Structural and Functional Effects of Pseudo-module Substitution in Hemoglobin Subunits
NEW STRUCTURAL AND FUNCTIONAL UNITS IN GLOBIN STRUCTURE*

Kenji InabaDagger , Koichiro IshimoriDagger , Kiyohiro Imai§, and Isao MorishimaDagger

From the Dagger  Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501 Japan and the § Department of Physicochemical Physiology, Osaka University Medical School, Suita, Osaka 565-0871, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Functional and structural significance of the "module" in proteins has been investigated for globin proteins. Our previous studies have revealed that some modules in globins are responsible for regulating the subunit association and heme environmental structures, whereas the module substitution often induces fatal structural destabilization, resulting in failure of functional regulation. In this paper, to gain further insight into functional and structural significance of the modular structure in globins, we focused upon the "pseudo-module" in globin structure where boundaries are located at the center of modules. Although the pseudo-module has been supposed not to retain a compactness, the beta alpha (PM3)-subunit, in which one of the pseudo-modules, the F1-H6 region, of the alpha -subunit is implanted into the beta -subunit, conserved stable globin structure, and its association property was converted into that of the alpha -subunit, as the case for the module substituted globin, the beta alpha (M4)-subunit. These results suggest that modules are not unique structural and functional units for globins. Interestingly, however, the recent reconsideration of the module boundary indicates that the modules in globins can be further divided into two small modules, and one of the boundaries for the new small modules coincides with that of the pseudo-module we substituted in this study. Although it would be premature to conclude the significance of the modular structure in globins, it can be safely said that we have found new structural units in globin structure, probably new modules.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Recent structural studies of proteins have revealed that many protein structures are constructed by the compact structural unit, "modules," which correspond to the exons on the gene structure (1, 2). The gene of globin is made up of three exons interrupted by two introns, and exons 1-3 correspond to the modules M1, M2 + M3, and M4, respectively. The correlation of globin structure and function with its modular structure was exemplified by the observation that specific functions of globins are attributed to the specific modules (3). As found in Fig. 1A, the amino acid residues associated with the heme contacts and the alpha 1-beta 2 contacts are concentrated in module M2 + M3, whereas the alpha 1-beta 1 contact cluster is located in module M4.


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Fig. 1.   A, module and pseudo-module boundaries and residues with well defined functional roles in hemoglobin subunits proposed by Eaton (3). B, amino acid sequence for hemoglobin alpha - and beta -subunits in the pseudo-module PM3 and the module M4. The identical residues are expressed in bold type. The underlines indicate the residues contributing to alpha 1-beta 1 or alpha 1-beta 2 contacts. The proximal histidine (F8) is marked by an asterisk.

To gain insights into the structural and functional significance of the module in globins, we have prepared a variety of the module substituted globins (4-6). The module M4 substituted hemoglobin beta -subunit (beta alpha (M4)-subunit), in which the module M4 was replaced by that of the alpha -subunit, exhibited the native beta -subunit-like heme environmental structure, while it preferentially associated with the native beta -subunit and not with the alpha -subunit (4). These findings indicate that the module substitution can convert the association property of the hemoglobin subunit without substantial structural changes in the heme vicinity, suggesting that the module is a structural and functional unit (4). However, the counterpart chimeric globin, the alpha beta (M4)-subunit, of which module M4 was derived from that of the beta -subunit, was quite unstable, and the association property was not affected by the module substitution (5). Such destabilization in globin structure was also encountered for other module substituted globins (6), which leads us to reconsider the structural and functional significance of the modular structures in globins and examine effects of "non-module substitution" on globin structure and function to compare with those of the module substitution.

In the present study, we have focused upon the "pseudo-module" in globin structure. The pseudo-module is defined as a segment starting at the center of one module and ending at the center of the adjacent module (Fig. 1A) and is supposed not to form a compact structural unit (7). Since the pseudo-modules do not statistically coincide with exons (8), they would have neither evolutionary nor functional meanings (9, 10). Herewith, we have prepared a pseudo-module substituted globin, the beta alpha (PM3)-subunit, as illustrated in Fig. 2 and compared its structural and functional properties with those of the corresponding stable module substituted globin, beta alpha (M4)-subunit. We are concerned here with the effects of the pseudo-module substitution on the globin structure. Furthermore, we have paid attention to the association property of the chimeric globins, since the amino acid residues contributing to the alpha 1-beta 1 contact are concentrated in the pseudo-module PM3 as well as in the module M4 (Fig. 1A). The heme environmental structure and oxygen affinity for the chimeric globin have been also examined by using various spectroscopic methods to describe the functional and structural effects of the pseudo-module substitution in globins.


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Fig. 2.   Novel globin subunits synthesized in this study. Restriction enzyme sites used in these preparations are noted in parentheses.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Expression Vector Construction-- The expression vectors of the beta alpha (M4)- and beta alpha (PM3)-subunits were constructed as illustrated in Fig. 2. The N-terminal valine residue was replaced by a methionine to initiate the peptide elongation for the module and pseudo-module substituted subunits. To obtain the genes of the beta alpha (PM3)-subunit, KpnI (GGTACC) and MluI sites (ACGCGT) were introduced at the start and end of the PM3, respectively, by polymerase chain reaction with silent mutation.

Protein Preparation-- All of the module and pseudo-module substituted subunits were purified as previously reported for recombinant Hb (4, 11-13). We confirmed the correct expression of the desired subunits by fast atom bombardment-mass spectroscopy (data not shown) (14), and no additional mutations were detected. We also synthesized "wild-type" beta -subunit1 that has a methionine residue at the N-terminal instead of the valine residue as a reference and confirmed that the structural properties of the wild-type beta -subunit are virtually the same as those of "native" beta -subunit isolated from human red blood cells.

Circular Dichroism Spectra-- CD spectra of the cyano-met subunits in far UV region were measured with Jasco J-760. Concentration of the samples was 5 µM, and the light path of the cell was 1 mm. The buffer used in the measurements was 20 mM sodium phosphate containing 0.1 M NaCl and 5 mM NaCN, pH 7.4, at room temperature.

Urea Denaturation Curves-- Reaction solutions contained 20 mM Tris, pH 7.4, 1 mM NaCN, and various concentrations of urea. Sample concentration was 5 µM. The ellipticity at 222 nm was monitored by Jasco J-760 CD spectrometer after ~10 h equilibration at room temperature. Cyano-met derivatives are used for the measurements to avoid aggregation of heme and reduce the irreversible denaturation2 (15). The fractional denatured population (fD) for various urea concentrations was estimated by Equation 1.
f<SUB><UP>D</UP></SUB>=([&thgr;]<SUB>222, <UP>N</UP></SUB>−[&thgr;]<SUB>222</SUB>)/([&thgr;]<SUB>222, <UP>N</UP></SUB>−[&thgr;]<SUB>222, <UP>D</UP></SUB>) (Eq. 1)
where [theta ]222, N, [theta ]222, D, and [theta ]222 represent ellipticities at 222 nm in the native (N) and denatured (D) states and in each urea concentration, respectively. The free energy of denaturation, Delta G, was calculated by Equation 2 (15).
&Dgr;G=<UP>−</UP>RT <UP>ln</UP>(f<SUB><UP>D</UP></SUB>/(1−f<SUB><UP>D</UP></SUB>)) (Eq. 2)
When Delta G varied linearly with urea concentration, [urea], Delta GH2O, extrapolated Delta G at [urea] = 0, can be estimated by the following equation:
&Dgr;G=&Dgr;G<SUB><UP>H<SUB>2</SUB>O</UP></SUB>−m<SUB><UP>urea</UP></SUB>[<UP>urea</UP>] (Eq. 3)
where murea is the slope of the linear relation between Delta G and [urea].

Gel Chromatogram-- Gel filtration measurements were performed by using a Sephacryl S-200 HR column (0.8 × 62 cm) at 4 °C. The buffer used for the chromatography was 50 mM Tris, in the presence of 0.1 M NaCl, and 1 mM Na2EDTA, pH 7.4, and the flow rate was 7 ml/h. The eluted fractions were monitored by absorption at the Soret band (16, 17). Dimer-tetramer dissociation constant of the samples was determined by concentration dependence of the centroid elution volume over the range from 0.5 to 200 µM (16, 17). The following functional dependence of the elution volume (Ve) versus protein concentration (CT) allows us to determine the dimer-tetramer equilibrium constants for the samples (16), see Equation 4.
V<SUB>e</SUB>=∑ jV<SUB>j</SUB>(m<SUB>j</SUB>)/C<SUB>T</SUB> (Eq. 4)
where Vj is the elution volume for the individual species pertaining to the various aggregates (j-mers), and the (mj) term represents molar concentrations for the respective species.

NMR Spectra-- 1H NMR spectra at 500 MHz were recorded on Bruker Avance DRX 500. We used a WaterGate pulse sequence for the diamagnetic region to minimize the water signal in the sample. For the measurements of the hyperfine-shifted proton resonances, we utilized a LOSAT pulse sequence. The probe temperature was controlled at 290 ± 0.5 K by a temperature control unit of the spectrometer. The volume of the NMR sample was 500 µl, and the concentration was 600 µM on the heme basis. Proton shifts were referenced with respect to the proton resonance of 2,2,-dimethyl-2-silapentane-5-sulfonate.

Oxygen Equilibrium Curves and Analysis-- Oxygen equilibrium curves were measured by using an improved version (18, 19) of an auto-oxygenation apparatus (20). The wavelength of the detection light was 560 nm, and the protein concentration was 60 µM on the heme basis. The temperature of the sample in the oxygenation cell was constant within ±0.05 °C. The hemoglobin reductase system (21) was added to the sample before each measurement to reduce oxidized subunits. To minimize the autoxidation of the sample during the measurements, catalase and superoxide dismutase were added to the sample, and the concentration was 0.1 µM (22, 23). The oxygenation data were acquired by use of a micro-computer (model PC-98XA, Nippon Electric Co., Tokyo), which was interfaced to the oxygenation apparatus (24).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Circular Dichroism Spectra-- As reported by our previous studies (5, 6), the module substitutions often induced severe destabilization in globin structure, which is characterized by the prominent decrease of the negative ellipticities at 222 and 208 nm in the CD spectra (5). In Fig. 3, however, the pseudo-module substituted globin, beta alpha (PM3)-subunit, exhibits two broad and large negative peaks around 222 and 208 nm characteristic of the alpha -helical structure. These negative peaks were also observed for the module substituted beta alpha (M4)-subunit and native hemoglobin (25), indicating that the secondary structure for the beta -subunit is almost insensitive to the substitution of the pseudo-module PM3 as the case for the M4 substitution.


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Fig. 3.   CD spectra in far UV region of the cyano-met native, module, and pseudo-module substituted globin subunits. Lines correspond to hemoglobin (---), beta alpha (M4)-subunit (- - -), and beta alpha (PM3)-subunit (--- - ---).

Urea Denaturation Curves-- Alterations in equilibrium stability upon the module and pseudo-module substitutions were quantified by the urea-induced denaturation experiment. As clearly delineated in Fig. 4A, the transition curve for the urea denaturation in the beta alpha (PM3)-subunit is quite similar to that for the native beta -subunit, indicating that the globular structure of the beta alpha (PM3)-subunit is as stable as that of the native beta -subunit. For the beta alpha (M4)-subunit, the denaturation curve was shifted to the right side from that of the beta -subunit and almost superimposed on that of tetrameric native hemoglobin. In Fig. 4B, the free energy of denaturation (Delta G) was plotted against urea concentrations, and a linear fitting procedure using Equation 3 determined the extrapolated Delta G in the absence of urea, Delta GH2O, and the slope of the linear relation (i.e. dDelta G/d[urea]), murea (Table I). Although the substitution of the module M4 increases Delta GH2O by about 25 kJ/mol and murea by 2.3 kJ/mol per M (M, urea concentration), the corresponding parameters for the substitution of the pseudo-module PM3 are almost unchanged from those for the beta -subunit. The effects of the pseudo-module PM3 substitution on the protein stability of the beta -subunit are minimal, compared with those of the module M4.


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Fig. 4.   A, urea-induced denaturation curves for hemoglobin (black-triangle), alpha -subunit (black-down-triangle ), beta -subunit (black-diamond ), beta alpha (M4)-subunit (triangle ) and beta alpha (PM3)-subunit (open circle ). Molecular ellipticities in the native and completely denatured states are normalized to 0 and 1, respectively. Experimental conditions were as follows: 20 mM Tris, 0.1 M NaCl, 5 mM NaCN, pH 7.4, at 290 K. Sample concentration was 5 µM on the heme basis. B, relationship between Delta G and [urea] for hemoglobin (black-triangle), alpha -subunit (black-down-triangle ), beta -subunit (black-diamond ), beta alpha (M4)-subunit (triangle ), and beta alpha (PM3)-subunit (open circle ). They were calculated from the respective urea denaturation curves in A.

                              
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Table I
Parameters of urea denaturation for tetrameric Hb, isolated alpha -, beta -, beta alpha (PM3)-, and beta alpha (M4)-subunits
Delta GH2O and murea were determined by fitting the Delta G-[urea] relations to Equation 3.

Association Properties-- However, the association property of the pseudo-module substituted globin, beta alpha (PM3)-subunit, clearly differs from that of the wild-type beta -subunit. Fig. 5A illustrates gel chromatogram of the carbonmonoxy chimeric globins in the presence and absence of the native subunits, and the centroid elution volumes of the samples as a function of protein concentration (16, 17) are shown in Fig. 5B. Under the condition employed here, the mixture of native alpha - and beta -subunits forms a tetramer, whereas the isolated alpha -subunit remains in a monomer (17). The wild-type beta -subunit is in the equilibrium between monomers and tetramers (17). The position of the elution peak for the beta alpha (PM3)-subunit is also between those of a tetramer and a monomer, but the peak position at 22.5 ml was significantly deviated from that of the wild-type beta -subunit at 22.0 ml. Since this elution peak was independent of the sample concentration from 5 to 80 µM (Fig. 5B), the beta alpha (PM3)-subunit forms a stable homodimer, not an equilibrium state between tetramers and dimers or monomers.


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Fig. 5.   A, chromatography of carbonmonoxy form of the beta alpha (PM3)-subunit on a Sephacryl S-200 HR column. Experimental conditions were as follows: 50 mM Tris, 0.1 M NaCl, pH 7.4, at 277 K. Sample concentration was 20 µM on the heme basis. B, centroid elution volumes of hemoglobin, the isolated beta alpha (PM3)-subunit, and the mixture of the beta - and beta alpha (PM3)-subunits as a function of protein concentration. Symbols correspond to hemoglobin (black-triangle), beta alpha (PM3)-subunit (open circle ), and the mixture of the beta - and beta alpha (PM3)-subunits (square ). Experimental conditions were as follows: 50 mM Tris, 0.1 M NaCl, pH 7.4, at 277 K.

In the chromatogram for the mixture of the beta alpha (PM3)- and alpha -subunits, two peaks were observed, each of which coincides with the peak for the isolated beta alpha (PM3)- and alpha -subunits, indicating no association of the beta alpha (PM3)-subunit with the alpha -subunit. On the other hand, the peak for the mixture of the beta alpha (PM3)- and beta -subunits showed a single broad peak, and the elution pattern for the mixture was not a simple addition of those of the corresponding isolated subunits. These elution patterns imply that the beta alpha (PM3)-subunit preferentially binds to the beta -subunit, not to the alpha -subunit, and the association property of the beta alpha (PM3)-subunit corresponds to that of the alpha -subunit as the case for the beta alpha (M4)-subunit (4). It should be noted here that the elution peak for the mixture was detected at the middle of those for tetrameric native Hb A and dimeric beta alpha (PM3)-subunits, suggesting that the complex of the beta alpha (PM3)- and beta -subunits is in the equilibrium between a heterodimer [beta alpha (PM3)]beta and a heterotetramer [beta alpha (PM3)2]beta 2. As shown in Fig. 5B, the elution peak for the mixture of the beta alpha (PM3)- and beta -subunits depends on the protein concentration, and the fitting curve of the mixture shifts to the right side, compared with that of native hemoglobin tetramer, indicating that the dissociation into dimers was enhanced in the complex of the beta alpha (PM3)- and beta -subunits. The tetramer-dimer dissociation constants, KD, were estimated as 1.4 (16) and 11 µM for Hb A and the complex of the beta alpha (PM3)- and beta -subunits, respectively.

Subunit Interface Structures-- To gain further insights into the subunit interface structure for the pseudo-module substituted globins and its complex of the beta alpha (PM3)- and beta -subunits, we have measured the 1H NMR spectra in the hydrogen-bonded proton region for the carbonmonoxy and deoxy forms (Fig. 6, A and B). Although no exchangeable proton signals were observed in the downfield region from 10 to 15 ppm for the isolated carbonmonoxy beta -subunits, a broad proton signal at 12.4 ppm was detected for the beta alpha (PM3)-subunit. This peak disappeared in 100% D2O (data not shown). Such an exchangeable proton signal in the downfield region was also encountered for carbonmonoxy Hb A, which has been assigned to the hydrogen bonds in the subunit interface (26-28). Similarly, the resonance at 12.4 ppm can be assignable to the hydrogen bond at the subunit interfaces of the homodimer, [beta alpha (PM3)]2. In the presence of the native beta -subunit, another exchangeable proton resonance appeared at 10.4 ppm (Fig. 6A), which would also originate from a hydrogen-bonded proton in the subunit interface of the complex of the beta alpha (PM3)- and beta -subunits.


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Fig. 6.   A, NMR spectra in the hydrogen-bonded proton region for carbon monoxygenated subunits. Proton resonance peaks for Hb at 10.4, 11.9, and 12.8 ppm have been assigned to the hydrogen-bonded protons between Asp-94alpha 1 and Asn-102beta 2 (27), His-103alpha 1 and Asn-108beta 1 (28), and Asp-126alpha 1 and Tyr-35beta 1, respectively (28). An asterisk represents a novel resonance peak which appears upon the mixture of the beta - and beta alpha (PM3)-subunits. Experimental conditions were as follows: 50 mM sodium phosphate, 0.1 M NaCl, pH 7.4, at 290 K. Sample concentration was 600 µM on the heme basis. B, NMR spectra in the hydrogen-bonded proton region for deoxygenated subunits. Proton resonance peaks for Hb at 11.0, 12.2, 12.9, and 13.9 ppm have been assigned to the hydrogen-bonded protons between Asp-94alpha 1 and Trp-37beta 2 (27, 30), His-103alpha and Asn-108beta (28), Asp-126alpha and Tyr-35beta (28), and Tyr-42alpha 1 and Asn-99beta 2, respectively (27). An asterisk represents a novel resonance peak which appears upon the mixture of the beta - and beta alpha (PM3)-subunits. Experimental conditions were as in A.

Structural alterations in the subunit interface by the pseudo-module substitution are also evident in the NMR spectra of the deoxygenated state of the chimeric globin. By the dissociation of the ligands from Hb A, the rearrangements in the alpha 1-beta 2 subunit interface are induced (29), which is reflected in the NMR spectra in the downfield region. For deoxy-Hb A, the characteristic T-state marker resonances were observed at 13.9 and 11.0 ppm (Fig. 6B) (27, 30), whereas in the beta alpha (PM3)-subunit, these marker signals were not detected and a new peak appeared at 11.3 ppm by deoxygenation. Although the appearance of the resonance at 11.3 ppm by deoxygenation suggests the rearrangements in the hydrogen bonds at the subunit interface in the pseudo-module substituted globin, its spectral pattern is quite different from that of hemoglobin. In addition to the resonance peaks at 11.4 and 12.4 ppm, the complex of the beta alpha (PM3)- and beta -subunits in the deoxy state exhibited an exchangeable proton signal at 10.7 ppm (Fig. 6B), which was not observed for the isolated deoxygenated subunits. The spectral features for the complex are still quite different between the carbonmonoxy and deoxy states, but the resonance positions for the complex are not identical with those for hemoglobin. This implies that the quaternary structural changes accompanied by deoxygenation for the complex would not fully correspond to those for native Hb A.

Heme Environmental Structures-- The structural perturbation was also manifested in the heme environmental structure, as revealed by 1H NMR spectra of the carbonmonoxy and deoxy form of the chimeric globins (Figs. 7 and 8). In the carbonmonoxy isolated subunits, a peak from the gamma 1-methyl proton of Val(E11) appeared at -2.0 and -2.2 ppm for the alpha - and beta -subunit, respectively, which has served as a marker for the tertiary structure in the heme vicinity (31, 32). The corresponding signal for the beta alpha (PM3)-subunit was detected at the same position as that of the native beta -subunit, implying that the heme environmental structure near Val(E11) residue was not so perturbed by the pseudo-module substitution (33). However, the resonance peak for the beta alpha (PM3)-subunit is significantly broadened and asymmetric,3 suggesting conformational changes in the heme vicinity upon the pseudo-module substitution. Such conformational changes in the heme distal site are supported by the spectral changes in the spectrum between -0.5 and -1.4 ppm.


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Fig. 7.   Proton NMR spectra (500 MHz) for carbon monoxygenated subunits. Experimental conditions were as follows: 50 mM sodium phosphate, 0.1 M NaCl, pH 7.4, at 290 K. Sample concentration was 600 µM on the heme basis. The resonance peak around -2.0 ppm has been assigned to gamma 1-methyl protons of Val(E11). The signals for the alpha -subunit at -0.9, -0.7, and -0.6 ppm are from the delta 2-methyl of Leu(B10), delta 1-methyl of Leu(FG3), and delta 1-methyl protons of Leu(B10) (53). The signals for the beta -subunit at -1.2, -1.0, and -0.8 ppm are from the delta 1- and delta 2-methyls of Leu(H19) and delta 2-methyl of Leu(B10) (54).


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Fig. 8.   Proton NMR spectra (500 MHz) for deoxygenated subunits. Experimental conditions were as follows: 50 mM sodium phosphate, 0.1 M NaCl, pH 7.4, at 290 K. Sample concentration was 600 µM on the heme basis. I, hyperfine-shifted proton resonances of proximal His Ndelta -H. II, hyperfine-shifted proton resonances of heme methyl groups.

Here, noteworthy is that the spectral pattern for the complex of the beta alpha (PM3)- and beta -subunits is a simple addition of that for the corresponding isolated subunits, regardless of rather stable complex formation of these two subunits. The association of the beta alpha (PM3)- and beta -subunits would not affect the heme distal structure of the counterpart subunit, which is in sharp contrast to the complex formation of the native alpha - and beta -subunits.

Fig. 8 shows the 1H NMR spectra for the deoxy state of the chimeric and native globins. In the NMR spectra of the isolated native alpha - and wild-type beta -subunits, a hyperfine-shifted exchangeable proton resonance was observed at 78 and 88 ppm in the far downfield hyperfine-shifted region (Fig. 8I), respectively, which had been assigned to the Ndelta -H proton of the proximal histidine (F8) (34, 35). A couple of hyperfine-shifted resonances in the region between 12 and 28 ppm (Fig. 8II) originated from the protons of heme peripheral groups including heme methyl groups (36). By the substitution of the pseudo-module PM3 in the beta -subunit, the resonance position of the proximal Ndelta -H proton was obviously shifted to the position for the alpha -subunit. The spectral pattern for the resonances of the heme peripheral group is also highly perturbed by the substitution of the pseudo-module PM3. These prominent spectral changes suggest that the substitution of the pseudo-module PM3 alters the heme proximal structure of the beta -subunit and converts the coordination structure of the beta -subunit into that of the alpha -subunit. On the other hand, the substitution of the module M4 induced a much smaller upfield shift for the resonance of the proximal histidine Ndelta -H,4 although the spectral pattern for the heme peripheral groups are rather close to that of the pseudo-module substituted subunit.

In addition to the isolated native and chimeric subunits, we have measured the NMR spectra for the complex of the beta alpha (PM3)- and beta -subunits in the deoxy state. As shown in Fig. 8, native hemoglobin subunits show a large upfield shift of the proximal histidyl Ndelta -H proton by formation of the alpha 2beta 2 tetramer, indicating that significant structural changes are induced by the tetramer formation of the native subunits. The resonance positions of the heme peripheral groups in the NMR spectra of tetrameric hemoglobin are quite different from those of the two isolated subunits, supporting the structural changes by formation of the functional tetrameric hemoglobin (36). However, such remarkable spectral changes were not accompanied by the association of the beta alpha (PM3)- and beta -subunits. Only a slight downfield shift of the proximal histidyl Ndelta -H proton resonance was detected for the beta alpha (PM3) subunit. Such a small shift for the resonance positions of the proximal histidines implies that the subunit association of the beta alpha (PM3)- and beta -subunits does not induce the large structural rearrangements around their heme vicinity.

Oxygen Binding Property of the beta alpha (PM3)-Subunit-- To evaluate the effects of the pseudo-module substitution on the oxygen affinity and cooperativity for the oxygen binding of globins, oxygen equilibrium curves for the chimeric and native globins were examined. Fig. 9A delineates the oxygenation curves, expressed by saturation versus log P plots, and the Hill plots for the isolated subunits and their mixtures are illustrated in Fig. 9B. The P50 value of the beta alpha (PM3)-subunit was estimated to be 1.37 mm Hg, corresponding to the lower affinity than that for the isolated wild type beta -subunit (Table II).


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Fig. 9.   A, Y versus log P plots of oxygen equilibrium curves. B Hill plots. Symbols correspond to hemoglobin (black-triangle), beta -subunit (black-diamond ), alpha -subunit (black-down-triangle ), beta alpha (PM3)-subunit (open circle ), and the mixture of the beta - and beta alpha (PM3)-subunits (square ). Experimental conditions were as follows: 50 mM Tris, 0.1 M NaCl, pH 8.4, at 25 °C. Sample concentration was 60 µM on the heme basis.

                              
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Table II
Oxygen equilibrium parameters for tetrameric Hb, isolated alpha -, beta -, beta alpha (PM3)-subunits and mixture of the beta - and beta alpha (PM3)-subunits
Experimental conditions are listed in Fig. 9.

For the complex of the beta - and beta alpha (PM3)-subunits, the P50 value is 0.97 mm Hg, which is middle of those of the isolated subunits. Since the oxygen curve for the complex of the beta - and beta alpha (PM3)-subunits was not biphasic, the difference of the oxygen affinities of beta alpha (PM3)- and beta -subunits in the complex are indistinguishable (37). The nmax value for the complex is unity, implying that it does not show any cooperative oxygen binding as the case for the isolated chimeric and native alpha - and beta -subunits (Table II).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Effects of Pseudo-module Substitution on Structure and Function of Globins-- As illustrated in the CD spectra, the pseudo-module substituted subunit, beta alpha (PM3)-subunit, can be folded to a stable globin structure as observed for native globins. These minimal effects of the pseudo-module substitution on the protein structure were also supported by the urea denaturation curve for the chimeric subunits (Table I). The free energy change (Delta GH2O) for the denaturation of the beta alpha (PM3)-subunit was minimally affected by the substitution of the pseudo-module PM3. These findings clearly indicate that the replacement of the pseudo-module PM3 in hemoglobin beta -subunit with that of the alpha -subunit did not perturb the globin structure of the native protein.

However, the effects of the pseudo-module substitution on the heme environmental structure were prominent. The characteristic features of the heme environment for the beta alpha (PM3)-subunit is that the resonance of the proximal Ndelta -H proton for the deoxy beta alpha (PM3)-subunit was observed at the position for the isolated alpha -subunit. For the module substituted beta alpha (M4)-subunit, the coordination structure of the proximal His is still a beta -like structure as shown in Fig. 8I (4). Since the pseudo-module PM3 shares the amino acid residues from FG4 to H6 with the module M4, this different coordination structure between the two chimeric subunits would originate from the region from F1 to FG3 which is included in PM3 and not in M4. In fact, the amino acid sequence from F1 to FG3 constructs most parts of the heme proximal structure and includes the proximal histidine, which strongly suggests that this segment regulates the heme coordination structure.

Contrary to the coordination structure, the structure of the heme distal site of the chimeric beta alpha (PM3)-subunits is rather close to the beta -subunit. The NMR resonance from methyl group of Val(E11) in the carbonmonoxy form appeared at the position for the native beta -subunit as found for the module substituted beta alpha (M4)-subunit, although minor conformational changes around Val(E11) were detected in the NMR spectrum. Based on the amino acid sequences for the beta -subunit, the distal cavity of globins is formed by the B, C, D, and E helices which are not involved in the pseudo-module PM3 or module M4. Thus, we can conclude that the substantial structural changes induced by the pseudo-module PM3 substitution are rather localized in the proximal site.

The subunit association is another property that depends on the pseudo-module PM3. As Fig. 5A shows, the beta alpha (PM3)-subunit can associate with the beta -subunit but not with the alpha -subunit, which also resembles that of the native alpha -subunit. Such a conversion of the association property was also experienced for the module substituted subunit, beta alpha (M4)-subunit (4). As depicted in Fig. 2, the common region in the pseudo-module PM3 and the module M4 is from FG4 to H6 in the amino acid sequence, and these amino acid residues would play key roles in the subunit association of hemoglobin. Inspection of the x-ray crystal structure also supports the functional role of this region, since most parts of the alpha 1beta 1 subunit interface dominating subunit assembly in hemoglobin consist of these residues as revealed in Fig. 1A.

In contrast to the effects of the PM3 substitution on the coordination structure of the proximal histidine and on the association property of the subunits, the oxygen binding properties for beta alpha (PM3)-subunit are not so simple to interpret. As found in Fig. 9, the isolated beta alpha (PM3)-subunit shows a lower oxygen affinity than the isolated beta -subunit. The NMR spectra of its deoxygenated state (Fig. 8) could also support the lower oxygen affinity of the beta alpha (PM3)-subunit; the upfield bias of the proximal His Ndelta -H resonance corresponds to the increased strain of the iron-histidyl bond, which would be responsible for the decreased oxygen affinity (35). However, the oxygen affinity of the beta alpha (PM3)-subunit is still lower than that of the alpha -subunit in which the resonance position of the proximal histidine Ndelta -H is the same as that in the beta alpha (PM3)-subunit. As discussed in the extensive NMR studies on natural mutant hemoglobins by Ho and co-workers (38, 39), the correlation between the position of the hyperfine-shifted NMR signal from the proximal histidine Ndelta -H and the strain of the iron-proximal histidine bond (38-41) is not simple, and the resonance position is also dependent on the subunit interface structure, salt bridges within the subunits, and conformation of the heme pockets as well as on the strain imposed on the iron-proximal histidyl bond (38, 39). In the beta alpha (PM3)-subunit, therefore, the amino acid replacements by the pseudo-module substitution would affect those structural factors, which might lead to the lower oxygen affinity.

On the other hand, the oxygen affinity of the complex of the beta alpha (PM3)- and beta -subunits was much higher than that of Hb A, and allosteric cooperativity was not detected for the complex. As revealed by the NMR spectrum for the hydrogen-bonded region, the hydrogen bonds in the subunit interface of the complex are quite different from that of native Hb A, and the spectral feature characteristic of the T-state was missing for the complex. The NMR spectrum in the hyperfine-shifted region at 12-28 ppm is also suggestive of absence of the specific interactions between these two subunits (36). On the basis of x-ray structural studies, two sliding contact regions, C2-CD1 and FG3-G7, are essential for the allostericity in hemoglobin A (16, 42-48). However, the C2-CD1 region of the beta alpha (PM3)-subunit is derived from the beta -subunit (Fig. 2). The incomplete adjustment of the alpha 1beta 2 subunit interface would reduce the subunit interactions in the complex of the beta - and beta alpha (PM3)-subunits, resulting in non-cooperative oxygen binding.

Structural and Functional Significance of the Pseudo-module PM3 in Globins-- In this study, we have shown that the substitution of the structural segment other than the module can also produce a stable "chimeric" globin, which leads us to re-examine the structural and functional significance of the modular structure in globins. Comparison of the chimeric property of the beta alpha (PM3)-subunits with that of the native and the module substituted subunit can reinforce the structural and functional significance of the pseudo-module PM3. One of the possible reasons for formation of the stable and functional chimeric globin by the pseudo-module substitution would be high structural homology between the alpha - and beta -subunits. Although the amino acid sequence homology is not so high between the two subunits (~40%), their globin structures are quite similar except for the deletion of the D helix in the alpha -subunit. However, our previous study on the module substituted alpha beta (M4)-subunit, in which the module M4 of the beta -subunit was implanted into the alpha -subunit, showed that the globin structure of the alpha beta (M4)-subunit is highly perturbed and destabilized probably due to loss of the stable intramolecular helix packing (5), concluding that the structural similarity is not enough to produce a stable chimeric protein. It is thus likely that the PM3 substitution would not accompany substantial failures in the helix packings. According to the previous study by Jennings and Wright (49), the packing between the A and H helices is formed in the first step of the folding process in apo-myoglobin, and these helices are crucial for protein folding of globins. Since the pseudo-module PM3 contains only a part of H helix as shown in Fig. 1B and most of the A and H helices are derived from the parent beta -subunit, the PM3 substitution would not substantially perturb the helix packing.

The stable structure and functional conversion for the beta alpha (PM3)-subunit suggest that the pseudo-module PM3 might have some structural significance in the globin structure like the modules, which leads us to re-examine the boundary of the module in globins. It should be noted here that the modules in globins, which have been defined on the basis of the diagonal plots of the inter-Calpha atom distances for the main chain (1), are much larger in size than those in other proteins (50). That is, the modules in globins consist of 30-40 amino acid residues (1), whereas the numbers of the amino acid residues for the modules in most of other proteins are 10-25 (7, 50). Such a large modular structure in globins may allow us to infer that the current modules in globin are not minimal units for the structure and function of globin and can be further divided into some "sub"-modules. In fact, this speculation is strongly supported by the recent revised module assignment with a centripetal profile (7) which indicates that each of the modules in globins can be decomposed into the two "small" modules, and consequently, globins has eight small modules.5 Interestingly, the new identified module boundaries in the modules M3 and M4 almost coincide with the boundaries of the pseudo-module PM3. In other words, the substitution of the pseudo-module PM3 might be a type of module substitution. Although it is still premature to conclude that the pseudo-module PM3 is a combination of the two small modules, it can be safely said that the pseudo-module PM3 of globins has some structural and functional significance.

In summary, the substitution of the pseudo-module PM3 which is originally supposed to have no structural and functional significance can retain the structural stability comparable to the native globins. Moreover, the structural and functional properties of the PM3 substituted subunit were chimeric between the parent subunits. The coordination structure of the proximal histidine and the association property for the beta alpha (PM3)-subunit was rather close to that for the alpha -subunit, whereas the structure of the distal site was still beta -subunit like. These findings strongly suggest that the pseudo-module has some structural and functional significance. Together with the structural and functional properties of the native and module substituted subunits, we can propose that the former half of the pseudo-module PM3 and the latter one would correspond to the sub-modules regulating the heme proximal structure and subunit assembly, respectively. To strengthen our argument on structural and functional significance of the sub-module in globins, the preparation and characterization of the novel chimeric globin subunits in which one of these sub-modules is replaced by that of the partner subunit are now in progress.

    ACKNOWLEDGEMENT

We thank Dr. Yoshinao Wada for fast atom bombardment-mass spectrometry. We are indebted to the reviewers' suggestions.

    FOOTNOTES

* This work was supported by Grants-in-aid for Scientific Research (A) and by Grants 08249102 (to I. M.) and 07280101 (to K. I.) by Specially Promoted Research from the Ministry of Education, Science, Sports and Culture.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 and reprint requests should be addressed. Tel.: 81-75-753-5921; Fax: 81-75-751-7611; E-mail: morisima{at}mds.moleng.kyoto-u.ac.jp.

1 Wild-type subunit represents the protein expressed in Escherichia coli, and a methionine residue is located at the N-terminal. Native subunit corresponds to the protein purified from human red blood cells. In the wild-type beta -subunit, we confirmed that the mutation of Val to Met does not seriously perturb the globular structure and heme environmental structure of the beta -subunit. The CD and NMR spectra for the wild-type beta -subunit were virtually the same as those of the native beta -subunit.

2 We have also tried to carry out the urea denaturation experiment in the absence of the heme group. However, the apo-chimeric beta alpha (PM3)- and beta alpha (M4)-subunits are extremely unstable to aggregate under the following conditions: pH 7.4, urea concentration of below 2 M, sample concentration of 5 µM. Therefore, we cannot exactly measure molar ellipticity at 222 nm and estimate equilibrium stability for the apo-globins.

3 We confirmed the homogeneity of the purified beta alpha (PM3)-subunit by native and SDS-polyacrylamide gel electrophoresis. Thus, the spectral change would not be due to inhomogeneous preparation.

4 The line width for the resonance from the beta alpha (M4)-subunit was broad compared with the one for other globins (51). Such a line broadening was also observed for the native isolated subunit in high pH region due to the enhanced exchange rate of the Ndelta -H of the proximal histidine (52). Although the reasons for the line broadening in the chimeric globin are not yet clear, some structural changes around the proximal histidine induced by the module substitution might affect the exchange rate of the Ndelta -H of the proximal His.

5 M. Go, submitted for publication.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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