Temperature adaptation in Gillichthys (Teleost: Gobiidae) A4-lactate dehydrogenases : identical primary structures produce subtly different conformations
1 Hopkins Marine Station, Biological Sciences Department, Stanford
University, Pacific Grove, CA 93950, USA
2 School of Pharmacy, Department of Pharmaceutical Sciences, University of
Colorado Health Sciences Center, Denver, CO 80262, USA
* Author for correspondence and present address: Department of Biology, Franklin and Marshall College, PO Box 3003, Lancaster, PA 17604, USA (e-mail: p_fields{at}fandm.edu )
Accepted 8 February 2002
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Summary |
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Key words: A4-LDH, alternative conformation, conformational flexibility, Gillichthys mirabilis, Gillichthys seta, temperature adaptation, lactate dehydrogenase
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Introduction |
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These studies reveal that single polypeptides with multiple
three-dimensional conformations are numerous. Few studies, however, have shown
instances in which such modifications of protein structure might be adaptive,
i.e. where the alternative conformers function optimally in different
environments and are produced differentially as environmental parameters
dictate. Somero (1969) showed
that pyruvate kinase from the Alaskan king crab (Paralithodes
camtschatica) occurs as both a `warm' and `cold' variant, depending on
measurement temperature, and that these variants have different apparent
Michaelis constants for the substrate phosphoenolpyruvate. Similarly, Ozernyuk
et al. (1994
) found that the
muscle-type LDH (A4-LDH) of the loach (Misgurnus fossilis)
occurs in two forms with differing kinetics depending on season and that both
could be converted to a single form with intermediate characteristics by brief
exposure to 3 mol l-1 urea.
A recent study by Fields and Somero
(1997) showed that the
A4-LDHs (EC 1.1.1.27; NAD+:lactate oxidoreductase) of
two closely related species of goby, Gillichthys mirabilis and G.
seta, have identical primary sequences despite four synonymous
differences in the coding regions of their mRNAs. However, these orthologs
have different substrate affinities, as measured by apparent Michaelis
constants for the substrate pyruvate (KmPYR),
as well as different thermal stabilities. The two species live in disparate
habitats: G. seta occupies the high rocky intertidal zone in the
northern Gulf of California (latitude approximately 31°N) and experiences
broader and warmer temperatures (approximately 5-41 °C) than its congener
G. mirabilis (approximately 9-30 °C), which lives in sloughs and
estuaries from the Gulf of California north along the west coast of North
America to Tomales Bay (latitude 38.16°N)
(Miller and Lea, 1972
).
The KmPYR values measured for the
A4-LDH orthologs of the two species reflect these environmental
differences: at any measurement temperature, the A4-LDH of G.
seta has a lower KmPYR value than does the
A4-LDH of G. mirabilis, indicating greater substrate
affinity. Further, G. seta A4-LDH
KmPYR is less affected by temperature change
within the range 10-40 °C than is the G. mirabilis form. The two
orthologs were shown to have an identical mass using ion-spray mass
spectrometry (Fields and Somero,
1997), thus discounting the possibility of post-translational
covalent modification. Moreover, on the basis of the work of Ozernyuk et al.
(1994
) described above, gentle
denaturation with urea (3 mol l-1 for 15 min) resulted in an
increase in the KmPYR of G. seta
A4-LDH, rendering it indistinguishable from that of G.
mirabilis A4-LDH. We concluded, therefore, that the
differences in kinetics between the two native forms must be due to
modifications in tertiary or quarternary interactions unique to the ortholog
of each species. Interestingly, maintaining populations of the two species for
extended periods at the same temperature (>3 months at 15 °C)
(Fields and Somero, 1997
) did
not change the KmPYR or thermal stability of
either A4-LDH ortholog. This suggests that the conformational
differences between the orthologs is not a result of temperature acclimation
and is, instead, a permanent phenotypic characteristic.
To describe better the conformational differences between the G. mirabilis and G. seta orthologs that may underlie the measured differences in substrate affinity and thermal stability, we have examined each using circular dichroism (CD) and fluorescence spectroscopy, which provide measures of secondary and tertiary protein structure, respectively. We have also used hydrogen/deuterium exchange monitored by Fourier-transform infrared spectroscopy (H/D-FTIR), which measures the rate at which amide hydrogens buried within the native fold of the protein are transiently exposed to the solvent medium, to examine in greater detail the differences in molecular flexibility that might explain the changes we found in substrate affinity and thermal stability between the two orthologs. We performed CD and H/D-FTIR spectroscopy at 20 and 40 °C, temperatures within the range that G. seta experiences but extending above those that G. mirabilis experiences, to determine how temperature change affects the structure of A4-LDH in these species.
In this paper, we show (i) that the orthologs have identical fluorescence
and far-ultraviolet CD spectra, indicating that their secondary and tertiary
structures are identical; (ii) that G. seta A4-LDH has a
faster H/D exchange rate than the G. mirabilis form at 20 °C,
suggesting greater conformational flexibility in the former; (iii) that
differences in conformational flexibility can be associated with
-helical, but not ß-sheet, structure and (iv) that, on the basis
of a comparison of second-derivative ultraviolet spectra, at least one of the
five tyrosyl residues, but none of the six tryptophyl residues, in the G.
mirabilis LDH-A monomer is in a more hydrophobic environment than the
corresponding residue in the G. seta ortholog. Using this evidence,
we argue that the conformational differences between the forms responsible for
the potentially adaptive differences in KmPYR
probably occur in a small region of the molecule bounding helix
1G-
2G, a structure whose mobility is closely tied to the
catalytic rate and substrate affinity of A4-LDH
(Fields and Somero, 1998
;
Dunn et al., 1991
).
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Materials and methods |
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Purification of A4-LDH
White epaxial muscle was dissected from freshly killed individuals;
contamination from the skin and viscera was minimized to avoid contamination
by heart-type (B4) LDH. Muscle tissue was homogenized and
centrifuged, and A4-LDH was purified from the supernatant using
oxamate-affinity chromatography (Yancey
and Somero, 1978), as described previously
(Fields and Somero, 1997
).
Lactate dehydrogenase binds tightly to oxamate in the presence of NADH, so an
extended wash with NADH-containing buffer ensured the removal of other
proteins together with any small molecules that might have been loosely bound
to LDH. The purity of the A4-LDH was confirmed using sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver
staining; only one protein band was seen for each species of fish.
An initial goal of the study was to compare the structures of the wild-type
A4-LDHs with those of the urea-denatured A4-LDHs, which
showed identical KmPYR values in the two
species (Fields and Somero,
1997). We were unable, however, to purify enough urea-treated
A4-LDH for the structural studies described below because of
irreversible loss of activity of the enzyme during the brief denaturation
process. Thus, we describe only a comparison of the wild-type
A4-LDHs from the two congeners.
Samples of purified A4-LDH were precipitated and stored in 90 % ammonium sulfate until analyses were performed. The ammonium sulfate was removed, and the samples were concentrated by repeated washing with 10 mmoll-1 potassium phosphate buffer (pH 7.0) in a Centricon-30 (Amicon, Beverly, MA, USA) centrifugal separator.
Fluorescence spectroscopy
Intrinsic tryptophan fluorescence spectra for A4-LDH from both
fish species were measured from 310 to 400 nm in an Aviv ATF 105
spectrofluorometer using an excitation wavelength of 295 nm. A4-LDH
concentration was 20 µg ml-1 in 10 mmoll-1 potassium
phosphate buffer (pH 7.0), and cuvette temperature was maintained at 20
°C.
To monitor thermal unfolding of the A4-LDH orthologs,
fluorescence intensity was monitored at 347 nm as cuvette temperature was
stepped from 20 to 85 °C in increments of 0.5 °C. At each temperature,
samples were equilibrated for 30 s, followed by signal-averaging for 10 s. The
resultant data were analyzed by complex sigmoid, non-linear least-squares
fitting to obtain the fraction of protein molecules unfolded at each
temperature and the apparent midpoint of the nativedenatured transition
region (Pace, 1986
;
Kim et al., 2000
). The latter
datum is used as an estimate of the apparent melting temperature,
Tm, which is an indicator of global protein stability.
Circular dichroism spectroscopy
All CD spectra were produced using an AVIV 60DS spectropolarimeter.
Far-ultraviolet spectra of G. mirabilis and G. seta
A4-LDHs were collected from 190 to 260 nm using a 0.1 cm
path-length quartz cuvette; buffer spectra were also collected and subtracted
from each protein spectrum. The protein concentration used for far-ultraviolet
spectroscopy was approximately 0.1 mg ml-1. Ellipticity data thus
obtained were converted to mean residue ellipticity, MRW, using the
equation:
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Thermal denaturation profiles were obtained by monitoring ellipticity at
222 nm while increasing cuvette temperature in increments of 1 °C from 25
to 85 °C, equilibrating at each temperature for 12 s and signal-averaging
for 5 s. Values of Tm for each sample were calculated
using the method of Pace
(1986) and Kim et al.
(2000
) described above.
In the near-ultraviolet CD experiments, absorbance spectra were collected
between 270 and 300 nm, buffer absorbance was subtracted and values were
converted to mean residue ellipticity as described above. Second-derivative
ultraviolet spectra were calculated using Grams 386 software (Galactic
Industries); such second-derivative spectra are sensitive to the global
environment of aromatic amino acids and are especially useful in reporting
changes in the hydrophobicity of the environment surrounding Tyr residues.
According to the method of Servillo et al.
(1982), the ratio
r=a/b, where a is the difference between the
minimum of the second-derivative spectrum at approximately 283 nm and the
maximum at approximately 287 nm (Tyr signal) and b is the difference
between the minimum at approximately 291 nm and the maximum at approximately
296 nm (Trp signal) (see Results), is indicative of the relative number of Tyr
and Trp residues as well as the hydrophobicity of the Tyr environment. Values
of r were calculated for each form of A4-LDH at 20, 30 and
40 °C.
Although r should be independent of protein concentration
(Ragone et al., 1984), we
performed the following analysis to ensure that minor differences in protein
concentration did not affect the results of the second-derivative ultraviolet
study. The spectra were eight-times-interpolated and area-normalized between
250 and 310 nm to correct for potential differences in protein concentration.
A new second-derivative spectrum was calculated from each of these
area-normalized ultraviolet spectra and compared with the corresponding
original second-derivative spectrum. The differences between second-derivative
spectra of A4-LDH within each species were negligible compared with
the differences found between the A4-LDH orthologs.
Hydrogen/deuterium exchange
All H/D exchange assays were performed in a Bomem MB-series FTIR
spectrometer with a temperature-controlled cell holder. A4-LDH
samples were diluted to a concentration of approximately 0.75 mg
ml-1 in aqueous or 75 % D2O buffer (10
mmoll-1 potassium phosphate, pH 7.0) and were immediately injected
into a CaF2 cell with a 25 µm Teflon spacer. Spectra were
collected 2, 7, 12, 17, 25, 40 and 60 min after injection into the sample cell
and every 30 min thereafter up to 360 min for 20 °C assays and up to 300
min for 40 °C assays. Reference spectra were collected with buffer only in
the sample cell before each experimental assay. The spectra of liquid and
gaseous water were subtracted from each sample spectrum according to the
method of Dong et al. (1992),
and the resulting spectrum was smoothed with a seven-point Savitsky-Golay
function as implemented by the Grams 386 software.
The ratio of the amide II peak (centered at approximately 1550 cm-1) to the amide I peak (centered at approximately 1655 cm-1) for each spectrum was determined, and the change in this ratio with time exposed to D2O was used as an indication of the rate of exchange of internal amide hydrogens with solvent deuterons. The resultant decay plots were fitted with a double-exponential equation to produce a smoothed curve, and the exponents of these curves were taken as the time constants of H/D exchange in subpopulations of amide hydrogens.
To determine H/D exchange rates in specific secondary structures,
second-derivative spectra were calculated from the aqueous spectrum and the
H/D spectra over time for both A4-LDH forms at 20 and 40 °C.
Each second-derivative spectrum was baseline-corrected
(Dong and Caughey, 1994) and
area-normalized under the amide I region (Kendrick et al.,
1996
,
1997
). Because peaks in the
second-derivative infrared spectrum within the amide I region correspond to
specific secondary structures (i.e.
-helix, ß-sheet, turn), this
analysis allowed H/D exchange in distinct areas of the A4-LDH
molecule to be compared.
Homology modeling
To examine the positions of Tyr residues within the three-dimensional fold
of Gillichthys A4-LDH, we used the SWISS-MODEL program
(Guex and Peitsch, 1997) to
create a structural homology model. We overlaid the primary structure of the
Gillichthys protein on two templates consisting of dogfish
A4-LDH (Protein Data Bank code 1LDM)
(Abad-Zapatero et al., 1987
)
and pig A4-LDH (9LDT) (Dunn et
al., 1991
). An alignment of the three A4-LDH sequences
is available from the authors upon request. The crystallographically-derived
three-dimensional conformations of the pig and dogfish orthologs are very
similar in secondary and tertiary structure and, because their phylogenetic
divergence precedes that of pig and teleost, we are confident that they are
appropriate templates for the development of the Gillichthys
A4-LDH model. The resulting coordinate file was used to visualize
the positions of the residues of interest in the molecule, their exposure to
solvent and their role in inter-subunit interactions.
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Results |
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Circular dichroism spectroscopy
Fig. 2A shows far
ultraviolet CD spectra for G. mirabilis and G. seta
A4-LDHs at 20 and 40 °C. At both temperatures, the spectra are
indistinguishable. This indicates that the secondary structures of the two
A4-LDH forms are similar and that they maintain their similarity at
temperatures at which differences in conformational flexibility are
significant (see H/D exchange below). Coupled with the Trp fluorescence data
described above, these findings suggest that the A4-LDHs of the two
Gillichthys species possess secondary and tertiary structures that
are identical within the sensitivities of the two techniques.
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Thermal denaturation profiles were followed with farultraviolet CD at 222 nm (Fig. 2B) and produced results similar to the thermal denaturation assays followed by Trp fluorescence (Fig. 1B). A4-LDH from G. mirabilis had an apparent Tm of 60.2±0.1 °C and the G. seta form had an apparent Tm of 58.4±0.1 °C. These values are higher than the Tm values derived by Trp fluorescence. This difference probably reflects the loss of secondary structure, which CD monitors, at temperatures higher than the quarternary and tertiary structure to which Trp fluorescence is sensitive. Nevertheless, both methods produce the same result: G. mirabilis A4-LDH maintains its native structure to temperatures higher than does G. seta A4-LDH.
Near-ultraviolet CD spectra for the two A4-LDH forms at 20 °C are shown in Fig. 3A. Near-ultraviolet spectra include absorbance signals from Trp and Tyr residues at approximately 295 and approximately 275 nm, respectively, and the subtraction spectrum (G. mirabilis minus G. seta) indicates that the Trp absorbance does not differ significantly between the two forms but that the Tyr absorbance is higher for G. seta A4-LDH. These results suggest that the environments in which the six Trp residues are found are similar in the two A4-LDHs, which is consistent with the Trp fluorescence data (Fig. 1A), but that at least one of the five Tyr residues occupies a different environment between the two forms.
|
Fig. 3B shows the
second-derivative near-ultraviolet absorbance spectra of the two
Gillichthys A4-LDHs at 20 °C. Second-derivative
ultraviolet spectra are useful for resolving overlapping peaks, such as the
peaks caused by Trp and Tyr absorbance at approximately 295 and approximately
275 nm. The ratio r between values a (the difference between
the trough at approximately 283 and the peak at approximately 287 nm) and
b (the difference between the trough at approximately 291 and the
peak at approximately 296 nm) provides information regarding both the relative
number of Tyr and Trp residues in the protein and the environment in which the
Tyr residues occur (Ragone et al.,
1984). For G. mirabilis A4-LDH at 20 °C,
r=0.51; for the G. seta form, r=0.71. Because the
numbers and positions of Tyr and Trp residues are identical between the two
forms, the difference in r values indicates that at least one Tyr in
the G. mirabilis LDH-A monomer is in a more hydrophobic environment
than the corresponding residue in the G. seta ortholog. Values of
r at 30 and 40 °C for G. mirabilis A4-LDH are
0.49 and 0.43, and for the G. seta form are 0.66 and 0.61,
respectively. The reduction in r with increasing temperature
indicates that the environment around the variable Tyr residue(s) is becoming
more hydrophobic, but the effect is comparable between species. Because
thermal denaturation monitored by Trp fluorescence
(Fig. 1B) or CD spectroscopy
(Fig. 2B) shows that both
A4-LDH forms remain in the native state above 40 °C, and both
forms of A4-LDH are active at 40 °C
(Fields and Somero, 1997
),
this increase in hydrophobicity cannot be due to denaturation and aggregation.
Instead, we speculate that the source of increased hydrophobicity at higher
temperatures may be either the formation of soluble oligomers or a subtle
rearrangement of subunits within the A4-LDH tetramer. Further
research is necessary to determine the underlying cause of the changes in the
Tyr environment with increasing temperature.
Hydrogen/deuterium exchange
When a protein is placed in deuterated water, amide hydrogens on the
exterior of the molecule exchange with solvent deuterons almost
instantaneously. Those amide hydrogens that are protected from solvent
exposure by their location in the interior of the protein will exchange only
when they too become exposed to solvent. Because the conditions of our H/D
exchange experiments, pH 7.0 and 20 or 40 °C, maintain the native state of
the protein (i.e. there is no thermal or acid denaturation), exchange of
internal amide hydrogens occurs predominantly through small-scale (local)
fluctuations rather than through partial or global unfolding
(Kendrick et al., 1997;
Miller and Dill, 1995
).
Different groups of amide hydrogens within the protein are exposed at
different rates, depending on the type, rigidity and location of the
structures to which they belong (Hvidt and
Nielsen 1966
). It is the exchange of these internal amide
hydrogens that we have measured using H/D-FTIR spectroscopy. The rates at
which these amide hydrogens exchange under different experimental conditions
can then be correlated with differences in flexibility between the two
A4-LDH forms or with flexibility changes within one form at two
different temperatures.
Infrared spectroscopy is used to monitor H/D exchange by following the
ratio of the amide II (approximately 1550 cm-1) peak to the amide I
(approximately 1650 cm-1) peak, which decreases during H/D exchange
as the amide II peak is replaced by a red-shifted amide II' peak
(approximately 1450 cm-1) (Dong
et al., 1996). Fig.
4 shows changes in the amide II/amide I ratio for
A4-LDH from G. mirabilis and G. seta at both 20
and 40 °C. Double-exponential decay curves were fitted to the data using
Sigmaplot non-linear regression software (Jandel Scientific, San Rafael, CA,
USA), and rate constants were derived from these regressions.
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It is clear from an examination of Fig. 4 that there are two separate exchange rate constants for each of the A4-LDHs at the two test temperatures; these rate constants represent the major populations of amide hydrogens exchanging during the time course of the experiment (360 min at 20 °C, 300 min at 40 °C). At 20 °C, the fast exchange constant for G. seta A4-LDH is greater than that for G. mirabilis A4-LDH (4.00e-2 min-1 versus 3.28e-2 min-1), but the slow exchange constants are similar (1.82e-4 min-1 versus 1.95e-4 min-1). At 40 °C, all exchange rates have increased, which is expected given the greater conformational flexibility of proteins at higher temperature. However, there is no longer a difference in the fast exchange constants between the two A4-LDHs (5.93e-2min-1 versus 6.00e-2min-1 for G. seta and G. mirabilis A4-LDHs, respectively); at 40 °C, the slow exchange constant of G. mirabilis A4-LDH is higher than that of G. seta (3.22e-4min-1 versus 2.64e-4min-1).
From these data, we conclude that at 20°C the most rapidly exchanging subpopulation of amide hydrogens is replaced more quickly in G. seta A4-LDH than it is in G. mirabilis A4-LDH. As temperature is raised to 40°C, this difference is lost. These results suggest that at 20°C the outermost secondary structural components of the G. seta A4-LDH molecule are more flexible (and more often transiently exposed to the deuterated solvent) than the same regions in the G. mirabilis molecule. However, the G. mirabilis form shows a greater increase in conformational flexibility with temperature, so that at 40°C the difference in rapid H/D exchange between the two forms has been lost.
In addition, Fig. 4 shows that the rate of change of the amide II/amide I ratio of G. mirabilis A4-LDH shifts from being dominated by the fast exchange constant to being dominated by the slow exchange constant at a higher amide II/amide I value than does that of G. seta A4-LDH. That is, in G. mirabilis A4-LDH, the slope of the curve of amide II/amide I versus time `flattens out' at a smaller degree of overall exchange. This suggests that, in G. mirabilis A4-LDH, the population of fast-exchanging amide hydrogens is smaller and that there are fewer residues in the more flexible, rapidly exchanging regions in this form than in the G. seta A4-LDH molecule.
To determine which structures within the A4-LDH molecule
contribute to the difference in H/D exchange seen between the two forms, we
used second-derivative analysis to examine the amide I (1600-1700
cm-1) region of the infrared spectra. This method allows separation
and narrowing of adjacent bands within the amide I peak. The amide I peak is
due mainly to C=O stretching vibrations along the peptide backbone, and within
this peak subtle differences in vibration due to the effects of different
secondary structures result in bands that can be visualized by
second-derivative analysis (Dong et al.,
1995; Jackson and Mantsch,
1995
). Fig. 5 shows
area-normalized, inverted second-derivative amide I spectra of
A4-LDH from G. mirabilis and G. seta at 20 and
40°C in 75% D2O over the first 60 min of H/D exchange. Because
of the area normalization, this analysis does not provide information about
the total amount of H/D exchange per unit time. Instead, these data indicate
relative levels of exchange within each type of secondary structure.
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Examination of these spectra shows that, for each isoform and at each
temperature, the major band shifts occur at approximately 1655 cm-1
and approximately 1639 cm-1 (arrows in
Fig. 5). The former has been
ascribed to -helical structure, while the latter corresponds to
ß-strand (Susi and Byler,
1986
). Fig. 6A,B shows the change in height of these peaks over time after exposure to 75%
D2O. For the ß-strand peak at approximately 1639
cm-1 (Fig. 6A),
there is a clear change in the rate at which H/D exchange occurs as
temperature increases from 20 to 40°C, but there is little difference
between the two species at either temperature. This suggests that there are no
substantial differences in ß-strand conformational flexibility between
the two A4-LDH forms. A different conclusion is reached upon
examination of the
-helix peak at approximately 1655 cm-1
(Fig. 6B). Here, there is
little difference between the species in the rate at which peak height changes
at 20°C or in G. mirabilis A4-LDH at 20 and 40°C.
The A4-LDH of G. seta measured at 40°C, however, does
show a more rapid change in peak height and, thus, a greater rate of H/D
exchange. This indicates that at least part of the difference in
conformational flexibility between the G. seta and G.
mirabilis A4-LDHs must be due to differences in exposure of
-helical structures. Furthermore, because these H/D exchange
experiments were relatively short, the
-helices that differ between the
two forms must be close to the surface of the protein, where rapid exchange
can occur.
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Discussion |
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Temperature adaptation of protein function appears to rely in large part on
the maintenance of an appropriate balance between structural stability,
allowing the maintenance of appropriate ligand-binding geometry, and
conformational flexibility, allowing catalytic conformational changes to occur
without inordinately high energy barriers
(Fields, 2001;
Feller and Gerday, 1997
;
Somero, 1995
;
Jaenicke, 1991
). Assuming the
validity of this flexibility/stability model of protein adaptation to
temperature, and confronted by the lack of differences in the primary
structures of the Gillichthys A4-LDHs, we hypothesized
that subtly different conformations between the two forms lead to changes in
both local and global protein stability and that these differences are
manifest in the measured differences in KmPYR.
Although we recognize the possibility that large-scale conformational
rearrangements, such as domain swapping, may better explain the apparently
high activation energy needed to switch from one conformation to the other (as
demonstrated by the absence of interconversion on the time scale of these
experiments), we argue that such changes would necessarily bring about larger
modifications in KmPYR than we have measured.
Thus, we favor more subtle, local conformational shifts as the likeliest
source of differences in the properties of the two orthologs.
Gerstein and Chothia (1990) described the catalytic conformational changes that A4-LDH undergoes and divided the molecule into a static inner core and a more flexible outer shell of `major mover' structures whose mobility is important for catalysis. The Tm data we have derived from our Trp fluorescence and CD spectroscopy measurements indicate that the G. mirabilis A4-LDH molecule has a more stable static core: the entire molecule `melts' at a higher temperature than does the G. seta form. In addition, our H/D exchange data show that the flexible outer region of the A4-LDH molecule is more stable in G. mirabilis A4-LDH at 20°C than it is in the G. seta form, but this difference is lost as temperature is increased to 40°C.
Interestingly, the greater sensitivity to temperature of G.
mirabilis A4-LDH H/D exchange can be correlated with the
greater sensitivity of this ortholog's KmPYR to
temperature. We showed previously (Fields
and Somero, 1997) that between 10 and 20 °C the
KmPYR values of G. mirabilis and
G. seta A4-LDHs are similar, but that between 25 and 40
°C the KmPYR of G. mirabilis
A4-LDH rises rapidly while that of G. seta
A4-LDH remains relatively constant. Such insensitivity to
temperature in the G. seta form is a hallmark of eurythermality
because it allows A4-LDH to function optimally across a broad range
of temperatures, including those (>30 °C) at which G.
mirabilis A4-LDH is losing affinity for substrate. Because
G. seta lives in an environment with a broader temperature range
(5-41 °C) than its congener (9-30 °C), the relative insensitivity to
temperature of its A4-LDH H/D exchange rate
(Fig. 4) may be evidence of
adaptation to a highly variable thermal environment. In other words, the
temperature-insensitivity of both the KmPYR and
the H/D exchange rate of G. seta A4-LDH may arise from the
same cause, the relative lack of change in the mobility of the outer shell of
the G. seta A4-LDH molecule with temperature. This is
plausible because movement of secondary structural elements in this outer
shell has been shown to be necessary for catalysis
(Dunn et al., 1991
; Gerstein
and Chothia, 1990).
The results we have reported here allow us to draw inferences regarding
where the differences in structure between the orthologs may lie. The
far-ultraviolet CD and Trp fluorescence spectroscopy indicate that the
secondary and tertiary structures of these A4-LDH orthologs are
very similar. This is not surprising considering the large number of studies
of LDH across taxonomic domains, which show that the structure of this enzyme
is strongly conserved regardless of environmental temperature or taxonomic
affinity (Read et al., 2001;
Auerbach et al., 1998
), but,
combined with the identity in primary structure between the
Gillichthys A4-LDHs, it does suggest that the
conformational differences between these enzymes must be slight. In addition
to the differences in the flexibility of parts of the outer shell between the
two A4-LDH forms discussed above, we have shown that at least one
Tyr is in a different microenvironment in the two orthologs, as demonstrated
by the different r values (Fig.
3B) at 20, 30 and 40 °C. Because it is unlikely that two
independent conformational differences are responsible for these two
observations, it is parsimonious to assume that the modification in fold or
subunit association responsible for the change in hydrophobicity of the Tyr
environment is also responsible for changes in flexibility and for the lower
Tm and relatively temperature-insensitive
KmPYR of G. seta A4-LDH in
comparison with G. mirabilis A4-LDH. These findings,
combined with an analysis of the three-dimensional structure of
Gillichthys A4-LDH, allow us to identify one region of the
A4-LDH molecule that is likely to be involved in the conformational
shift.
To determine the positions of Trp residues in Gillichthys
A4-LDH, we examined a homology model of the protein based on
dogfish and porcine A4-LDHs.
Fig. 7A shows one LDH-A monomer
of the Gillichthys A4-LDH homotetramer. There are five Tyr
residues at positions 82, 126, 144, 238 and 246 in the protein sequence. In
the context of the static core and mobile outer shell of A4-LDH
(Gerstein and Chothia, 1990), three of the five Tyr residues, Tyr82, Tyr126
and Tyr144, are located in the static core of the molecule. Because these Tyr
residues do not move appreciably during catalysis (Gerstein and Chothia,
1990), it is unlikely that they are structurally associated with
conformational modifications responsible for differences in
KmPYR and, according to the parsimony argument
outlined above, to the other physical differences we have measured between the
two A4-LDHs. Tyr238 is located in the outer shell, on a `major
mover' secondary structural element whose conformational rearrangement is
necessary for catalysis (Dunn et al.,
1991; Gerstein and Chothia, 1990). This structure, helix
1G
2G, closes down over the catalytic vacuole after entry
of substrate and cofactor (the other major movers, helix
H and the
`catalytic loop,' also close down over the catalytic vacuole from other
directions) (Gerstein and Chothia, 1990). Tyr238 is necessarily
solvent-exposed; Abad-Zapatero et al.
(1987
) have argued that
rotation of the Tyr238 side chain away from the solvent and into the active
site would strongly inhibit enzyme activity. Thus, minor changes in structure
should not alter the hydrophobicity of the environment surrounding Tyr238
it must continue to project into the polar medium for catalysis to
occur.
|
Immediately C-terminal to the mobile helix 1G
2G is the
relatively static helix
3G, which is involved in subunit binding
through interactions with helix
C on the complementary monomer
(Abad-Zapatero et al., 1987
).
The final Tyr residue, Tyr246, sits at the junction between helices
1G
2G and
3G, a `hinge' between the flexible and
static portions of one side of the active site. Tyr246 residues in two
monomers hydrogen-bond through a water molecule trapped within the Q-axis
interface of the A4-LDH homotetramer
(Abad-Zapatero et al., 1987
)
(Fig. 7B). Thus, Tyr246 seems
to be in an appropriate location to experience changes in hydrophobicity if
its microenvironment is altered slightly. That is, if shifts in the
interaction between the two subunits were to modify the intersubunit contact
region and alter the relative positions of contact residues and trapped water
molecules, they would modify the absorbance of Tyr246 in the near-ultraviolet
region and be seen as differences in r value between the two
forms.
In addition, Figs 5 and
6 show that a surface
-helix is involved with differences in H/D exchange rates between
G. mirabilis and G. seta A4-LDHs. A
conformational modification centered around helix
1G
2G
and helix
3G would be consistent with these results and would explain
parsimoniously both the difference in r and the difference in
second-derivative infrared spectra.
Tyr246 can also be associated with changes in
KmPYR between the two Gillichthys
A4-LDH molecules. It has been argued that the structural changes
underlying changes in enzyme kinetic parameters must be associated with those
regions of the molecule that move during catalysis
(Fields and Somero, 1998)
because it is the energy barriers to conformational change that ultimately
control the rates of binding of substrate to and release of product from
enzymes, such as A4-LDH, where the chemical modifications of
catalysis are not rate-limiting (Dunn et
al., 1991
). In the A4-LDHs of Antarctic notothenioid
fishes, helices
1G
2G and
H
(Fig. 7A) were identified as
targets of temperature adaptation (Fields
and Somero, 1998
) because changes in their flexibility would
necessarily alter the energy barriers to substrate binding, catalysis and
product release. In the notothenioid enzyme, the region N-terminal to helix
1G
2G was noted as having a number of amino acid
substitutions (e.g. Ser
Gly and Thr
Gly) that could lead to greater
flexibility in the cold-adapted form. Within the Gillichthys
A4-LDHs studied here, Tyr246 sits in the opposite, C-terminal
`hinge' of helix
1G
2G. Thus, changes in conformation and
mobility of this `major mover' structure, which is closely associated with
active site residues (e.g. Thr248), would result in changes in not only
r or H/D exchange rates but also in substrate affinity.
To summarize, the results obtained using H/D exchange reinforce the
argument that 1G
2G is associated with the conformational
and functional differences between the two Gillichthys
A4-LDHs. It is the only
-helix near the surface of the
protein that both has a nearby buried Tyr and is involved in motions necessary
for catalysis. It is only in this area that a minor change in conformation
could explain the three differences we have noted between G.
mirabilis and G. seta A4-LDHs: alterations in
KmPYR (necessarily linked to changes in
mobility of the `major mover' structures), changes in Tyr microenvironment
hydrophobicity (i.e. r value) and differences in H/D exchange
constants associated with
-helical structure.
We therefore identify the region immediately surrounding Tyr246, at the
C-terminal end of the 1G
2G helix, as the area most likely
to show conformational differences between the A4-LDHs of G.
mirabilis and G. seta. We argue that the data we have presented
further support the idea that minor changes in the conformation of enzymes, in
the absence of any change in amino acid structure, may be a source of adaptive
differences in function. The mechanism by which G. seta and G.
mirabilis produce A4-LDHs with subtly different conformations,
either during or after translation, remains to be described.
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Acknowledgments |
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References |
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