Formation and carbon monoxide‐dependent dissociation of Allochromatium vinosum cytochrome c′ oligomers using domain‐swapped dimers

Abstract

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c′ from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four‐helix bundle structure containing a covalently bound five‐coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer–monomer transition according to the absence and presence of CO. Herein, domain‐swapped dimeric AVCP was constructed and utilized to form a tetramer and high‐order oligomers. The X‐ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain‐swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain‐swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit–subunit interactions at the interfaces of the domain‐swapped dimer and monomer subunits in the tetramer were also similar to the subunit–subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain‐swapped dimer and two monomers upon CO binding. Without monomers, the domain‐swapped dimers formed tetramers, hexamers, and higher‐order oligomers in the absence of CO, whereas the oligomers dissociated to domain‐swapped dimers in the presence of CO, demonstrating that the domain‐swapped dimer maintains the CO‐induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer–monomer transition protein.

Short abstract

PDB Code(s): 5GYR

Abbreviations

AA

Aquifex aeolicus

AVCP

Allochromatium vinosum cytochrome c′

CD

circular dichroism

CO

carbon monoxide

CP

cytochrome c′

cyt

cytochrome

cyt cb₅₆₂

c‐type mutant of cytochrome b ₅₆₂

FPLC

fast protein liquid chromatography

HT

hydrogenobacter thermophilus

Mb

myoglobin

PA

Pseudomonas aeruginosa

rmsd

root‐mean‐square deviation

SEC

size exclusion chromatography

Introduction

Protein oligomers for biomaterials have been constructed by various methods,1, 2, 3 such as chemical modification,4 disulfide bonding,5, 6 computational design,7 metal coordination,8, 9, 10, 11 host–guest interaction,12, 13 hydrophobic interaction,14 electrostatic interaction,15 and protein fusion.16, 17, 18, 19 3D domain swapping (i.e., simply, domain swapping) is an oligomerization phenomenon, which has been suggested as a general property of proteins.20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 In domain swapping, oligomers are formed by exchanging the same structural regions between molecules. We have previously shown that many hemoproteins form oligomers by domain swapping. Class I monomeric globular c‐type cytochromes [e.g., horse cytochrome (cyt) c, mesophile Pseudomonas aeruginosa (PA) cyt c ₅₅₁, thermophile Hydrogenobacter thermophilus (HT) cyt c ₅₅₂, and hyperthermophile Aquifex aeolicus (AA) cyt c ₅₅₅] form domain‐swapped oligomers by exchanging the N‐ or C‐terminal region between molecules.31, 32, 33, 34 The c‐type mutant of Escherichia coli cyt b ₅₆₂ (cyt cb ₅₆₂) exhibiting a four helix bundle structure, as well as horse myoglobin (Mb) possessing a noncovalently bound heme, form domain‐swapped dimers by exchanging half of the structure between molecules.35, 36 Domain swapping has also been used to construct various protein oligomers. We have constructed an artificial Mb heterodimer, which possessed two different heme coordination sites: His/H₂O and bis‐His coordinated hemes.37 A protein nanoring has been constructed by domain swapping a hinge loop‐elongated HT cyt c ₅₅₂ mutant,38 whereas a protein nanocage has been constructed with three domain‐swapped cyt cb ₅₆₂ dimers.35

Cytochrome c′ (CP) is a class II c‐type cyt found in various Gram‐negative bacteria.39, 40 CP typically forms a four‐helix bundle structure with a covalently bound five‐coordinate heme as a gas‐binding site.41, 42, 43, 44 A conserved His is coordinated to the heme iron, whereas various noncoordinating residues occupy the sixth coordination site of the heme.44 CP is a monomeric protein or forms a homodimer by the surface interaction between subunits in its native form.43, 44 Allochromatium vinosum CP (AVCP) forms a homo‐dimer in its oxidized form as well as in its reduced form in the absence of CO, whereas it exhibits a reversible transition to monomers upon CO binding to its reduced form.45, 46, 47 The sixth coordination site, possibly the CO binding site, is occupied with the side chain of Tyr16 which is tightly constrained by neighboring residues, and thus Tyr16 has been suggested to be a trigger for the dimer‐to‐monomer transition upon CO binding.46 There are some other heme proteins exhibiting ligand‐binding dissociation/association characters. Hemoglobins from organisms in five phyla (molluscs, echinoderms, annelids, phoronids, and chordates) undergo oxidation‐induced dissociation to subunits and ferric iron ligand‐binding reassociation,48 whereas monomeric, ligated lamprey hemoglobin associates to dimers and tetramers upon deoxygenation.49

Although various protein oligomers have been constructed, it is difficult to control formation of protein oligomers.50, 51 Proteins exhibit structural changes upon changes in conditions, such as pH, salt concentrations, atmosphere component(s), and light irradiation, which may be used for controlling protein oligomer formation. A biomaterial that could be degraded by a trigger, such as an addition of a ligand, may be potentially useful. In this study, we constructed an AVCP tetramer and its high‐order oligomers using domain swapping, and investigated the quaternary structural changes of the oligomers upon CO binding/dissociation.

Results

Oligomerization of AVCP

AVCP native dimer in 50 mM potassium phosphate buffer, pH 7.0, was treated with ethanol, lyophilized, and dissolved in the same buffer. By analyzing the ethanol‐treated AVCP with size exclusion chromatography (SEC), several peaks corresponding to the tetramer and high‐order oligomers (≥ hexamer, ∼80 kDa) were observed in the chromatogram in addition to the dimer peak (Fig. 1). These results indicate that oligomeric AVCP was formed by the ethanol treatment, similar to the properties in other c‐type cyts.31, 32, 33, 34, 38 The purified tetrameric AVCP converted to native dimers at low salt concentrations, whereas the dissociation was inhibited in the presence of 160 mM sodium sulfate (Supporting Information Fig. S1). Thus, subsequent measurements for the AVCP tetramer and oligomers were performed in the presence of 200 mM sodium sulfate.

Figure 1.

Size exclusion chromatograms of oxidized AVCP. A: The chromatogram of the native dimer. B: The chromatogram of the oligomers obtained by ethanol treatment. C: The calibration curve of the protein molecular weight for the column used. The calibration curve (black line) was obtained by least‐square fitting the partition coefficients (K _av) plots of standard proteins (black square); cyt c (12 kDa), Mb (17 kDa), ovalbumin (44 kDa), bovine serum albumin (BSA; 66 kDa), and disulfide dimer of BSA (132 kDa). The plots of the AVCP oligomers (red square) are depicted with the estimated molecular weights (in parentheses) obtained with the calibration curve. Measurement conditions: column, HiLoad 26/600 Superdex 75 pg; flow rate, 2.5 mL/min; monitoring wavelength, 280 nm; solvent, 50 mM potassium phosphate buffer, pH 7.0; temperature, 4°C.

Absorption and circular dichroism (CD) spectra of tetrameric AVCP

Absorption and far‐UV CD spectra of the purified tetrameric AVCP were measured to investigate its heme environment and secondary structures, respectively. The Soret band wavelengths of oxidized (399 nm) and reduced (426 nm) tetrameric AVCP were same to those of the corresponding forms of the native dimer (Fig. 2). The Soret band wavelength of reduced tetrameric AVCP red shifted from 426 to 418 nm with an increase in its intensity by exchanging the atmosphere from N₂ to CO at pH 7.0, similar to the case of the native dimer (Fig. 2). These results indicate that tetrameric AVCP possesses a heme environment and CO binding ability similar to those of the native dimer.

Figure 2.

Absorption spectra of oxidized and reduced AVCP: (A) native dimer and (B) tetramer. Oxidized state was measured under a N₂ atmosphere (red), and the reduced state was measured under N₂ (blue) and CO atmospheres (green). Measurement conditions: sample concentration (heme unit), 3 μM; solvent, 50 mM potassium phosphate buffer, pH, 7.0, containing 200 mM sodium sulfate; temperature, 25°C.

The CD spectrum of oxidized tetrameric AVCP at pH 7.0 exhibited negative peaks at 208 and 222 nm similar to that of the oxidized native dimer under the same conditions (Supporting Information Fig. S2), indicating that the secondary structures with α‐helices of tetrameric AVCP were similar to those of the native dimer.

X‐ray crystal structure of tetrameric AVCP

The X‐ray crystal structure of tetrameric AVCP at 1.6 Å resolution (PDB code: 5GYR) was obtained to investigate its detailed structure. There were two independent tetramers in the asymmetric unit of the tetrameric AVCP crystal (Supporting Information Fig. S3). Both tetramers consisted of one domain‐swapped dimer subunit and two monomer subunits (Fig. 3). In the domain‐swapped dimer, the region containing helices αA and αB were exchanged between protomers, and a helix‐to‐loop transition was observed at the hinge region (Glu79–Gly85; Fig. 4). A tetramer with a domain‐swapped dimer and two monomer subunits, similar to tetrameric AVCP, has been reported for the heterotetrameric EsxRS complex.52 In the EsxRS complex, two EsxS proteins formed a domain‐swapped dimer subunit possessing two long antiparallel α‐helices by loop‐to‐helix transitions at the short loops between α‐helices of the monomers. Two EsxR proteins interacted with the antiparallel α‐helices of EsxS, where each monomeric EsxR subunit interacted at each end of the domain‐swapped EsxS dimer.

Figure 3.

X‐ray crystal structure of tetrameric AVCP (PDB code: 5GYR)_. Tetrameric AVCP comprised a domain‐swapped dimer subunit (red and blue) and two monomer subunits (green). The red and blue regions in the domain‐swapped dimer correspond to different protomers. The structure of the AVCP native dimer (orange, PDB code: 1BBH) is also shown for comparison. The hemes are shown as stick models. Side chain atoms of heme‐binding Cys121 and Cys124 and heme iron‐coordinating His125 are also shown as stick models. The N‐ and C‐termini are labeled as N and C, respectively. The α‐helices are labeled from the N‐terminus as αA, αB, αC, and αD.

Figure 4.

Changes in the hinge region structure of the domain‐swapped dimer subunit of tetrameric AVCP (PDB code: 5GYR) from that of the monomer subunit of the AVCP native dimer (PDB code: 1BBH). The red and blue regions in the domain‐swapped dimer correspond to different protomers. Subunits of the native dimer are depicted in light orange, and superimposed to the corresponding regions of the domain‐swapped dimer. The hemes, side chain atoms of heme‐binding Cys121 and Cys124, and heme iron‐coordinating His125 are shown as stick models. The first and last residues of the hinge region, Glu79 and Gly85, are indicated with arrows. The N‐ and C‐termini are labeled as N and C, respectively.

The overall protein structure of the domain‐swapped dimer subunit of tetrameric AVCP corresponded well to that of the monomer subunit of the native dimer (Supporting Information Fig. S4). The root‐mean‐square deviation (rmsd) values for the Cα atoms of the region containing helices αA and αB (Ala1 − Phe78) and those of the rest of the protein excluding the hinge region (Lys86 − Lys131) between the protomer of the domain‐swapped dimer and the subunit of the native dimer were calculated as 0.37 − 0.96 Å (Supporting Information Table SI). These results indicate that the structures of the region containing helices αA and αB, as well as those of the region containing helices C and D, were similar between the domain‐swapped dimer and the subunit of the native dimer. The rmsd values for the Cα atoms between the monomer subunits of the tetramer and native dimer were calculated as 0.43–0.87 Å (Supporting Information Table SII), indicating that the monomer subunit structures in the tetramer and native dimer were also similar.

The hydrogen‐bonding network and hydrophobic packing in the subunit of the native dimer were mostly preserved in the domain‐swapped dimer subunit of the tetramer except for those in the hinge region (Fig. 4 and Supporting Information Figs. S5 and S6). The hydrogen bond (<3.2 Å) at Asn51(Oδ1)/Asn96(Nδ2), hydrogen‐bonding network among Arg12(Nη1), Gln13(Nɛ2), Leu57(O), Lys70(O), Arg72(N), and heme 13‐propionate, and the hydrophobic interactions between the α‐helices in the subunit of the native dimer were maintained in the domain‐swapped dimer (Supporting information Figs. S5 and S6). However, rearrangement of the hydrogen bonds was observed around the hinge region by domain swapping; the intramolecular 13 hydrogen bonds in the native dimer were broken in the domain‐swapped dimer, and 5 new hydrogen bonds within the protomer and 4 new hydrogen bonds between protomers were formed in the domain‐swapped dimer (Supporting Information Fig. S7 and Table SIII).

The heme environments of the monomer and domain‐swapped dimer subunits of the tetramer were similar to those of the native dimer (Fig. 5). However, the heme iron of AVCP was presumably reduced to the ferrous state by the accumulated X‐ray dose. The Fe − His125 distances in the monomer and domain‐swapped dimer subunits of the tetramer were 2.2–2.3 Å (PDB code: 5GYR), similar to the corresponding distances in the subunits of the native dimer (2.0 Å; PDB code: 1BBH). Additionally, Tyr16 of helix αA occupied the sixth coordination position of the heme iron in the monomer and domain‐swapped dimer subunits of the tetramer as in the native dimer, although Tyr16 originated from the other protomer to which the heme belonged in the domain‐swapped dimer (Fig. 5).

Figure 5.

Superimposed figure of the active site structures of the AVCP native dimer (PDB code: 1BBH) and tetramer (PDB code: 5GYR). The monomer and domain‐swapped dimer subunits of the tetramer are depicted in green and red/blue, respectively. The red and blue regions in the domain‐swapped dimer correspond to different protomers. The native dimer is depicted in orange. The hemes and the side chain atoms of Tyr16, heme‐binding Cys121 and Cys124, and heme iron‐coordinating His125 are shown as stick models. The sulfur atoms of the side chains of Cys121 and Cys124 are depicted in yellow, the nitrogen atoms of the side chain of His125 are depicted in blue, and the oxygen atom of the side chain of Tyr16 is depicted in red.

The subunit–subunit interaction between the regions containing helices αA and αB in the native dimer were conserved in the tetramer except for an additional inter‐subunit Thr11(Oγ1)/Glu17(Oɛ2) hydrogen bond (2.5–2.7 Å) at the interfaces between the monomer and domain‐swapped dimer subunits (Supporting Information Fig. S8). Intersubunit hydrogen bonds were formed between Lys25(Nζ) and Ser52(O) (2.5–2.7 Å) at the interfaces of the monomer and domain‐swapped dimer subunits in the tetramer, similar to that in the native dimer. The hydrophobic packings of Leu3, Phe18, Trp21, Val45, Ile49, Met54, and Leu57 at the subunit interface in the native dimer were also preserved in the interfaces between the monomer and domain‐swapped dimer subunits in the tetramer (Supporting Information Fig. S9).

Domain‐swapped AVCP oligomerization and effect of CO binding

Quaternary structural changes of domain‐swapped AVCP upon CO binding/dissociation were investigated by SEC analysis (Fig. 6). The oxidized native dimer exhibited a peak at elution volume 11.3 mL in the SEC chromatogram, whereas the peak was shifted to 12.5 mL corresponding to the monomer under CO saturated conditions in the presence of 5 mM sodium dithionite [Fig. 6(A)]. For the oxidized and reduced AVCP tetramer, peaks were detected at elution volume 9.8 mL in the chromatograms [Fig. 6(B) and Supporting Information, Fig. S10]. By an addition of CO to reduced tetrameric AVCP, two peaks were detected in the chromatogram at elution volumes 11.2 and 12.3 mL, corresponding to the dimer and monomer, respectively, in a molecular ratio of about 1:2 (heme absorption ratio of about 1:1) [Fig. 6(B)]. These results indicate that tetrameric AVCP dissociates to a dimer and two monomers by CO binding, where the dimer‐to‐monomer ratio corresponded well to that in the tetramer. Since the positions of Tyr16 and helix αA relative to the heme in the monomer and domain‐swapped dimer subunits are identical to those in the native dimer, the tetramer may dissociate to a domain‐swapped dimer and monomers in a similar way as the native dimer dissociate to monomers upon CO binding. However, the dimer‐to‐monomer ratio was <0.5—the absorption intensity of the dimer and monomer peaks should be the same when the dimer‐to‐monomer ratio is 0.5—after the reduction of the heme and CO addition, indicating that some domain‐swapped dimers have dissociated to monomers by the treatment.

Figure 6.

Size exclusion chromatograms of the AVCP native dimer, tetramer, and oligomers. The oligomers were constructed from the domain‐swapped dimers. Elution curves of the AVCP native dimer (A), purified tetramer (B) and oligomers comprising domain‐swapped dimers in the oxidized forms under air (solid line) and their reduced forms under CO atmospheres (dashed line) are depicted. (C) Elution curves of domain‐swapped dimeric AVCP under air (solid line) and reduction under a CO atmosphere (dashed line) are shown. Measurement conditions: column, Superdex 75 10/300 GL; flow rate, 0.5 mL/min; monitoring wavelength, 399 and 418 nm for oxidized and CO form, respectively; solvent, 50 mM potassium phosphate buffer, pH 7.0, containing 200 mM sodium sulfate; temperature, 4°C. The elution curves were normalized according to the total intensities.

The domain‐swapped dimer was purified by SEC using CO‐saturated buffer containing 5 mM sodium dithionite, and subsequently air‐oxidized allowing the CO to dissociate from the heme iron. In the SEC chromatogram of air‐oxidized domain‐swapped dimers, peaks corresponding to tetramers and higher‐order oligomers were observed at elution volumes 9.8 and 7.7 mL, respectively, in addition to the dimer peak at 11.2 mL [Fig. 6(C)]. The oligomer peaks of AVCP were detected periodically when using a column for higher molecular weight analysis, and thus the peaks may correspond to tetramers, hexamers, and higher‐order oligomers (Supporting Information Fig. S11). By reducing and retreating CO to the resulting oligomer mixture, the tetramer and high‐order oligomer peaks disappeared and the intensity of the dimer peak increased in the chromatogram [Fig. 6(C)]. These results indicate that the CO‐dependent quaternary structural changes of the domain‐swapped dimer are reversible, similar to the case of the AVCP native dimer. However, some dimers were detected at 11.2 mL in the chromatogram of the domain‐swapped dimer after reoxidation [Fig. 6(C), solid line] and a shoulder peak was observed in the chromatogram around 12.3 mL for the CO‐treated oligomers [Fig. 6(C), dashed line], indicating the presence of monomers. The domain‐swapped dimers did not produce unlimited oligomerization, presumably due to the binding of the existing monomers to the domain‐swapped dimers as termini of the oligomers.

Discussion

Many proteins with domain‐swapped structures have been reported, including class I cyts c and cyt cb ₅₆₂.20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 AVCP is the first example of the domain swapping oligomerization of a class II cyt c. Oligomers formed by domain swapping are frequently metastable and dissociate to monomers irreversibly.31, 32, 33, 34, 35, 36, 53, 54 In these proteins, the stability of domain‐swapped oligomers correlates well to that of its monomer, since most of the interactions in the monomer are reproduced in the domain‐swapped oligomer, although some of the interactions may convert from within a molecule to between protomers. The denaturation temperature of AVCP in its homodimeric form has been reported as 52°C,55 and thus the domain‐swapped dimer of the AVCP tetramer may dissociate to monomers relatively easily, producing native dimers (Supporting Information Fig S1). However, AVCP retained its oligomeric structure under the presence of 200 mM sodium sulfate. The domain‐swapped oligomer of RNase S has also been stabilized by an addition of salts.54 Nondomain‐swapped oligomeric proteins have been stabilized by salts through electrostatic and hydrophobic interactions,56, 57 and domain‐swapped oligomers may be stabilized by salts through similar interactions.

Domain swapping oligomerizations of four helix bundle proteins have also been reported for WA2058 and cyt cb ₅₆₂.35 The domain‐swapped dimer of WA20 exhibited a long four helix bundle structure with loop‐to‐helix transitions at the short loops between helices αA and αB and between helices αC and αD upon dimerization. However, AVCP and cyt cb ₅₆₂ possess long loops between helices αB and αC,46, 59 and a part of the long loop becomes the hinge loop in domain‐swapping.35 As a result, dimeric WA20 forms a rod structure, whereas domain‐swapped dimeric cyt cb ₅₆₂ and AVCP form dogleg structures, demonstrating that the length of the loop containing the hinge region may affect the domain‐swapped dimer structures in four helix bundle proteins.

In some hemoproteins, the properties may change by domain swapping oligomerization. Domain‐swapped dimeric horse cyt c exhibited a different active site structure compared to that of the monomer (i.e., heme ligating Met80 in the monomer was dissociated from the heme iron in the dimer).31 As a result, dimeric horse cyt c exhibited an enhanced peroxidase reactivity.60 Additionally, domain‐swapped dimeric AA cyt c ₅₅₅ exhibited binding abilities for exogenous ligands, such as CO and CN^‐, whereas its monomer did not under the same conditions, although the active site structures were similar between the domain‐swapped dimer and monomer.33 In the domain‐swapped dimer of these c‐type cyts, the hinge loop was in proximity to the active site. However, some hemoproteins do not change their properties by domain swapping. The domain‐swapped dimers of HT cyt c ₅₅₂, PA cyt c ₅₅₁, and cyt cb ₅₆₂ exhibited similar active site structures compared to their monomers, and the dimers exhibited similar redox potentials as their monomers.32, 34, 35 The active site structure of Mb did not change significantly by domain swapping, and the domain‐swapped dimer exhibited an oxygen binding property similar to the monomer.36 Tetrameric AVCP possessed the same active site structure for gas binding as the native dimer (Fig. 5), and the AVCP oligomers comprising the domain‐swapped dimers exhibited the CO binding ability as in the native dimer (Fig. 2). For these hemoproteins exhibiting similar properties before and after domain swapping, the hinge loop was located relatively far from the active site. Taken together, the property of a hemoprotein may change by domain swapping when the hinge loop is located close to the active site, whereas the property may not change by domain swapping when the hinge loop is far from it.

Control of protein oligomer formation has also been of interest. A variant of Doronpa, a photochromic fluorescence protein, has been reported to exhibit a reversible tetramer–monomer transition upon 500/400 nm light irradiation.51 The N‐terminal region‐deleted variant of Bacillus stearothermophilus dihydrolipoyl acetyltransferase formed a cage structure at pH 7.4, whereas it was in a monomeric state at pH 5.0.50 Oligomerization by successive domain swapping (Runaway domain swapping) for RNase A, serpin, and horse cyt c have been reported,26, 31, 61 but oligomer formation has not been controlled. The domain‐swapped dimer subunit interacted with two monomer subunits in tetrameric AVCP through subunit–subunit interactions similar to that in the native dimer. The AVCP native dimer exhibits a unique dimer–monomer transition upon CO binding/dissociation.45, 46, 47 The coordination site of the heme of AVCP is occupied with the side chain of Tyr16, which is tightly constrained by neighboring residues of helix αA.46 When CO binds to the heme iron, CO may push Tyr16 away from the heme and generate a movement of helix αA, affecting the interface of the monomers in the native dimer. Since the subunit–subunit interface was conserved in the domain‐swapped dimer, domain‐swapped dimers in the absence of the monomers assembled continuously forming oligomers in the absence of CO, whereas the oligomers dissociated to domain‐swapped dimers upon CO binding (Figs. 6 and 7). These results provide an example for control of protein oligomer formation using domain swapping together with the quaternary structural changes of a gas‐binding protein.

Figure 7.

Model view of CO‐dependent oligomer formation and dissociation of domain‐swapped dimers. The tetrameric AVCP comprising a domain‐swapped dimer (red and blue) and two monomer subunits (green) is also shown in the center. The red and blue regions in the domain‐swapped dimer correspond to different protomers. Additional domain‐swapped dimers (pink and light blue) are aligned to the subunit–subunit interfaces of the tetramer, and each domain‐swapped dimer in the oligomer is indicated with a square. The oligomer dissociate to domain‐swapped dimers in the presence of CO.

Conclusion

Gas‐binding AVCP, a dimeric CP protein, formed a non‐native tetramer comprising two monomer subunits and one domain‐swapped dimer subunit, in which the region containing helices αA and αB swapped between protomers. The domain‐swapped dimer interacted with two monomer subunits through helices αA and αB in the tetramer, and the subunit–subunit interfaces were similar to that of the native dimer. The domain‐swapped dimer formed oligomers successively by intermolecular interactions, whereas the native dimer did not oligomerize. The oligomers dissociated to and formed from domain‐swapped dimers reversibly by CO binding and dissociation, respectively. These results demonstrate that the combination of domain swapping oligomerization and structural changes in a sensor protein may lead to greater control of protein oligomer formation.

Materials and Methods

Preparation of tetrameric AVCP

Expression and purification of the AVCP native dimer were performed as reported previously.55 Ethanol was added to the purified AVCP native dimer solution to a final concentration of 60% (v/v) at room temperature. The solution was lyophilized, and the resulting lyophilized precipitate was dissolved in 50 mM potassium phosphate buffer, pH 7.0, at room temperature. Tetrameric AVCP in the solution was separated from the dimer and high order oligomers with a HiLoad 26/600 Superdex 75 pg gel filtration column (GE Healthcare) using a fast protein liquid chromatography (FPLC) system (Biologic DuoFlow 10, Bio‐rad, CA) with 50 mM potassium phosphate buffer, pH 7.0, at 4°C. The fraction of tetrameric AVCP was further purified with a HiTrap Q anion exchange column (GE Healthcare) using the FPLC system with a sodium sulfate concentration gradient (0–500 mM) and the same buffer. The concentrations of the proteins were calculated from the absorbances of the Soret band using the coefficients of oxidized AVCP native dimer (86,000 M⁻¹cm⁻¹) and tetramer (85,000 M⁻¹cm⁻¹) obtained by the pyridine hemochrome method.62

Optical absorption measurements

Optical absorption spectra of the AVCP native dimer and tetramer (heme unit, 3 μM) in the oxidized and reduced forms in 50 mM potassium phosphate buffer, pH 7.0, containing 200 mM sodium sulfate were obtained with a UV‐2450 spectrophotometer (Shimadzu, Japan) using a 1‐cm‐pathlength quartz cell at 25°C. The reduced AVCP native dimer and tetramer were obtained by an addition of 5 mM sodium dithionite under a N₂ atmosphere. Binding of CO to the reduced AVCP native dimer and tetramer was performed by incubation of the reduced sample at 25°C under a CO atmosphere.

CD measurements

CD spectra of oxidized AVCP native dimer and tetramer (heme unit, 10 μM) were obtained with a JASCO J‐725 spectrometer (JASCO, Japan) using a 0.1‐cm‐path‐length quartz cell in 50 mM potassium phosphate buffer, pH 7.0, containing 200 mM sodium sulfate at 20°C.

X‐ray crystallographic analysis

Crystallization of tetrameric AVCP was performed at 4°C using the sitting drop vapor diffusion method with crystal plates (CrystalClear D Strips, Douglas Instruments, Hampton Research, CA). Tetrameric AVCP was dissolved in 50 mM potassium phosphate buffer, pH 7.0, containing 200 mM sodium sulfate at a protein concentration of 2.0 mM (heme unit). Droplets prepared by mixing 1 μL of the protein solution with 1 μL reservoir solution were equilibrated. The best reservoir solution was found to be 100 mM sodium acetate, pH 4.6, containing 200 mM ammonium acetate and 30% (w/v) polyethylene glycol 3350.

The diffraction data were collected at the BL38B1 beamline at SPring‐8, Japan, using a MX225HE detector (Rayonix). The crystal treated with a cryoprotectant (100 mM sodium acetate, pH 4.6, containing 200 mM ammonium acetate, 30% (w/v) polyethylene glycol 3350, and 10% (v/v) glycerol) was mounted on a cryo‐loop and flash‐frozen at 100 K in a nitrogen cryo system. The crystal‐to‐detector distance was 125 mm, and the wavelength was 1.0000 Å. The oscillation angle was 0.5°, and the exposure time was 3 s per frame. The total number of frames was 360. The diffraction data were processed using the programs, iMosflm63 and SCALA.64 The preliminary structure was obtained by a molecular replacement method (MOLREP) using the atomic coordinates of the structure of the AVCP native dimer (PDB code: 1BBH) as a starting model.46, 65 The structure refinement was performed using the program, REFMAC 5.8.66 The work using iMosflm, SCALA, MOLREP, and REFMAC 5.8 was carried out within CCP4i.67 The molecular model was manually corrected, and water molecules were picked up in the electron density map using the program, COOT.68 The data collection and refinement statistics are summarized in Table 1. The three‐dimensional structures of tetrameric AVCP and native dimer were compared using the molecular graphics program, PyMOL.69

Table 1.

Statistics of Data Collection and Structure Refinement of Tetrameric AVCP (PDB code: 5GYR)

Data collection
X‐ray source	SPring‐8 (BL38B1)
Wavelength (Å)	1.0000
Space group	P21
Unit cell parameters
a, b, c (Å)	82.9, 48.6, 130.6
α, β, γ (°)	90.0, 92.3, 90.0
Resolution (Å)	41.4−1.60 (1.69−1.60)
Number of unique reflections	135,869 (19,444)
R merge a	0.069 (0.325)
Completeness (%)	98.6 (97.2)
<I/σ(I)>	7.9 (2.9)
CC1/2	0.998 (0.947)
Redundancy	3.7 (3.6)
Refinement
Resolution (Å)	50.0−1.60 (1.64–1.60)
Number of reflections	129,158 (9788)
R work b	0.2040 (0.270)
R free b	0.232 (0.326)
Completeness (%)	98.6 (97.2)
Number of atoms in an asymmetric unit
Protein	7736
Water	808
Heme	344
Average B factors (Å2)
Protein	27.0
Water	34.5
Heme	18.5
Ramachandran plot (%)
Favored	97.7
Allowed	2.2
Outlier	0.1

Statistics for the highest‐resolution shell are given in parentheses.

^a R _merge = Σ_hkl ‖I–‖ (Σ_hkl‖I‖)⁻¹.

^b R _work = Σ_hkl ‖‖F _obs‖–k‖ F _calc ‖‖(Σ_hkl‖F _obs‖) ⁻¹, k: scaling factor. R _free was computed identically, except where all reflections belong to a test set of 5% of randomly selected data.

SEC analysis

The solution containing the AVCP native dimer or tetramer was analyzed with a Superdex 75 10/300 GL gel filtration column (GE Healthcare) using the FPLC system (Biologic DuoFlow 10, Bio‐rad) (flow rate, 0.5 mL/min; monitoring wavelength, 399, 418, and 426 nm, corresponding to the wavelength of the Soret band of the oxidized, CO, and reduced forms, respectively; solvent, 50 mM potassium phosphate buffer, pH 7.0, containing 200 mM sodium sulfate; temperature, 4°C). The CO form of AVCP was analyzed with the same conditions except for the solvent of 50 mM potassium phosphate buffer, pH 7.0, containing 200 mM sodium sulfate, and 5 mM sodium dithionite, and saturated with CO.

Supporting information

Supporting Information