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Biochimica et Biophysica Acta 1787 (2009) 1009–1015
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Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a b i o
Site-directed mutagenesis of the His residues of the rat mitochondrial carnitine/
acylcarnitine carrier: Implications for the role of His-29 in the transport pathway
Annamaria Tonazzi a,b,1, Nicola Giangregorio a,b,1, Cesare Indiveri c,⁎, Ferdinando Palmieri a,b,⁎
a
b
c
Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, 70125 Bari, Italy
CNR Institute of Biomembranes and Bioenergetics, via Orabona 4, 70125 Bari, Italy
Department of Cellular Biology, University of Calabria, 87036 Arcavacata di Rende, Italy
a r t i c l e
i n f o
Article history:
Received 22 December 2008
Received in revised form 20 February 2009
Accepted 23 February 2009
Available online 9 March 2009
Keywords:
Carnitine
Mitochondria
Transport
Acylcarnitine
Site-directed mutagenesis
a b s t r a c t
The mitochondrial carnitine/acylcarnitine carrier (CAC) of Rattus norvegicus contains two His, His-29 and
His-205. Only the first residue is conserved in all the members of the CAC subfamily and is positioned before
the first of the three conserved motifs. In the homology model of CAC, His-29 is located in H1 close to the
bottom of the central cavity. His-205 is the first amino acid of H5 and it is exposed towards the cytosol. The
effect of substitution of the His residues on the transport function of the reconstituted mutant CACs has been
analysed, in comparison with the wild-type. H29A showed very low activity, H29K and H29D were nearly
inactive, whereas H205A, H205K and H205D showed activities similar to that of the wild-type. His-29 has
also been substituted with Gln, Asn, Phe and Tyr. All the mutants showed very low transport function and,
similarly to H29A, higher Km, reduced Vmax and altered selectivity towards (n)acylcarnitines, with the
exception of H29Q, which exhibited functional properties similar to those of the wild-type. The experimental
data, together with a comparative analysis of the carnitine acyltranferase active sites, indicated that His-29
forms an H-bond with the β-OH of carnitine. The substitution of His-205 led to a change of response of the
CAC to the pH. The results are discussed in terms of relationships of His-29 with the molecular mechanism of
translocation of the CAC.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
The carnitine/acylcarnitine carrier (CAC) belongs to the mitochondrial carrier protein family; it plays a central role in the β-oxidation of
fatty acids [1]. The amino acid sequence of the CAC shows all the specific
properties of the carrier family, as the tripartite structure and the threefold repeated signature motif PX[DE]XX[RK] [2]. It shows also the typical
hydropathy profile of the mitochondrial carriers, which is characterized
by six hydrophobic transmembrane segments (H1–H6) connected by
five hydrophilic loops, two of which are exposed towards the cytosolic
side (h23, h45) and three towards the matrix side (h12, h34, h56) of the
protein; the three matrix loops are much larger than the cytosolic ones.
The CAC is encoded in Homo sapiens by the gene SLC25A20 [3] that maps
Abbreviations: DTE, dithioerythritol; NEM, N-ethylmaleimide; PLP, pyridoxal 5phosphate; Pipes, 1,4-piperazinediethanesulfonic acid; SDS-PAGE, sodium dodecyl
sulfate polyacrylamide gel electrophoresis; CAC, carnitine/acylcarnitine carrier
⁎ Corresponding authors. C. Indiveri is to be contacted at Department of Cellular
Biology, University of Calabria, 87036 Arcavacata di Rende, Italy. F. Palmieri, Department
of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of
Bari, 70125 Bari.
E-mail addresses: [email protected] (C. Indiveri), [email protected]
(F. Palmieri).
1
These authors contributed equally to this work.
0005-2728/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbabio.2009.02.026
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to chromosome 3p21.31, in Saccharomyces cerevisiae by the gene CRC [4]
and in Aspergillus nidulans by the gene acuH [5]. The CAC proteins are
grouped in a sub-family characterized by the RXXPANAAXF motif which
is located in the H6 [5]. The CAC from rat liver has been widely
characterized on the functional point of view after the purification and
reconstitution in liposomes [6 and see 7 for Refs.]. This carrier catalyses
efficient carnitine/acylcarnitine and carnitine/carnitine antiports and,
in the absence of counter-substrate, a 10-times slower carnitine uniport.
The transport protein inserts itself into the liposomal membrane rightside out compared to mitochondria; thus, the extraliposomal and
intraliposomal sides correspond to the cytosolic and matrix sides,
respectively. The substrate antiport catalysed by the CAC occurs via a
ping-pong transport mechanism, implying that one substrate-binding
site is alternatively exposed to the cytosol (c-state) or the matrix
(m-state) [8]. Sulfhydryl reagents strongly inhibit the transport function
of CAC [9]. To investigate the structure/function relationships by sitedirected mutagenesis, the transport protein has been over-expressed in
Escherichia coli and reconstituted in liposomes in active state. The
recombinant CAC resembles all the main functional and kinetic
properties of the native protein [10]. Using reconstituted wild-type
and mutant over-expressed proteins, it was revealed that Cys-136 is
responsible for the inhibition of the CAC by the SH reagents. This residue
is accessible from the external (cytosolic) side of the proteoliposomes to
hydrophilic SH reagents [11]. Further site-directed mutagenesis studies
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showed that Cys-136, which is located in H3, and Cys-155, which is
located in h34, can come in contact during some steps of the catalytic
cycle [7]. The homology structural model of the CAC [7] obtained on the
basis of the tertiary structure of the ADP/ATP carrier [12], is in
agreement with the experimental findings. Very recently it was found
that the accessibility of Cys-136 and Cys-155 from the external
(cytosolic) side of the reconstituted CAC in the proteoliposomes
depends on the conformational state of the protein. The Cys residues
are located in a mobile structure of the protein that become more
accessible to the external (cytosolic) aqueous environment when the
transporter is in m-state [13]. Further information on the structure/
function relationships could be obtained by mutagenesis of His residues,
which are often involved in the function of membrane transporters [14–
20]. The rat CAC has two His residues, one of which is conserved in all the
members of the CAC subfamily. In the present paper the relationships
between the function of the carrier and the two His residues have been
investigated by site-directed mutagenesis. These studies, in agreement
with multialignment and homology structure predictions, revealed that
the conserved His-29 is involved in the transport pathway, whereas the
non-conserved His-205 does not play any major role in the CAC.
2. Experimental procedures
2.1. Materials
Sephadexes G-50, G-75 and G-200 were purchased from Pharmacia, L-[methyl-3H]carnitine from Amersham, egg-yolk phospholipids
(L-α-phosphatidylcholine from fresh turkey egg yolk), Pipes, Triton
X-100, cardiolipin, L-carnitine, L-acetylcarnitine, DL-(n)acylcarnitines
and N-dodecanoylsarcosine (sarkosyl) from Sigma. All other reagents
were of analytical grade.
2.2. Site-directed mutagenesis, overexpression and isolation of the
CAC proteins
The previously constructed pMW7-WTratCAC recombinant plasmid was used to introduce the mutations in the CAC protein [11]. The
amino acid replacements were performed with complementary
mutagenic primers using the overlap extension method [21] and the
High Fidelity PCR System (Roche). The PCR products were purified by
the Gene Clean Kit (La Jolla), digested with NdeI and HindIII
Fig. 1. Alignment of proteins of the mitochondrial carnitine carrier subfamily. Proteins identified (R. norvegicus NP446417 [10], H. sapiens NP000378 [26], A. thaliana NP568670 [47],
S. cerevisiae NP014743 [4], A. nidulans AJ011563 [5]) or predicted (M. musculus NP065266, B. taurus XP001253588, D. rerio NP957153, C. elegans NP501223, D. melanogaster NP477221,
O. sativa NP001065471) as CAC, have been aligned using the Clustal W software. The upper boxes and lines indicate the hydrophobic transmembrane segments (H1–H6) and the
hydrophilic loops (h12, h23, h34, h45, h56), respectively. The asterisks indicate identities and the dots or colons indicate conservative or highly conservative substitutions. The black
rectangles highlight the His residue (His-29 in rat) and the RXXPANAAXF motif (starting from Arg-275 in rat) conserved in all the aligned proteins.
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(restriction sites added at the 5′ end of forward and reverse primers,
respectively) and ligated into the pMW7 expression vector. All
mutations were verified by DNA sequencing, and, except for the
desired base changes, all of the sequences were identical to that of rat
CAC cDNA. The resulting plasmids were transformed into E. coli C0214.
Bacterial overexpression, isolation of the inclusion body fraction,
solubilization and purification of the wild-type CAC and mutant CAC
proteins were performed as described previously [11].
2.3. Reconstitution of wild-type and mutant CAC proteins in liposomes
The recombinant proteins were reconstituted into liposomes as
described previously [11]. The concentration of intraliposomal
carnitine was 13 mM. The external substrate was removed from
proteoliposomes on Sephadex G-75 columns.
2.4. Transport measurements
Transport at 25 °C was started by adding 0.1 mM [3H]carnitine to
proteoliposomes and terminated by the addition of 1.5 mM NEM [6].
In controls, the inhibitor was added together with the labelled
substrate, according to the inhibitor stop method [22]. This strategy
allows to subtract, from the experimental samples, the aliquot of
radiolabelled carnitine diffusing through the liposomal membrane.
Finally, the external substrate was removed by chromatography on
Sephadex G-50 columns, and the radioactivity in the liposomes was
measured [22]. The experimental values were corrected by subtracting control values. All of the transport activities were determined by
taking into account the efficiency of reconstitution (i.e. the share of
successfully incorporated protein).
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cavity, slightly above the charged network that closes the cavity at the
bottom. His-205 is localized at the beginning of the α-helix H5 after
the short loop h45 which connects H4 and H5 and faces towards the
external aqueous environment (ref. [7] and see Fig. 6).
Both residues have been substituted with the small hydrophobic
Ala or with the charged Lys and Asp. The functional consequence of
the mutations have been analysed in proteoliposomes reconstituted
with the different mutant proteins over-expressed in E. coli. The
incorporation of the proteins into the liposomal membrane was tested
as previously described [11,14] and no significant difference of
incorporation was found for the various mutants (not shown). In
Fig. 2 A the time courses of the [3H]carnitine/carnitine antiport in
liposomes reconstituted with wild-type or with the mutant proteins
were compared. The substitution H29A caused a strong reduction of
the CAC transport function. The uptake of [3H]carnitine at 30 min was
about 20% of the uptake mediated by the wild-type protein. The
mutants H29D, H29K and, not shown, H29R were nearly inactive (less
than 5% of the wild-type activity). On the contrary, the uptake of [3H]
carnitine mediated by the H205A, H205K or H205D mutants was
similar or even slightly higher (H205K) than that of wild-type. The
effect of the double substitutions H29A/H205A, H29A/H205K and
H29A/H205D was also analysed (Fig. 2A). The transport activity of
these mutants was similar or lower (H29A/H205A) than that of
the single mutant H29A. All the data suggested that His-29, but not
His-205, should play an important role in the transport function of
the CAC. To evaluate whether His-29 could undergo aromatic or
H-bonding interactions, which are typical of His residues [19,27,28,
29], it has been substituted with the aromatic Phe and Tyr or with Asn
3. Other methods
SDS-PAGE was performed according to Laemmli [23] as previously
described [11]. The amount of recombinant protein was estimated on
Coomassie blue-stained SDS-PAGE gels by the Bio-Rad GS-700
Imaging Densitometer equipped with the software Bio-Rad MultiAnalist, using bovine serum albumin as standard. The extent of
incorporation of the recombinant protein into liposomes was
determined as described in Phelps et al. [14], with the modifications
reported in ref. [11]. N-terminal sequencing was carried out as
described before [24]. The homology model of the rat CAC was built
based upon the structure of the bovine ADP/ATP carrier [12] by using
the computer application Swiss PDB Viewer [25].
4. Results
4.1. Functional analysis of the mutant CAC proteins
It was previously reported that some members of the mitochondrial carrier family share the RXXPANAAXF sequence motif, which is
located in the sixth hydrophobic segment H6 [5]. It was proposed that
the proteins containing this motif constitute the CAC subfamily [5,26].
Indeed, the RXXPANAAXF motif is conserved in the CAC of R.
norvegicus, H. sapiens, A. nidulans, C. elegans, D. melanogaster, as
previously described [5] and also in the CACs of M. musculus, B. taurus,
D. rerio, S. cerevisiae, A. thaliana and O. sativa as depicted in Fig. 1. The
multialignment highlights that one of the two His of the rat CAC,
His-29, is conserved in all the CACs. This residue is positioned before
the first of the three repeated PX[DE]XX[RK] motifs. As the
RXXPANAAXF motif, this His residue is found in the CAC subfamily
and in few other members of the mitochondrial carrier family (see
Discussion section). Differently, the second His of the rat CAC, His-205,
is not conserved within the CAC subfamily. In the homology model of
CAC [7], which was built using the structure of the ADP/ATP carrier in
c-state as template [12], His-29 is located in the central water filled
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Fig. 2. Time course of [3H]-carnitine uptake in liposomes reconstituted with
recombinant CAC proteins. Transport was started by the addition of 0. 1 mM [3H]carnitine to proteoliposomes containing 13 mM carnitine and stopped at the indicated
times. In each panel the data relative to the wild-type CAC (●) are shown with those
relative to the mutants. In (A): H29A (▴), H29D ( ), H29K (Δ), H205A (◊), H205D (♦),
H205K (○), H29A/H205A (▽), H29A/H205D (▾), H29A/H205K (□); in (B): H29F (Δ),
H29N (□), H29Q (○), H29Y (▴). The data represent means ± SD of at least three
independent experiments.
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Table 1
Kinetic constants of reconstituted wild-type and mutant CACs.
Protein
Km (mM)
Vm (mmol/min/g protein)
WT
H29A
H29Y
H29F
H29N
H29Q
H205A
H205K
H205D
0.54 ± 0.10
0.93 ± 0.15
0.76 ± 0.11
0.98 ±.031
1.80 ± 0.28
0.82 ± 0.16
0.38 ± 0.16
0.53 ± 0.14
0.47 ± 0.19
1.90 ± 0.61
1.27 ± 0.43
1.32 ± 0.46
1.36 ± 0.32
1.15 ± 0.37
1.85 ± 0.55
1.93 ± 0.58
2.01 ± 0.79
1.87 ± 0.65
The Km and Vmax values were calculated from double-reciprocal plots of the rates of
carnitine/carnitine antiport under variation of the external substrate concentration.
Transport was started by the addition of 0.12–2. 0 mM [3H]carnitine to proteoliposomes
reconstituted with the wild-type CAC or one of the indicated mutants and containing
13 mM carnitine. The reaction time was 4 min. The data represent the means ± S.D. of
four different experiments.
and Gln, the N amides of which can form H-bonds. The uptake of
[3H]carnitine mediated by the H29F or H29Y reconstituted mutants
(Fig. 2B) was very similar to that of the H29A mutant. The uptake of
[3H]carnitine mediated by H29N was about 10% of the wild-type,
whereas the uptake mediated by H29Q was more than 80% of the
wild-type. These data indicated that the H-bonding property of His-29
is the most important for the function and is very critical since only
Gln, not Asn, restored the activity of the transporter. It cannot be
excluded that His-29 may also form hydrophobic interaction, which
may explain the residual activity of the mutants in which His-29 had
been replaced with hydrophobic or aromatic residues. However, the
residual activity may be more likely due to the ability of these residues
to form weaker H-bonds in proteins [30].
The effect of the substitution of His-29 and His-205 on the kinetics
of the CAC was further investigated (Table 1). The substitution of
His-205 did not lead to significant variations of the Km and Vmax.
Whereas, the substitution of His-29 led to an increase of the Km for
carnitine which, in the H29N mutant, reached a value more than three
fold that of the wild-type. The Vmax values of the His-29 mutants
were lower than that of the wild-type with the exception of H29Q
which showed the same value of the wild-type. The inactive mutants
H29K, H29D and H29R could not be tested for kinetic properties since
their transport activity was too low even in the presence of very high
(60 mM) intraliposomal substrate concentrations, a condition
Fig. 3. Influence of substrate on the inhibition of the wild-type and mutant CACs by
DEPC. Proteoliposomes were incubated, at time zero, with 30 mM PLP and 10 μM
mersalyl. After 10 min, 20 mM carnitine plus 2 mM DEPC (grey bars) or DEPC alone
(white bars) was added. After 2 min all the proteoliposome samples were passed onto
G-75 Sephadex columns (see Experimental procedures section) and then the transport
activity was started by adding 0. 1 mM [3H]carnitine and 0. 5 mM DTE and stopped after
20 min. Percent inhibition was calculated for each sample with respect to the control
(referred to as 0%), i.e., proteoliposomes not treated with DEPC; the values are means± S.D.
of the percentage of three experiments. In the experiments analysed, the transport
activity of the sample without the addition of DEPC was 3.05± 0.65 mmol/10 min/g
protein.
previously used to study mutants of the mitochondrial citrate
transporter (CTP) with low activity [31].
4.2. Substrate protection against the inhibition by DEPC
Since the CAC functions according to rapid-equilibrium mechanism [8], the Km increase of the His-29 mutants highlighted a
decrease of affinity towards carnitine; this might indicate the
occurrence of a direct interaction of His-29 with the substrate. This
hypothesis was further investigated by inhibition studies. The
sensitivity of the CAC to DEPC, an His-specific reagent [32], was
tested. 2 mM DEPC inhibited 90% of the transport activity of the wildtype. However, besides H29A (62% inhibition) and H205A (82%
inhibition) also the H29A/H205D mutant (i.e., no His in the
recombinant protein) was inhibited (56%), indicating that DEPC also
reacted with residues different from His, as it was previously found for
other proteins [32]. To prevent the unspecific reaction, proteoliposomes were pre-treated with PLP and mersalyl to protect the Lys and
Cys residues which could react with DEPC [32]. After the following
incubation with DEPC, the proteoliposomes were treated with DTE to
remove the mersalyl and subsequently passed through Sephadex G-75
columns to remove the PLP [33] and the unreacted DEPC; the residual
activity was then measured. As expected (Fig. 3), a lower extent of
inhibition was generally found after these treatments. The absence of
inhibition of the mutant H29A/H205D demonstrated that DEPC had
reacted specifically with His residues. The activities of wild-type and
H205A proteins were inhibited about 30% and 25%, respectively. The
activity of the H29A mutant was inhibited less than 15%, i.e., H29A
showed a strong decrease of the sensitivity to DEPC compared to the
Fig. 4. Specificity of the CAC mutants for acylcarnitines and comparison with the wildtype. The carnitine acyl derivatives with the indicated (n) carbon chain length were
added to the reconstituted proteoliposomes together with 0.1 mM [3H]carnitine. The
concentration used for acylcarnitines were: 1 mM acetylcarnitine (n = 2); 0.05 mM
octanoylcarnitine (n = 8); 0.02 mM decanoylcarnitine (n = 10), lauroylcarnitine
(n = 12) and myristoylcarnitine (n = 14). After 2 min the transport was stopped as
indicated in Experimental procedures. Percent residual activity is reported with respect
to the control, i.e., without the addition of acyl-carnitine. In each panel the data relative
to the wild-type CAC (black bars) are shown with those relative to the mutants. In (A):
H29A (dark grey), H29F (light grey), H29Y (white); in (B): H29N (dark grey), H29Q
(light gray), H205A (white). The data represent means ± S.D. of three independent
experiments.
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that of the wild-type. The same pH dependence was also found for
H29Y, H29F and H29Q (not shown). The H205A transport activity
increased with the pH, but, surprisingly, reached a maximum at pH 8.0
instead of pH 7.0, and, then, decreased.
5. Discussion
In this paper the structure/function relationships of the two native
His residues of the rat CAC have been investigated by site-directed
mutagenesis. According to the bioinformatic predictions, the mutation
of the conserved His-29, not of His-205, severely impaired the
transport function, except in the case of the H29Q mutant, whose
Fig. 5. pH dependence of the H29A and H205A mutants in comparison with the wildtype. Transport was started by adding 0.1 mM [3H]carnitine to proteoliposomes
(containing 13 mM carnitine) reconstituted at the indicated pH (see legend panel) and
stopped after 10 min as described in Experimental procedures. The percent of activity
was calculated with respect to the transport in 10 min at pH 7.0 for each mutant. The
data represent means ± S.D. of three independent experiments. The transport activity at
pH 7.0 were 1.92 ± 0.53, 0.32 ± 0.069 and 2.0 ± 0.66 mmol/10 min/g protein for the
wild-type, H29A and H205A mutants, respectively.
wild-type and H205A proteins, indicating that most of the inhibitory
effect was due to His-29. The presence of 20 mM carnitine during the
incubation with DEPC mostly prevented the inhibition of the wildtype and of the H205A mutant (containing His-29), whereas had not
significant effect on the mutant H29A. The addition of 0.5 mM
carnitine (close to the Km value) instead of 20 mM, during the
incubation with DEPC of the H205A mutant, resulted in inhibition of
13.5 ± 4.1% (three experiments; not shown), i.e., the protection was
about half of the maximal effect. These findings indicated a possible
interaction of His-29 with the substrate.
4.3. Selectivity of CAC mutants towards acylcarnitines
It is known that CAC accepts carnitine acyl derivatives as
physiological substrates, with higher affinities towards acylcarnitines
with a longer carbon chain. These compounds behave as competitive
inhibitors towards carnitine since they bind to the same site [10,34].
The effect of the substitution of His-29 on the sensitivity towards
acylcarnitines has been tested measuring the inhibition of the [3H]
carnitine transport in proteoliposomes reconstituted with H29A,
H29N, H29Q, H29Y, H29F and H205A, in comparison with the wildtype. For a reliable estimation of the variations of sensitivity, the
concentration of each inhibitor was chosen (see legend to the Fig. 4) to
obtain, in the wild-type, residual activities of roughly 50%. As shown in
Fig. 4A, the mutant H29A showed a slight reduction of sensitivity
towards acetylcarnitine (n = 2) with respect to the wild-type;
whereas it showed a more evident reduction of sensitivity towards
longer acylcarnitines. Some reduction of sensitivity towards acylcarnitines was also found for H29F and H29Y, but shifted towards longer
acyl chains. In the case of H29N a strong decrease of sensitivity
towards all the acyl derivatives was found. Differently, H29Q exhibited
sensitivity towards acylcarnitines similar to that of the wild-type,
except for the C2 derivative. H205A showed sensitivity to the
acylcarnitines overlapping that of the wild-type.
4.4. pH dependence of H29A and H205A mutants
The pH dependence of the H29A and H205A mutants was studied
in comparison with the wild-type. Fig. 5 shows the influence of the pH
on the transport activity expressed as percent of the activity at pH 7.0.
The pH dependence of the wild-type resembled the pH dependence of
the native protein [34], i.e., the transport activity increased with
increasing the pH from pH 6.0 to pH 7.0 and, then, it decreased at more
alkaline pH. The H29A mutant showed a pH dependence similar to
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Fig. 6. Structural model of the CAC. Ribbon diagrams viewing the carrier from the lateral
side (A) or from the cytosolic side (top view; B). The transmembrane α-helices are
coloured as followed: H1, red (partly transparent in A); H2, light brown (partly
transparent in A); H3, green; H4, emerald-green; H5, cyan; H6, blue. White or light blue
(E132) surfaces highlight the residues of the salt bridge network, D32, K35, E132, K135,
D231 and K234, which are numbered in black. H29 and H205 (purple), R275 and F284
(blue), D179 (red) are highlighted by stick representation and numbered with the same
colour of the residues. The substrate carnitine is depicted in a ball and stick
representation in red with the oxygen atoms in light brown and it is visualized in
two hypothetical sites, U (upper) and L (lower). The homology structural model has
been represented using the molecular visualization program VMD.
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activity, was comparable to that of the wild-type. This finding
indicated that the main structure/function relationship of His-29 is
the formation of an H-bond, most likely with the substrate. It is well
known that the N amide of Gln structurally corresponds to the τ-N
(distal), whereas the N amide of Asn corresponds to the π-N (vicinal)
of the His imidazole [28]; thus, only the distal N of His-29 is involved
in the H-bonding. To predict which functional group of the substrate
interacts with His-29, we have viewed the interactions between
substrate and active site in carnitine acyl-transferases, whose
structures have been resolved with X-ray crystallography [35–37]. In
these enzymes, a conserved His residue (His-343 in CAT) interacts via
an H-bond between the distal N of the His and the β-OH of carnitine or
with the β-O- of acylcarnitines. In addition, besides other residues, a
conserved Arg (Arg-518 in CAT) interacts with the carboxyl group of
carnitine; the distance between the conserved His and Arg residues in
the enzymes is about 10 Å (from 8.9 to 10.5 Å, calculated from the
deposited PDB structures [35–37] between the distal N of His and the
guanidino N groups of Arg, using the computer application Swiss PDB
viewer [25]). The transfer reaction is allowed in the enzymes by a
vicinal Ser residue. In the structural model of CAC, His-29 lies at a
distance of 9.8 Å from a conserved Arg (Arg-275 in rat, see Figs.1 and 6)
which is located in the central cavity too. No Ser residues in the
vicinity of His-29 are present in the CAC in line with absence of
transferase activity. Interestingly, Arg-275 is important for function in
the human CAC [26]. Thus, in analogy with the CAT enzymes, His-29
will interact via an H-bond with the alcoholic β-OH of carnitine or
with the esterified β-O- of acylcarnitines, playing a role in the
positioning of the substrate preceding or concomitant with the
translocation towards the opposite side of the membrane. However,
the binding site of carnitine would be deeper in the central cavity of
the protein (Fig. 6, L site) respect to the predicted position of the
binding sites of yeast mitochondrial carriers previously proposed
([38] and see Fig. 6, U site). This discrepancy may be interpreted
according to an alternate interaction of the substrate with an upper
and a lower binding site. Even though this is a speculative hypothesis,
it is in agreement with the two binding site model proposed for the
yeast CTP [31]. On the basis either of this model and of the
bioinformatic predictions [38–40], in the upper binding site, carnitine
should interact with Arg-275 and Asp-179 (homologues of the yeast
CAC Arg-303 and Asp-204 [38]); in the lower binding site, carnitine
will interact with Arg-275 and His-29. In this site, the positioning of
γ-trimethylammonium close to charged residue of the second repeat,
Glu-132 (Fig. 6), will induce the opening of the channel by
perturbation of the salt bridge network, according to the translocation
mechanism of carriers previously proposed [39]. The decrease of
affinity for carnitine and the reduction of Vmax, which cause the
reduction of transport function in H29A, H29F and H29Y mutants, and
the variation of selectivity towards acylcarnitines can be explained by
anomalous fluctuations of the substrate(s) which is weakly anchored
to the amino acid residue-29 (see Results section). Whereas, the more
evident kinetic and selectivity variations observed in H29N may be
due to the formation of a strong but wrongly placed H-bond between
Asn-29 and the substrate which forces it in a distorted conformation
and impairs the interaction of the γ-trimethylammonium with
Glu-132. The loss of transport function of the H29K and H29D
mutants may be due to the formation of electrostatic interactions
between the positively or negatively charged residue, introduced in
the place of His-29, with the vicinal Asp-32 or Lys-135 (see Fig. 6). This
anomalous electrostatic interaction will interfere with the network of
salt bridges of the CAC.
A role in positioning of the substrate prior to the reaction, similar
under some aspects to that of His-29, was previously proposed for the
invariant His-1069 of the copper-transporting ATPase [17]. Among the
mitochondrial carriers with known function, a conserved His is
present in the corresponding position of His-29, i.e., immediately
before the first repeated motif, in DIC [41], FLX1 [42], GTP/GDP [43],
oxodicarboxylate [44] and folate [45] carriers. No data on mutations of
these specific His residues in mitochondrial carriers have been
reported so far with the exception of the conserved His-41 of the
folate transporter whose mutation in Ala led to some impairment of
function. However, no specific role was proposed for that residue [46].
His-205 has not a major role in the function of the CAC as
demonstrated by the lack of effect on the transport activity following
its substitution. However, it was found that the substitution of His-205
altered the pH dependence of the transporter, shifting the optimal
activity towards more alkaline pH (Fig. 5). Since it is known that His
and Phe residues can interact in proteins leading to a stabilization of
vicinal helical structures [28,29], the shift may be explained by the loss
of an interaction between His-205 and a Phe located on a vicinal
α-helix. Indeed, Phe-284 is located on the H6 and lies at a predicted
distance of 3.7 Å from His-205 in the c-state of the protein (Fig. 6). In
the wild-type the interaction His-205/Phe-284 will reduce the
reciprocal fluctuation of H5 and H6, which may be associated with
the conversion of the protein from the c-state to the m-state [7,13]. This
interaction, which is stronger at alkaline pH, at which the imidazole
(pKa, 6.5) is deprotonated, should contribute to the decrease of activity
observed in the wild-type at alkaline pH. The substitution of His-205
abolishes the interaction His-205/Phe-284 leading to the shift of the
pH optimum. The hypothesized aromatic His-205/Phe-284 interaction
should be a specific feature of the rat protein. In fact, the carnitine
transporters of other species (Fig. 1) have neither His nor other
aromatic residues on H5 in the vicinity of Phe-284.
Acknowledgements
This work was supported by grants from Ministero dell'Università
e della Ricerca (PRIN, FIRB and the Italian Human ProteomeNet No.
RBRN07BMCT_009), CNR, Center of Excellence in Genomics (CEGBA),
University of Bari and University of Calabria.
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