Alteration of the α1β2/α2β1 subunit interface contributes to the increased hemoglobin-oxygen affinity of high-altitude deer mice

Background Deer mice (Peromyscus maniculatus) that are native to high altitudes in the Rocky Mountains have evolved hemoglobins with an increased oxygen-binding affinity relative to those of lowland conspecifics. To elucidate the molecular mechanisms responsible for the evolved increase in hemoglobin-oxygen affinity, the crystal structure of the highland hemoglobin variant was solved and compared with the previously reported structure for the lowland variant. Results Highland hemoglobin yielded at least two crystal types, in which the longest axes were 507 and 230 Å. Using the smaller unit cell crystal, the structure was solved at 2.2 Å resolution. The asymmetric unit contained two tetrameric hemoglobin molecules. Conclusions The analyses revealed that αPro50 in the highland hemoglobin variant promoted a stable interaction between αHis45 and heme that was not seen in the αHis50 lowland variant. The αPro50 mutation also altered the nature of atomic contacts at the α1β2/α2β1 intersubunit interfaces. These results demonstrate how affinity-altering changes in intersubunit interactions can be produced by mutations at structurally remote sites.


Introduction
Hemoglobin is a heterotetrameric protein consisting of two α subunits and two β subunits, and each subunit has an iron-centered heme that reversibly binds oxygen [1]. Hemoglobin transports heme-bound oxygen from the lungs to all of the tissues in the body via oxygenation-linked shifts in the conformational equilibrium between the tense state (T state; deoxygenated) and the relaxed state (R state; oxygenated) [1][2][3]. This structural transition is controlled allosterically, as the binding of oxygen to one subunit affects the oxygen-affinity of PLOS  the other subunits in the same tetrameric assembly [3]. Therefore, studies of structure-function relationships in hemoglobin have focused on heme-ligand binding at the active site [4,5] as well as the oxygenation-linked conformational changes between the R and the T states [6][7][8]. Recent studies suggest the presence of several intermediate states between structural transitions [9][10][11]. When air-breathing vertebrates are exposed to environmental hypoxia, an increase in hemoglobin-oxygen affinity can compensate for the reduced partial pressure of oxygen in inspired air by safeguarding arterial oxygen saturation [12][13][14]. To investigate the structural basis of evolved changes in hemoglobin-oxygen affinity, we chose to focus on functionally well-characterized hemoglobin variants of the deer mouse, Peromyscus maniculatus. Populations of deer mice that are native to high-altitudes in the Rocky Mountains have evolved a genetically based increase in hemoglobin-oxygen affinity relative to lowland conspecifics in the prairie grassland. The hemoglobin of the highland mice has a higher intrinsic oxygen-affinity, and this affinity difference is further accentuated in the presence of chloride ions and 2,3-disphophoglycrate (DPG) [15][16][17][18][19]. The most common hemoglobin variants of high-and low-altitude deer mice are distinguished by a total of 12 amino acid substitutions-eight in the in the α subunit and four in the β subunit ( Fig 1A). The oxygenation properties of these alternative variants are well-characterized [15][16][17][18][19], but the structural basis of the observed differences in oxygen affinity has not been fully elucidated.
We solved the crystal structure of the highland hemoglobin variant to gain further structural insights into the molecular mechanism responsible for the evolved increase in oxygenbinding affinity. A comparative analysis of the highland and lowland deer mouse hemoglobin variants revealed that main chain displacements were present between α41 and α54, the loop region near heme, which resulted from a α50His!Pro mutation in the highland variant. The difference was also highlighted by the interaction between αHis45 and heme in the highland hemoglobin variant. In addition, the highland hemoglobin variant had a reduced intersubunit contact surface, particularly at the α 1 β 2 /α 2 β 1 switch region.
These analyses suggest that the loose intersubunit interfaces in the highland hemoglobin molecule confers a flexible structure that shifts the allosteric equilibrium towards the R state by lowering the free energy of the R$T transition, thereby increasing oxygen affinity. crystals 65 days after they were formed, and UV-visible spectroscopy revealed the spectrum expected for carboxy hemoglobin. Diffractable crystals were obtained in fiber clusters with 5 mM glutathione, 0.1% (v/v) 2,4-pentanediol and 7 mM calcium chloride (S1 Fig). The lowland hemoglobin variant was crystalized under the same conditions as the highland variant with the exception that 28% (w/v) PEG 3350 was used; however, the same fibrous format was not observed. Crystals of lowland hemoglobin were produced only with 14 mM calcium chloride without glutathione and 2,4-pentanediol [20]. Increased hemoglobin-oxygen affinity of high-altitude deer mice Preliminary X-ray diffraction analysis At least two types of crystals were grown. However, in both cases, the crystals were fragile and had a high mosaic spread in the diffraction images. Multiple attempts were required to obtain an interpretable diffraction image by HKL2000 (HKL Research, Charlottesville, VA, USA) [23]. The first crystal (type F) was subjected to the diffraction experiment at BL41XU at the SPring-8. The type F crystal diffracted up to a 3.0 Å resolution, and the crystal belonged to the space group C222/C222 1 with the unit cell dimensions a = 53, b = 91 and c = 507 Å. The second crystal (type S) was analyzed at the 14-BM-C, BioCARS of the Advanced Photon Source (APS) at Argonne National Laboratory. The type S crystal diffracted beyond 2.2 Å resolution and belonged to the space group P2 1 with the unit cell dimensions a = 53.18, b = 229.62, c = 53.29 Å and β = 119.12˚ (Table 1). Considering the compactness of the unit cell, we solved the structure of highland hemoglobin using the type S crystal. Data collection and structure refinement The diffraction data for the type S crystal were collected from a flash frozen single crystal under the cold stream at 90 K using 10% (v/v) glycerol as the cryo-protectant. Diffraction images were recorded by the ADSC Q315 detector at the 14-BM-C using the X-ray wavelength 0.978 Å at 14-B-C, APS BioCARS. We used the oscillation method with a swing width of 1˚. We collected two data sets to cover 180˚of the spindle for each using κ angles of 0˚and 30˚. During data collection, the crystal suffered immense radiation damage. After iterative image data processing using HKL2000 [23], only the first dataset with κ = 0˚was usable, in which only 128 images out of 180 images were interpreted. The collected diffraction data exhibited anisotropy, limiting data collection beyond 3.0 Å resolution along the L-axis. This resulted in 71% completeness, but we were still able to achieve a higher signal to noise ratio (I/σ >9) and a resolution up to 2.2 Å (Table 1). Despite the weak diffraction data statistics, we proceeded to the structure analysis, taking advantage of the consistent folding of hemoglobin. The phase was recovered using the molecular replacement method with lowland deer mouse hemoglobin as the search model (PDB ID: 4H2L) in Phenix [24]. The obtained solution was manually fitted to the electron density using Coot [25] and was then further refined by running phenix.refine. The fitting between the electron density map and the model was good. However, the last residue in all of the α chains, α141Arg, was not assigned in the final model because of weak electron density. The crystallographic statistics are listed in Table 1. The structure was deposited in the Protein Data Bank with the ID code 5KER. The obtained highland hemoglobin structure had two heterotetrameric hemoglobin molecules in the asymmetric unit. The chain ID allocations in this PDB entry were as follows: one tetramer consisted of chains A (α 1 ), B (β 1 ), C (α 2 ) and D (β 2 ), and the other of chains E, F, G and H in the same order. PyMOL (Version 1.8 Schrödinger, LLC.) was used for structural mining.

Structure of the highland hemoglobin variant
The type S highland hemoglobin crystal contained 2 tetramers in the asymmetric unit ( Fig  1B) that were connected by intersubunit hydrogen bonds between subunits C, D, E and F ( Table 2). Although the UV-visible spectrum of the dissolved crystal solution indicated the presence of carboxyhemoglobin, we were only able to assign a water molecule at each heme based on the obtained electron density map. In addition, chain H (β subunit), did not have an assigned ligand, as there was no electron density supporting the presence of a molecule of carbon monoxide, oxygen, or water (S2 Fig). Lowland hemoglobin was also in waterbound form, but it had one α and one β subunit in the asymmetric unit [20], as is often observed in liganded tetrameric hemoglobin structures including human hemoglobin [26]. At the tetramer interface, chain F and C had the highest subunit interaction area. One of the identified hydrogen bonds between α 1 C-Ala115 (N) and β 1 F-Asp121 (OD1) could be unique, as the lowland hemoglobin variant has Ser at α115. However, none of the identified interactions was located near the distal heme pocket. The two tetramers folded similarly with an r.m.s. value of 0.54 between ABCD and EFGH according to the PyMOL alignment function. Of note, in the sickle-cell deoxyhemoglobin (deoxyHbS) fiber using the same chain designation as the present structure, the head to tail tetramer interactions were α 1 C-Pro114: β 1 F-Glu121 [27]. The interactions observed during polymerization were different between deoxyHbS and the present structure. Main chain traces between each subunit of the highland and lowland hemoglobin variants were highly similar, with r.m.s.d. values falling within 0.3-0.8 Å, except for the terminal residues using the CCP4 superposing program [28]. Higher displacements in the main chain atoms were found in the α subunit residues between 41 and 54 with r.m.s.d. values of 1.2-2.6 Å. This was caused by the amino acid mutation at position α50 (Fig 1C; see the following section). β subunit main chain traces between highland and lowland hemoglobin showed no detectable displacements.
Effects of the α50 mutation on the configuration of αHis45 and the α 1 β 2 / α 2 β 1 switch interface Among the 12 amino acid mutations that distinguish the lowland and highland hemoglobin variants, one especially noteworthy change involved the replacement of αPro50 in highland hemoglobin for αHis50 in lowland hemoglobin (Fig 1A and 1C). Based on the main chain atom displacements, this αHis50Pro mutation changed the coordinates of the nearby loop region as well as that of αHis45. The α50 and αHis45 residues are located in the loop region leading to the E helix, and this loop is relatively rigid because it is stabilized by hydrogen bonds according to the Dictionary of Secondary Structure of Proteins (DSSP) [29,30] (data not shown). In the lowland hemoglobin variant, αHis50 interacts with αGlu30 (Fig 1C, [19]), which pinches the amino end of the loop and prevents a stable αHis45-heme interaction. This resulted in αHis45 interacting with nearby residues in the crystal symmetry relationship [20]. In the highland hemoglobin variant, this pinching was not observed because αPro50 has no interaction with αGlu30. As a result, the highland hemoglobin variant could have a stable αHis45-heme interaction. In fish hydrated hemoglobin, the contribution of αHis45 in heme retention reported [31], which supports our assumptions.
The difference in the loop region also affected intersubunit interactions. Hemoglobin exhibits allosteric motion in the transition between the T and R states, and this oxygenationlinked transition alters a set of subunit contacts at the α 1 β 2 /α 2 β 1 switching region (Fig 2). In human hemoglobin, the highlighted residues at this switching region are Thr38, Thr41 and Pro44 from the α 1 /α 2 subunit C/D helix and His97 from the β 2 /β 1 subunit [32,33]. In deoxygenated hemoglobin (T), β 2 His97 is located close to α 1 Pro44, whereas in oxygenated hemoglobin (R), β 2 His97 faces away from α 1 Pro44 [10,32,33]. In the case of deer mouse hemoglobin (Fig 1A), both highland and lowland hemoglobin variants have the same amino acid residues at the switch region as the case of human hemoglobin. Although the superimposed structure showed that the distance between α 1 Thr38 C α from highland and lowland hemoglobin was within a 0.3 Å difference, the distance comparisons between the α 1 Thr41 pairs and α 1 Pro44 pairs increased to 1.1 Å and 1.4 Å, respectively (Fig 2). In addition, the distance between α 1 Thr38, α 1 Thr41 and α 1 Pro44 with respect to β 2 His97 was shorter in the lowland hemoglobin variant than in the highland variant (Table 3).
Although the C α coordinates of residues at each switching region from highland hemoglobin were the same, the relative orientation of the β 2 His97 imidazole ring was different, particularly at chain G and F (α 2 β 1 interface, Fig 2).
Comparisons of subunit interfaces between the highland and lowland hemoglobin variants using the PISA webserver [34] showed other differences in the α 1 β 2 /α 2 β 1 interface interactions.
For example, the interaction between α 1 Arg92 and β 2 Asp43 was different due to the change in the orientation of α 1 Arg92. This particular interaction was not observed between chains G (α subunit) and F (β subunit) (Fig 3) in highland hemoglobin. Nevertheless, the α 1 Arg92 / β 2 Asp43 interaction was found in chain H, the subunit without assigned ligand (Fig 1B and S2  Fig) Since chain H had a planar heme like a typical in liganded hemoglobin, alterations of α 1 β 2 /α 2 β 1 interface appear to affect hemoglobin-oxygen affinity.
In addition, highland hemoglobin has a reduced subunit contact surface at the α 1 β 2 /α 2 β 1 interface, and this difference in interface area between the lowland and highland hemoglobin variants is similar in magnitude to the difference between oxygenated and deoxygenated states of human hemoglobin (Fig 4). In particular, lowland hemoglobin has about 30% larger α 1 β 2 / α 2 β 1 interface that is expected for the T-state [20]. Just as the T-state quaternary structure has lower oxygen-binding affinity than the R-state quaternary structure [35,36], the highland hemoglobin variant has a higher oxygen-binding affinity than lowland hemoglobin variant.

Fig 2. Difference in the α 1 β 2 switching region interaction between highland and lowland hemoglobin.
The α subunit is shown in the cartoon, and the key residues are represented as a stick model. The residues unique to highland and lowland hemoglobin are labeled in cyan and pink, respectively. The highland hemoglobin α 1 subunit is colored in cyan, and its β 2 subunit is colored in gray. Both subunits from the lowland hemoglobin are colored in pink. The right side boxes indicate differences in the α 1 β 2 /α 2 β 1 interface coordinates, particularly the β 2 His 97 imidazole ring orientation relative to the α 1 residues.

Hemoglobin
Interface ( Increased hemoglobin-oxygen affinity of high-altitude deer mice α 1 β 1 interface changes induced by amino acid mutations Previous structural analysis of the lowland hemoglobin variant suggested that the α 1 β 1 interface interaction (in particular, the interaction between α 1 Cys34 and β 1 Ser128) differs from that of the highland variant due to a mutation at β 1 Ala128 [20]. Our structural analysis revealed that the highland hemoglobin variant lacked this interaction, and also showed that the α 1 Cys34 side chain was pointed toward the subunit interface, allowing the interaction with the β 1 Pro124 main chain oxygen atom (S3 Fig). Mutations at α 1 113, α 1 115 and α 1 116 might also affect this interface as the β 1 128 mutation. The lowland hemoglobin variant has α 1 Leu113, α 1 Ser115 and α 1 Asp116, whereas the highland hemoglobin has α 1 His113, α 1 Ala115 and α 1 Glu116. Structural analysis of the highland hemoglobin variant demonstrated that α 1 His113 and α 1 Glu116 had different coordinates among the four α subunits (Fig 3-3), resulting in different interactions with nearby residues. For example, α 1 Glu116 from chain C showed an interaction with α 1 Lys16, and chain G α 1 His113 interacted with α 1 Tyr24 and α 1 Glu116. In the lowland hemoglobin variant, these residues show no interactions with nearby residues. In addition, α 1 Trp14, which alters conformations between the deoxygenated and oxygenated states in human hemoglobin [37], seems to have a different set of interactions in deer mouse hemoglobin. In lowland hemoglobin, the α 1 Trp14 side chain was oriented toward residues α 1 113, α 1 115 and α 1 116, whereas the majority of the highland hemoglobin had an α 1 Trp14 side chain pointed toward α 1 Tyr24 (Fig 3-3). Only chain G from the highland hemoglobin had the same α 1 Trp14 coordinate as the lowland hemoglobin. Because chain G had the highest number of interactions between residues at α 1 113, α 1 115 and α 1 116, mutations at these sites can be expected to affect the packing of nearby helices in the α subunit. Such changes could account for differences in the nature of atomic contacts at the α 1 β 1 interface.

Discussion
The hemoglobin variants of highland and lowland deer mice are distinguished by 12 amino acid mutations. We compared structures of both variants to elucidate how these mutations account for the observed difference in hemoglobin-oxygen affinity. A previous study of the lowland hemoglobin variant revealed no atomic contact between αTrp14 and E helix residue α67, which enhances the mobility of the E-helix (which contains the distal histidine, αHis58) [20]. In the lowland hemoglobin variant, the interaction between αHis50 and αGlu30 might promote tight α subunit packing as well as helix mobility [19]. In addition, the evidence suggests that the α 1 Cys34-β 1 Ser128 interaction in the lowland hemoglobin variant produced a change in the α 1 β 1 interface [19,20]. Three mutations at α113, α115 and α116 highlighted changes in nearby helix packing that stem from changes in inter-helix interactions. Additionally, αSer71 in the highland variant interacts with αThr68; an interaction that is not possible in the lowland variant due to the presence of αAla71 (Fig 3-1).
The α 1 β 2 /α 2 β 1 interfaces were different because of the main chain atom displacements between α41 and α54 and the stability of the αHis45-heme interaction, and these were enhanced by the αHis/Pro50 mutation. Additionally, the α 1 β 1 interface was altered, as the highland hemoglobin β 1 Ala128 substitution prevented interaction with α 1 Cys34, and this promoted an interaction between α 1 Cys34 and the β 1 Pro124 main chain oxygen atom.
In conclusion, the amino acid mutations that distinguish the highland and lowland deer mouse hemoglobin variants affect oxygen-binding affinity by altering interface interactions, Increased hemoglobin-oxygen affinity of high-altitude deer mice which are mainly attributable to mutations in the α subunit. The highland hemoglobin variant has a looser α 1 β 2 /α 2 β 1 switch region than the lowland variant due to the α50 mutation, and the nature of atomic contacts at the α 1 β 1 interface is also different because of mutations at β128, α113, α115 and α116. In guinea pig hemoglobin [38], the lack of a salt bridge between αThr30 and αPro50 conferred an increased flexibility. Also, the guinea pig hemoglobin has αHis40 instead of αPro40 (as in human hemoglobin), and the bulkiness of histidine affects the α 1 β 2 / α 2 β 1 interface during the T$R transition in quaternary structure. These results suggest that the modulation of allosteric interactions via changes in protein flexibility may represent an important mechanism in the evolutionary fine-tuning of hemoglobin-oxygen affinity.