From the Institute of Molecular Biophysics, Johannes Gutenberg-University of Mainz, Welder-Weg 26, D-55128 Mainz, Germany
Received for publication, November 17, 2000, and in revised form, March 13, 2001
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ABSTRACT |
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Hemocyanins are multisubunit respiratory proteins
found in many invertebrates. They bind oxygen highly cooperatively.
However, not much is known about the structural basis of this behavior. We studied the influence of the physiological allosteric effector L-lactate on the oxygenated quaternary structure of
the 2×6-meric hemocyanin from the lobster Homarus
americanus employing small angle x-ray scattering (SAXS). The
presence of 20 mM L-lactate resulted in
different scattering curves compared with those obtained in the absence
of L-lactate. The distance distribution functions p(r) indicated a more compact molecule in presence of
L-lactate, which is also reflected in a reduction of the
radius of gyration by about 0.2 nm (3%). Thus, we show for the first
time on a structural basis that a hemocyanin in the oxy state can adopt
two different conformations. This is as predicted from the analysis of
oxygen binding curves according to the "nesting" model. A
comparison of the distance distribution functions p(r)
obtained from SAXS with those deduced from electron microscopy revealed
large differences. The distance between the two hexamers as deduced
from electron microscopy has to be shortened by up to 1.1 nm to agree
well with the small angle x-ray curves.
Hemocyanins are the oxygen transport proteins of most molluscs and
arthropods. They are enormous molecular structures, ranging in
molecular mass from 4.5 × 105 to more than
107 Da; some contain more than 100 oxygen binding sites
(1-3). Hemocyanins exhibit cooperative oxygen binding and respond to a
variety of allosteric effectors (4-8). As a consequence of their
structural complexity, hemocyanins have proved important in extending
our understanding of allostery. For example, explanation of the
cooperative oxygen binding by arthropod hemocyanin has required
extension of the classical
MWC1 model to the
"nesting" model (8, 9), which reflects the hierarchical structure
of these proteins. The nesting model requires four states (rT, rT, tR,
rR) instead of the two states (T, R) of the MWC model. It makes the
prediction that even fully oxygenated hemocyanin can exist in two
conformational states. Although the model has served well to explain
oxygen binding and its dependence on effector (see Ref. 8, for
example), it has never been tested directly. That is, no experiments
have been performed to detect the two oxy conformations predicted by
the nesting model. To be definitive, such experiments must be carried
out in solution, under rigorously controlled oxygenation levels and
with a sensitivity sufficient to detect even small conformational changes.
A suitable method to detect conformational changes of large proteins in
solution is small angle x-ray scattering (SAXS). This technique has
been applied successfully to monitor changes of the quaternary
structure for several cooperative proteins such as hemoglobin and a
number of allosteric enzymes (10-18). Different conformations have
also been reported for the oxy- and deoxyhemocyanin from the tarantula
Eurypelma californicum from SAXS measurements (19).
Here, we present SAXS data of fully oxygenated 2×6-meric hemocyanin
from the lobster Homarus americanus in the absence and presence of the physiological effector L-lactate. We will
show that the fully oxygenated 2×6-meric hemocyanin can adopt two
clearly distinguishable conformations, which differ in structural
details. This observation indicates that the allosteric influence of
L-lactate on the oxygen binding behavior of this hemocyanin
is based on a preferential binding of L-lactate to one of
the two possible conformations present in the oxy state.
Preparation of Hemocyanin--
Lobsters (H. americanus) were obtained from a local fish supplier. Hemolymph
was obtained from heart puncture as described elsewhere (8). The
cellular content of the hemolymph was removed from the sample by
centrifuging for 10 min at 20,000 × g. Hemocyanin (2×6-mer) was purified by size exclusion chromatography (Bio-Gel A1.5
m, Bio-Rad) in 0.1 M Tris/HCl buffer at pH 8.0 in presence of 20 mM CaCl2 and 20 mM
MgCl2 at 4 °C. The hemocyanin solution was dialyzed
overnight at 4 °C against 0.1 M Tris/HCl buffer, pH 7.2 (at 20 °C) in the presence of 20 mM CaCl2
and 20 mM MgCl2. Protein solution for the
measurement with L-lactate contained 20 mM
L-lactate. The pH was determined for all samples to be pH 7.2 at 20 °C. Under these condition the 12-meric lobster hemocyanin is stable at least for months. The degree of saturation with oxygen was
determined by comparing the absorption at 280 and 340 nm. The ratio
A280/A340 of about
4.5 shows that the hemocyanin was fully oxygenated under these conditions.
Protein concentrations were determined spectroscopically using
A280 nm = 1.34 (20). Tris and
MgCl2 were purchased from Roth (Karlsruhe, Germany).
CaCl2 was obtained from Fluka (Deisenhofen, Germany), and
L-lactate acid was from Sigma (Deisenhofen, Germany).
Small Angle X-ray Scattering--
Small angle x-ray scattering
experiments were performed at the SAXS camera JUSIFA at the synchrotron
beamline B1 at Hamburger Synchrotronstrahlungslabor, Hamburg,
Germany (21). The scattered intensities were recorded by a
two-dimensional multiwire proportional counter with 256 × 256 pixels. Two different distances between detector and sample (918 and
3618 mm) were used with a step in reciprocal space of
Calculation of the distance distribution functions p(r) and
desmearing of the intensity were performed with the method of indirect Fourier transformation using the program GNOM, which also
delivers errors for the p(r) curves (22, 23).
Model Calculations--
For comparison with the experimental
data, model distance distribution functions were calculated from atomic
models. For this the x-ray structure of the hexameric crustacean
hemocyanin from Panulirus interruptus was used (24). First,
an atomic model of the dodecameric hemocyanin was constructed using the
relative translations and rotations of the two hexamers as reported for the 2×6-meric hemocyanin from the closely related crab Cancer pagurus obtained by transmission electron microscopy (TEM) and three-dimensional reconstruction (25). From the atomic model a distance
distribution function p(r) was calculated by counting all
distances between the atoms, each weighted by the atomic form factor.
To fit the experimental p(r), the center of mass distance between the two hexamers was varied in the direction of the
z axes in a second step. The rotational parameters and the
other two translational parameters describing the relative position of
the hexamers were adjusted to the results obtained from TEM experiments. The radius of gyration of the molecular models was calculated directly from the coordinates using the distances between the atoms and the center of mass.
Fig. 1a shows the original smeared
scattering intensities of lobster hemocyanin in the presence and
absence of 20 mM L-lactate. The difference
curves between the scattered intensities without and with the effector
were calculated for both protein concentrations, 7.8 and 15.6 g/liter
(Fig. 1b). For both measurements with low and high protein
concentration, the differences exceed the experimental errors up to a
factor of five for q values between 0.15 and 0.4 nm
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
q = 0.017 nm
1
(q = 4
sin
/
) at a wavelength of
= 0.1553 nm. Scattering curves were measured at two protein
concentrations, 7.8 and 15.6 g/liter, both in the presence and absence
of 20 mM L-lactate. A quartz capillary flow
cell was used for all experiments. The scattered intensities were
corrected for detector sensitivity and instrumental background and
normalized for monitor counts and transmission. Scattering curves of
the buffer were measured under identical conditions and subtracted from
the protein scattering curves. The total measurement times for the
protein samples and the buffer were divided into several time slices to
monitor possible shifts. Since no differences of the scattered
intensity between the individual measurements were detected, the time
slices were added to decrease the statistical error.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1. The shape of the two difference curves
is very similar over the whole q range.
View larger version (15K):
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Fig. 1.
SAXS intensities and difference curves for
lobster hemocyanin with and without L-lactate.
a, lobster hemocyanin in the absence (filled
symbols) and presence of 20 mM L-lactate
(open symbols). b, difference of the scattered
intensities divided by the propagated experimental error,
Ãdifference = (Ã2 + 2lac) of the difference. Data are shown for
protein concentrations 15.6 g/liter (filled symbols) and 7.8 g/liter (open symbols). I and
are the
intensity and experimental error for lobster hemocyanin without
L-lactate, and Ilac and
lac are the corresponding values in the presence of
L-lactate.
The radius of gyration was determined from the distance distribution
function p(r) (Fig. 2). For
the low concentration of hemocyanin it was found to be 7.21 ± 0.05 nm in absence of L-lactate and 7.02 ± 0.07 nm in
presence of L-lactate. The corresponding values for the
high protein concentration are 7.19 ± 0.05 and 6.99 ± 0.04 nm. These values agree excellently with the radii of gyration
calculated from the desmeared Guinier plot (7.19 and 7.01 nm for
the low protein concentration; 7.17 and 6.98 for the high protein
concentration). Although small, the differences in the radii of
gyration in the absence and presence of L-lactate are
highly significant, given the precision of the data. The decrease of
the radius of gyration by about 0.2 nm in the presence of
L-lactate indicates that the hemocyanin molecule becomes
more compact.
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In addition, the maximum diameters of lobster hemocyanin were calculated from the p(r) curves (Fig. 2). Although the value of 23.5 ± 0.5 nm in the presence of L-lactate is only slightly shorter than that observed in the absence of L-lactate (24.0 ± 0.5 nm), the number of large distances in the range between 14 and 24 nm is significantly smaller, compensated by an increase of p(r) at shorter distances (Fig. 2). Because the maximum diameter of a hemocyanin hexamer is about 13 nm as calculated from the x-ray structures (24, 25), only interhexameric distances can contribute to p(r) above 13 nm. Thus, the observed differences in p(r) indicate that the centers of mass of the two hexamers are shifted together by the binding of L-lactate.
These observations are supported by the following consideration: based
on the subunit composition of the lobster hemocyanin, we can assume two
identical hexamers (26). Therefore it is possible to calculate the
distance between the centers of mass of the hexamers, Dhex-hex, from the RG values
when the radius of gyration for the hexamer is known (27).
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(Eq. 1) |
We compared data obtained for lobster hemocyanin in absence of
L-lactate by SAXS with a model based on the
three-dimensional reconstruction of the closely related hemocyanin from
another crustacean, the crab Cancer pagurus, obtained by
transmission electron microscopy TEM (25). The radius of gyration for
this TEM model was calculated to be 7.44 nm. The SAXS experiments, however, yielded values that are smaller in both cases, by 0.23 nm in
the absence of L-lactate and 0.42 nm in presence of
L-lactate. In addition, the distance distribution function
deduced from TEM showed an extremely pronounced shoulder (Fig.
3), which was not found in the SAXS
experiments. The following consideration supports the hexamer-hexamer
contacts in our SAXS measurements. Obviously, the TEM model does not
fit our experimental data. The closest C-C
distances between the hexamers in the
TEM model are in the range of about 1.2 nm, which is too large for
reasonable contacts between subunits. Usually values of about 0.4 nm
for the C
-C
distances between subunits
are found in x-ray structures. To obtain a good agreement between the
experimental distance distribution function and the p(r)
calculated from the models, we reduced the center of mass distance
between the hexamers from 11.5 nm as calculated from the TEM model to
10.4 nm (with L-lactate, Fig. 3) and to 10.9 nm (without
L-lactate). These two fitted values agree well with the
distances (10.3 and 10.8 nm, see above) obtained experimentally. In
addition, the closest C
contacts between the hexamers
were then determined to 0.3 nm in the presence of L-lactate
and to 0.6 nm in the absence of L-lactate.
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DISCUSSION |
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Small angle x-ray scattering demonstrates that oxygenated hemocyanin from H. americanus undergoes a conformational change in the presence of the allosteric effector L-lactate. Different SAXS intensities and values for the radius of gyration were obtained in the presence and absence of 20 mM L-lactate, a concentration that is higher than the values (5-10 mM) observed in crustacea after stress (1, 2, 6, 7). Concentrations higher than 20 mM do not effect the oxygen binding curve anymore (7). The effector L-lactate seems to bind preferentially to a more compact hemocyanin molecule based on the decrease of the radii of gyration and the distance between the two hexamers. The inter-hexamer distance decreases by 0.5 nm, which corresponds to 2% of the total molecular dimensions. Besides the change in the hexamer-hexamer distance, additional rearrangements within the hexamers may occur during the conformational transition but cannot be identified with this method. The observed differences seem to be small. However, even when the oxy and deoxy states of the 24-meric hemocyanin from the tarantula E. californicum were compared, differences of similar magnitude were obtained (19).
To our knowledge we have provided here the first direct evidence that an allosteric effector can induce changes in the quaternary structure of a hemocyanin even though it remains fully oxygenated. The existence of two distinct, albeit fully oxygenated, conformations unequivocally rules out the applicability of the simple MWC model (28), which in toto offers only two conformations: one high affinity (at high pO2) and one low affinity (at low pO2) conformation. The observed increase of the affinity upon addition of L-lactate for crustacean hemocyanin (6, 7) means that in the frame of a MWC model the R state is stabilized compared with the T state. Thus, under fully oxygenated conditions addition of L-lactate cannot, according to an MWC model, change the conformational distribution to an observable amount, since the most favored conformation is already present to a large extent.
Thus, both oxygen binding curves obtained at different pH values (8) and the SAXS experiments presented here rule out the simple MWC model. In principle extensions of the MWC model may be considered to explain these two conformations in the oxy state (9, 29, 30). However, the dependence of the oxygen binding curves on pH can be better explained in terms of the nested MWC model (8, 31, 32). In addition, the binding curves of the half-molecules can be predicted and confirmed by this model (8, 31, 32). We could not find any other model that was in comparable agreement with all of these experimental data.
The structural interpretation of the observed change in RG values is independent of the model used for the interpretation of the oxygen binding behavior. The models for cooperativity are based on functionally different conformations, which are distinguished on the basis of their oxygen affinities. No structural constraints are imposed by the SAXS data. But any structural difference between the hemocyanin in the absence and presence of L-lactate is in accordance with the existence of two functionally different conformations in the oxy state as required in the nested MWC model.
For other hemocyanins, the existence of different conformations within both the oxy and deoxy state was also supported by analysis of the oxygen binding (8, 9, 30-33). Other methods also confirmed the applicability of the nested MWC model for arthropod hemocyanins. For the 2×12-meric tarantula hemocyanin two different conformations in the oxy state were found using a fluorescence tag 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-chloride (34). This has also been confirmed by O2/CO replacement experiments (35). An isothermic titration calorimetry study of effector binding to 2×6-meric hemocyanin from Homarus vulgaris also indicates the existence of two conformations in the oxy state, which differ in their affinities for the effectors urate and caffeine (36, 37). However, our small angle x-ray results provide the first direct structural evidence for two different conformations in the oxy state and reveal that structural changes are involved.
The simple MWC model describes well the oxygen binding properties of hexameric hemocyanins. For 1×6-meric hemocyanin of P. interruptus sophisticated O2/CO replacement experiments confirmed that the simple MWC model is the appropriate model (38). Additionally, hexamers obtained from dissociation of 2×6-meric hemocyanin from H. americanus were also well described by the simple MWC model (8). The binding parameters obtained for this hexamer correspond well to the predicted binding parameters of the two states (rR, tR) in the R conformation according to the nested MWC model when applied to the native 2×6-meric molecule (8). The two additional states (rT, tT) of the T conformation as postulated by the nested MWC model for the 1×6-mers must have been created by the assembly of the two hexamers accompanied by a functional coupling. The two conformations observed by SAXS can be assigned to the two high affinity states, rR and rT. On the basis of these considerations the nested MWC model seems the simplest to account for all the data. Therefore, other models were not considered for describing arthropod hemocyanins, especially from H. americanus.
Two questions arise: why do larger aggregates than hexamers exist, and is there any advantage for respiratory proteins such as the hemocyanins to function according to the nested MWC model? Obviously, for a number of species the hexameric hemocyanin serves well for the oxygen delivery (1-5). The reason to develop higher aggregates may to be found in keeping the osmotic pressure low in the hemolymph with respect to the extracellular proteins (39). But no further advantage would result from increasing the size of the molecule or the allosteric unit while maintaining regulation by the simple MWC model. However, a more sophisticated regulation mechanism would also be established when the 1×6-meric allosteric units (each behaving according to the simple MWC model) will not only assemble structurally but will also establish a functional coupling according to the nested MWC model. As discussed in previous papers this offers the possibility to provide an influence of different allosteric effectors on the oxygen binding behavior at different levels of the quaternary structure (1, 8, 9, 40).
An additional advantage for multihexameric hemocyanins working according to the nested MWC model is the ability to create cooperative behavior over a broad range of saturation levels as discussed previously (8, 9, 40). Thus, extracellular hemocyanins working according to the nested MWC model posses a broader functional flexibility compared with a hypothetical molecule with a simple MWC mechanism. This might be a consequence of the fact that extracellular respiratory proteins in poikilothermic animals such as arthropods have to face more pronounced environmental fluctuations than do intracellular respiratory proteins in mammals. The larger functional flexibility may help to ensure the delivery of oxygen precisely to the tissues under a broad range of conditions.
The importance of applying different methods for structural
investigations is demonstrated in our study by a comparison of hemocyanin structures obtained from TEM and SAXS. The interhexameric distance as obtained by TEM had to be shortened to be in agreement with
the SAXS data. Similar deviations of calculated distance distribution
functions p(r) based on TEM from those obtained by SAXS have
been reported previously for the tarantula hemocyanin (19). As in the
case of the 2×6-meric lobster hemocyanin, the half-molecules of the
2×12-meric tarantula hemocyanin had to be shifted together to yield
contacts between the half-molecules and to be in reasonable agreement
with the SAXS data (Fig. 4). The
differences may arise from the fact that these TEM studies utilized
negative staining. It may be that the hemocyanin molecules are slightly
distorted by the preparation of the negatively stained specimen
resulting in larger differences between the two loosely connected
half-molecules. Such an artifact cannot arise when SAXS is applied,
since untreated proteins are investigated in solution. Thus, SAXS,
which analyzes molecules in solution, may be considered as an important
check for the three-dimensional reconstruction of macromolecules based
on negatively stained specimens by TEM. This seems to be important
especially for proteins with a large and complex quaternary
structure.
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ACKNOWLEDGEMENTS |
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We thank Dr. G. Goerigk at Hamburger Synchrotronstrahlungslabor/Deutsches Elektronen Synchrotron (Hamburg, Germany) and Dr. T. Nawroth, University of Mainz, for their experimental and organizational support as well as Dr. K. van Holde for his many very helpful comments.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft, the Center for Science and Medicine, and the Center for Material Science, Mainz, Germany.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
49-6131-3923570; Fax: 49-6131-3923557; E-mail:
decker@biophysik.biologie.uni-mainz.de.
Published, JBC Papers in Press, March 15, 2001, DOI 10.1074/jbc.M010435200
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ABBREVIATIONS |
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The abbreviations used are: MWC, Monod-Wyman-Changeax, SAXS, small angle x-ray scattering; TEM, transmission electron microscopy.
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