Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 ...

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Table 4 Characteristics of the three patients carrying dilated cardiomyopathy-associated TXNRD2 mutations
Patient TXNRD2 mutation Family history LVEDD EF (%) Coronary ECG Outcome
(gender)
of DCM (mm)
angiography
...............................................................................................................................................................................
Patient 1 (male) 175G . A (Ala59Thr) Positive 64 18 Normal Left bundle branch block Died at the age of 68
Patient 2 (male) 175G . A (Ala59Thr) Negative 66 25 Normal AV block 1st degree Died at the age of 65
Patient 3 (male) 1124G . A (Gly375Arg) Negative 68 27 Normal Right bundle branch
block
Died at the age of 83
DCM, dilated cardiomyopathy; EF, ejection fraction; LVEDD, left ventricular end-diastolic diameter; ECG, electrocardiogram.
answered, whether the mutations act in a dominant-negative
fashion, thus impairing the function of the heterozygous wt allele.
The protein structure of TXNRD2 and
the FAD-binding domain
To gain better insight into a possible adverse role of the mutations
for TXNRD2 function, we followed two complementary
approaches: structural modelling of TXNRD2 and integrated analysis
of the evolutionary conservation of FAD-binding domains in
thioredoxin reductases and GSH reductases (GR), which are evolutionary
highly related enzymes. Although the crystal structure of
murine Txnrd2 as well as rat and human Txnrd1 have been
resolved, 24 – 26 the molecular nature of the FAD-binding domain
has remained poorly defined in thioredoxin reductases. The FADbinding
domain is essential for enzyme function since the reducing
equivalents from NADPH are first transferred to FAD, from where
they are passed on to the N-terminal redox-reactive centre within
the same molecule and eventually to the Sec-containing C-terminal
catalytic site of the second monomer. 27 Close inspection of the
structure revealed that the human TXNRD2-binding pocket for
FAD is formed by the N-terminal parts of four helices (h1: 49–
61, h2: 92–106, h4: 228–240, and h6: 368–382), the N-terminally
adjacent amino acids, and eight non-contiguous, non-helical short
stretches of amino acids. The mutations A59T and G375R are
located in helices 1 and 6, respectively, that both contribute to
the formation of the FAD-binding pocket (Figure 2A).
As FAD binding is common to thioredoxin reductases and GR,
we reasoned that amino acids conserved among both types of
enzymes would participate in FAD binding. To address this, we
made two sequence alignments, one comparing human thioredoxin
reductase 2 with human GR (Figure 2B), and a second that
included 36 thioredoxin reductases (including 13 thioredoxin
reductase 2 genes) and 10 GR genes (see Supplementary material
online, Figure S1 and Table S1). Amino acids shared among all or
almost all enzymes across a large number of species were
defined and marked in the alignment of TXNRD2 and GR. The
overlay of the structural and evolutionary analysis clearly demonstrated
that the regions participating in FAD binding are evolutionary
highly conserved. We then compared the FAD-binding domain,
which had been previously determined for GR, 28 with the structural
information obtained from the analysis of the modelled
TXNRD2 structure. Notably, the FAD-binding domain of GR as
defined by Schulz et al. 28 is virtually identical to that of TXNRD2
as defined by inspection of the TXNRD2 structure (Figure 2B).
The four helices as well as the non-contiguous, non-helical
stretches of amino acids are highly conserved in evolution. In
GR, a fifth helix participates in the formation of the FAD-binding
pocket. This helix is somewhat disturbed in TXNRD2 (amino
acids 208–212), but the corresponding conserved residues contribute
to the binding pocket in a similar manner.
Implications of the mutations for
TXNRD2 protein structure
D. Sibbing et al.
G375R is located in the middle of helix 6. Not only is arginine
much larger than glycine, it is also a polar amino acid. Our
model indicates that the charged side chain points into the core
of the enzyme (Figure 2A). This makes it likely that the bulky
charged side chain disrupts the hydrophobic interaction with the
neighbouring helix 1 and thus destroys the FAD-binding pocket.
A59T (Figure 2A) is located at the end of helix 1 and is thus not
directly involved in FAD binding. Our sequence alignments (see
Supplementary material online, Figure S1) including 46 enzymes
revealed only alanine and valine at this position. Two explanations
may be given that are not mutually exclusive. First, an unpolar
amino acid at this position may be required in this region. In our
model, the side chain of threonine causes clashes with A55,
located on helix1 or with V65 in the adjacent b-sheet
(Figure 2A). Proper backfolding of this b-sheet towards FAD is
required for hydrogen bonding of D69 to the ribose of FAD (as
shown for E50 in GR 28 ) and for bringing helix 2 into the proper
position relative to the other helices forming the FAD-binding
pocket. Furthermore, the polar threonine may disturb the movement
of the second redox centre, which is located on the flexible
C-terminal part of the second subunit.
In addition, we took a close look at the position of the five nonsynonymous
variants that were identified in patients as well as in controls.
Four of them (A66S, R286S, S299R, and G384S) are located
more in peripheral regions of TXNRD2 facing the solvent and are
neither involved in FAD/NADPH binding nor enzymatic function
(Figure 2A). The fifth non-synonymous variant I370T is located at
the beginning of helix 6, but it points away from FAD and is thus
not involved in FAD binding. It points into a pocket formed by residues
P365, L367, M390, Y392, and V395 of one subunit and V494’ of
the other. All amino acids of this pocket are strictly conserved in
mouse Txnrd2, which harbours threonine at this position. This
shows that threonine is tolerated. Moreover, amino acid sequence
alignment of orthologous TXNRD2 sequences revealed that 8 of
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