysteine. However, extensive investigations of
the NMR conformers by simulations yielded consensus structures for
the -Cys-X1-X2-Cys- motif, with well-defined orientations for the
cysteines. p<i>K</i><sub>a</sub> calculations indicated
that the consensus structure stabilizes the thiolate by local hydrogen
bonds. The consensus structures of EcGrx2 and T4Grx formed the basis
for predicting low p<i>K</i><sub>a</sub> values for their
N-terminal cysteines. Subsequent experimental titrations showed that
these p<i>K</i><sub>a</sub> values are <5, supporting
the validity of the consensus structure. The simulations also revisited
the conformational dynamics of side chains around the -Cys-X1-X2-Cys-
motif, which allowed reconciliation of calculated and measured p<i>K</i><sub>a</sub> values for important hGrx1 mutants. The conformational
spread of these side chains, which differs between NMR and molecular
dynamics models, is likely to be relevant to substrate recognition.
The new structural models determined in this work should prove to
be valuable in future molecular studies of the glutaredoxins
