The functional similarity and structural diversity of human and cartilaginous fish hemoglobins1

doi:10.1006/jmbi.2001.4446 available online at http://www.idealibrary.com on

J. Mol. Biol. (2001) 307, 259±270

The Functional Similarity and Structural Diversity of Human and Cartilaginous Fish Hemoglobins Yukie Naoi1*, Khoon Tee Chong2, Kazuhiko Yoshimatsu1 Gentaro Miyazaki1, Jeremy R. H. Tame3, Sam-Yong Park4 Shin-ichi Adachi4 and Hideki Morimoto1 1

Division of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan 2 Institute for Protein Research Osaka University, Suita, Osaka 560-0871 Japan 3

Protonic Nanomachine Project, ERATO, Japan Science and Technology Corporation (JST) Hikaridai, Seika, Kyoto 619-0237, Japan 4 Institute of Physical and Chemical Research (RIKEN) RIKEN Harima Institute Mikazuki-cho, Sayo, Hyogo 679-5143, Japan

Although many descriptions of adaptive molecular evolution of vertebrate hemoglobins (Hb) can be found in physiological text books, they are based mainly on changes of the primary structure and place more emphasis on conservation than alterations at the functional site. Sequence analysis alone, however, does not reveal much about the evolution of new functions in proteins. It was found recently that there are many functionally important structural differences between human and a ray (Dasyatis akajei) Hb even where sequence is conserved between the two. We have solved the structures of the deoxy and CO forms of a second cartilaginous ®sh (a shark, Mustelus griseus) Hb, and compared it with structures of human Hb, two bony ®sh Hbs and the ray Hb in order to understand more about how vertebrate Hbs have functionally evolved by the selection of random amino acid substitutions. The sequence identity of cartilaginous ®sh Hb and human Hb is a little less than 40 %, with many functionally important amino acid replacements. Wider substitutions than usually considered as neutral have been accepted in the course of molecular evolution of Hb. As with the ray Hb, the shark Hb shows functionally important structural differences from human Hb that involve amino acid substitutions and shifts of preserved amino acid residues induced by substitutions in other parts of the molecule. Most importantly, bE11Val in deoxy human Hb, which overlaps the ligand binding site and is considered to play a key role in controlling the oxyÊ in both the shark and ray Hbs. Thus gen af®nity, moves away about 1 A adaptive molecular evolution is feasible as a result of both functionally signi®cant mutations and deviations of preserved amino acid residues induced by other amino acid substitutions. # 2001 Academic Press

*Corresponding author

Keywords: vertebrate hemoglobin; X-ray crystallography; molecular evolution; shark, structural comparison

Introduction Present address: Yukie Naoi, Research Institute for Food Science, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan. Abbreviations used: human Hb, adult human hemoglobin known as HbA; ATP, adenosine 50 triphosphate; DPG, 2,3-diphosphoglycerate; IHP, inositol hexaphosphate; rmsd or rms deviation, root mean square deviation; Tris, tris (hydroxymethyl) aminomethane; bis-Tris, 2-[bis(2-hydroxymethyl)amino]2-(hydroxymethyl)-1,3-propanediol. Non helical region after C helix is designated as CD (not CE) irrespective of the existence of the D helix. E-mail address of the corresponding author: [email protected] 0022-2836/01/010259±12 $35.00/0

Globins appear to have arisen very early in evolution and are found in organisms from bacteria to man. This selective success has been due to the functional ¯exibility of the protein, in particular, the ability of globins to interact with each other to give cooperative oxygen binding in a number of ways. Given the large number of known globin sequences and structures, the globins are also a useful family for examining protein evolution. The neutral theory suggests that the majority of amino acid substitutions in protein evolution are non-adaptive but harmless.1 This theory, however, fails to explain the adaptive evolution of protein # 2001 Academic Press

260 function. We have examined the structure and function of ®ve vertebrate hemoglobins (Hbs) from evolutionarily diverse organisms to see how these proteins have evolved. Since the Hbs from ®sh have diverged much more than those of air-breathing animals2 we have solved the structure of shark Hb and compared it to Hbs from a ray, two bony ®sh and human Hb. Human Hb binds oxygen cooperatively by an allosteric mechanism that has been very well conserved among all vertebrate Hbs except lamprey and hag®sh. Lamprey Hb forms a dimer with an interface unrelated to the subunit contacts found in human Hb.3 The homodimeric Hb from the arcid clam Scapharca inaequivalvis also dimerises but using a different interface.4 It appears that the mechanism of cooperativity in invertebrate and cyclostome Hbs is quite different from the jawed vertebrate a2b2-type Hbs. Perutz5 has suggested that the properties of a2b2type animal Hbs can be explained using the structure of human Hb as a model, but not all the known functional properties of animal Hbs are understood. Attempts to prove models for the Root effect (the very strong pH sensitivity) of teleost ®sh Hb by introducing mutations into human Hb have not been successful.6 The bicarbonate effect of crocodile Hb was successfully transplanted into HbA, however by introducing 12 amino acid substitutions to create a novel bicarbonate binding site,7 different from that proposed by Perutz5 on the basis of the neutral theory. A number of crystal structures of teleost ®sh Hbs have been solved in order to discover the mechanism of the Root effect, but so far without success.8 ± 11

Structural Comparison of Vertebrate Hbs

We have recently reported the primary structures and the crystal structures of both CO and deoxy forms of Hb from a ray, Dasyatis akajei.12 The main-chain atoms of the helical regions of the CO and deoxy forms of the D. akajei Hb tetramer could be superimposed on those of CO and deoxy human Hb, respectively, with rmsd values less Ê , indicating that the quaternary structure than 2 A change has been highly conserved among vertebrates except cyclostomes. There are, however, many signi®cant displacements for the atoms of conserved amino acids between D. akajei and human Hb which have been assigned important roles in the allosteric mechanisms of Hb proposed by Perutz.5 There are also many amino acid substitutions each of which would affect oxygen binding if introduced alone into human Hb. These unexpected but important differences between the ray and human Hb led us to study the structures of another cartilaginous ®sh Hb. By identifying the functionally signi®cant changes in vertebrate Hb we hope to understand better the evolution of protein function. Here, we present the crystal structures of deoxy and CO forms of Hb from a shark, Mustelus griseus, and compare them to the structures of four vertebrate Hbs.

Results and Discussions Primary structure M. griseus Hb has 140 amino acid residues in the a chain and 136 in the b chain (Figure 1). Two deleted residues (aCD2 and aGH3) and one insertion (between aCD8 and aCD9) were found by comparing the three-dimensional structures of human and M. griseus Hb. In the b chain, ten resi-

Figure 1. Comparison of the amino acid sequences between M. griseus and human Hb. The residue numbers and helix names are those of human Hb. The deleted and inserted residues are deduced by comparing the threedimensional structures: * means the same residue as that of human Hb and ÿ means a deletion. All the residues in M. griseus Hb between the b C and E helix are placed at the C helix side, and a ten residue deletion is just before the E helix, which starts at residue 49, corresponding to E3 of human Hb (see Figure 2). Therefore the ten residue deletion corresponds to between b CD8 and E2 of human Hb.

261

Structural Comparison of Vertebrate Hbs

dues are deleted between the C and E helices and the residues between bCD3 and bE2 have quite different conformations (Figure 2). In M. griseus Hb the bE helix starts at residue 49 (corresponding to 59 (E3) in human Hb). All known cartilaginous ®sh Hb sequences have lost four residues between the C and E helices and are thought to lose the D helix in the b chain.12 All known vertebrate a globins also lack the D helix. Since recombinant human Hbs containing b subunits without the D helix and a subunits with the D helix show a small change in oxygen af®nity,13 the lack of a bD helix in cartilaginous ®sh Hb is thought not to exert large functional effect. The highly preserved C-terminal sequences, Tyr-Arg in the a and Tyr-His in the b chain, are found also in M. griseus Hb. Although both human a and M. griseus b subunits lack the D helices, the sequence identity between them (30 %) is still lower than that between the b chains of the two proteins (34 %). Similarly, the M. griseus a chain has 40 % sequence identity with the human a chain and 30 % with the human b chain. The identity of the a and b chains of the shark Hb was con®rmed by superimposing each on the subunits of human Hb. Oxygen equilibrium properties Oxygen equilibrium parameters for M. griseus Hb are shown in Table 1. Despite only 37 % sequence identity (Table 2A) and the shortness of the bE helix, the oxygen af®nity of M. griseus and human Hb14 are approximately the same at pH 7.4 without organic phosphate. The Bohr effect between pH 6.5 and 7.4 in the absence of organic phosphate is about 25 % that of human Hb.14 The Hill coef®cient, n, is smaller than the value for human Hb but relatively large in cartilaginous ®sh Hbs.15 K4 is about four times smaller than that of human Hb at pH 7.4 without organic phosphate,

although K1 is similar for both proteins. The low cooperativity relative to human Hb results from the difference between K4 values. Both IHP and ATP act as organic phosphate effectors, IHP reducing oxygen af®nity more than ATP, in common with human Hb but different from D. akajei Hb.12

Overall structure of M. griseus Hb The rmsd values of the main-chain atoms of helical regions among ®ve vertebrate Hb tetramers are shown in Table 2B. They are largely consistent with the percentage sequence identity (Table 2A). Ê ) shows that The fact that they are all small (<2 A the tertiary and quaternary structures are separately very similar to each other in the CO forms and in the deoxy forms, which indicates the quaternary structure change is common from cartilaginous ®sh Hb to human Hb. From comparisons of main-chain atoms between human and M. griseus Hb (Figure 3), large deviations are seen around the A helix and the CD corner in both the a and the b subunits, the aFG corner (89-93), a115 (GH3), the aC-terminal residues, the bE helix (57-76), and the ®rst half of the bF helix (85-93). The large deviations of the A helix, the CD corner, and the C-terminal residues are seen also in other ®sh Hbs10,12,16 and are attributable to the ¯exible nature of these regions. The deletion of a115 (GH3) in M. griseus Hb results Ê at this point. The aFG in a deviation of about 2 A corner participates in the a1b2 interface and is called the joint region, playing an important role for the quaternary structure change. Its threedimensional structure is discussed below. The relatively large and periodical rmsd of the bE helix residues, larger at external residues and smaller at internal residues, is smaller than but similar to the same effect seen in D. akajei Hb.12

Figure 2. A stereoscopic comparison of the b CE regions of M. griseus Hb (gray) and human Hb (black) in the heme frame. Ca traces and the side-chains of CD1Phe (human and shark), CD4Phe (human), and CD7Phe (shark) are shown. CD1Phe is one of the universally conserved residues in the globin family and CD4Phe is also a highly conserved residues.17 A ten residue deletion in M. griseus Hb results in a quite different conformation in this region. While a seven residue D helix is apparent in human Hb, the D helix and the ®rst two residues of E helix are absent in M. griseus Hb.

262

Structural Comparison of Vertebrate Hbs

Table 1. Oxygen equilibrium parameters of M. griseus Hb pH

P50

Nmax

K1

K4

8.5 7.4 6.5 8.5 7.4 6.5 8.5 7.4 6.5

3.35 5.90 8.05 4.27 21.42 46.33 3.99 10.57 20.12

1.84 2.25 2.48 2.13 2.39 1.75 2.05 2.57 2.43

0.102 0.0558 0.0257 0.0505 0.0183 0.0126 0.0805 0.0224 0.0143

0.737 0.810 0.597 0.709 0.277 0.055 0.618 0.315 0.207

(‡IHP) (‡IHP) (‡IHP) (‡ATP) (‡ATP) (‡ATP)

Experiment conditions are 25  C, 60 mM (heme) Hb and 100 mM chloride without or with 2 mM organic phosphate. P50, oxygen partial pressure (mmHg) at half saturation. Nmax, the maximum slope of the Hill plots of oxygen equilibrium curves. K1 and K4, the ®rst and fourth stepwise Adair constant.

are shown in Figure 4. One signi®cant difference between human and M. griseus Hb is a water molecule in the deoxy a heme pocket (Figure 4(a)). The distal pocket water molecule, hydrogen bonded to Ne of E7His, is observed in deoxy myoglobin and deoxy a subunits of Hbs from several species. This water molecule must be displaced before ligation and, as a result, the rate of ligand movement into the distal pocket is greatly inhibited. Quillin and colleagues20 have shown that the distal pocket water molecule was displaced by larger side-chains at position 68(E11) in the crystal structure of deoxymyoglobin mutated E11Val to Leu or Ile. In the case of trout HbI, the substitution of aE11Val to Ile is consistent with loss of the water molecule.9 In M. griseus Hb, however, despite the highly conserved a heme pocket structure, the water molecule is absent. Another water molecule is found on the opposite side of the aE7His sidechain, as has been observed in a mutant human Hb.21 In order to form a hydrogen bond to this water molecule, the histidine imidazole group was rotated 180  . The importance of this water molecule is not obvious, but its occupancy appears to be low and it is not seen in the CO form. Another signi®cant difference from human Hb is the position of bE11Val. In human Hb, this residue

The b CD corner As mentioned earlier, a ten residue deletion occurs between the bC and E helices of M. griseus Hb (Figure 1). This deleted region starts a little after CD1, a universally conserved Phe residue. Figure 2 shows the Ca trace of this region and the side-chains of bCD1Phe (human and M. griseus), bCD4Phe (human), and bCD7Phe (M. griseus). Although one of the best conserved residues, CD4Phe,17 is replaced by Arg in the sequence of M. griseus b-chain, the side-chain of bCD7Phe, three residues away, lies close to the position of CD4Phe in human Hb. This conformational change suggests that the mutation or loss of important residues for function or stability can be accepted by changes in tertiary structure. Heme regions The alteration in heme geometry upon ligation of M. griseus Hb, which ¯attens and moves the Fe atom to an in-plane position, is similar to that of human Hb.18 The movements of the proximal His (F8His), FG5Val and F4Leu observed in human Hb19 are all common among tetrameric Hbs of vertebrates. The heme regions from human and M. griseus Hb, superimposed on the heme frame,

Table 2. Comparisons of ®ve vertebrate Hbs Human A. Amino acid sequence identities (%) Human Rock cod Trout Ray Shark

Rock cod

Trout

Ray

Shark

47

55 57

38 35 33

37 36 36 40

1.4 0.9

1.8 1.7 1.6

1.5 1.5 1.6 1.5

B. The rmsd (AÊ) of the main-chain atoms of helical regions of tetramer Human 1.3 Rock cod 1.2 Trout 1.1 0.9 Ray 1.5 1.5 Shark 1.4 1.5

1.4 1.5

0.9

As for B, helical portions used for the superposition are A1-16, B1-16, C1-7, E1-20, F1-9, G1-19, and H1-21 for the a-subunit; A1-15, B1-16, C1-7, E3-20, F1-9, G1-19, and H1-18 for the b-subunit. Upper right, CO forms; lower left, deoxy forms.

Structural Comparison of Vertebrate Hbs

263

Ê ) of mainFigure 3. The rmsd (A chain atoms between human and M. griseus Hb relative to the BGH frame. The residue numbers and the helix names are those of human Hb. Filled symbols, deoxy form; open symbols, CO form. (a) a1 subunit. The data points corresponding to an insertion between aCD8 and aCD9 and two deleted residues at aCD2 and aGH3 have been omitted. (b) b1 subunit. Data points corresponding to residues between b 44 and 58 have been omitted, since the sequence correspondence appears insigni®cant in this region because of the ten residue deletion (Figure 2).

lies close to the Fe atom and weakens ligand binding in the T state.18 In M. griseus Hb bE11Val lies further from the iron and moves less on ligation of the heme (Figure 4(b), Table 3A). bE11Val of D. akajei Hb is also found relatively far from the ligand binding site,12 suggesting high oxygen af®nity. In fact the oxygen af®nity of both M. griseus and D. akajei Hb is slightly lower than that of human Hb. Furthermore, in M. griseus Hb K1, the ®rst stepwise Adair constant is almost the same as that of human Hb (Table 1), suggesting that the oxygen af®nity of deoxy M. griseus Hb is not very different from that of human Hb. On the other hand, bE11Val does appear to inhibit ligand binding in bony ®sh Hbs.8,9 Presumably, other changes in the ray and shark Hb compensate for the movement of this distal residue so that oxygen af®nity does not increase. The most signi®cant compensating difference may be the displacement of bE7His, close to the ligand binding site (Table 3A). Other notable changes are summarized in Table 3. a 1b b1 interface The level of sequence identity between human and M. griseus Hb in the a1b1 interface (50 % in

the deoxy structure) is slightly higher than in the overall sequence (Table 2A) but lower than in the a1b2 interface (71 % in the deoxy structure). However the conformation of the a1b1 interface is highly conserved during evolution and so this region is usually used as a reference frame in comparing structures, both between liganded and unliganded forms and Hbs from different species.8,12,22 The a1b1 interface in M. griseus Hb buries a surÊ 2 in the CO and Ê 2 and 814 A face area of 833 A deoxy form, respectively, less than that of human Ê 2). HowÊ 2; deoxy form, 839 A Hb (CO form, 952 A ever eight hydrogen bonds are formed between the a1 and b1 subunits of the CO and deoxy forms of both Hbs. The C helix and the beginning of the G helix in both a and b chains do not participate in the interface of M. griseus Hb. The aGH corner shows relatively large deviations because of the deletion at aGH3 (Figure 3(a)). As a result, different contacts are formed between a1GH1Leu and b1GH2Lys, a1GH1Leu and b1G18Tyr, and a1GH3Glu and b1G18Tyr in M. griseus Hb. Similarly, a new salt-bridge is formed between the carboxyl group of a1H3Asp and the amino group of b1B15Lys. The bB15 residue is Val in human Hb, and sits in a hydrophobic environment covered by

264

Structural Comparison of Vertebrate Hbs

Table 3. Some of the functionally signi®cant structural differences between human and M. griseus Hb atom1

atom2

A. Distances between distal Val or His residues and the oxygen atoms of CO in the CO form structure Deoxy form a1E7His (Ce) a1CO (O) a1CO (O) a1E11Val (Cg) b1E7His (Ne) b1CO (O) g b1CO (O) b1E11Val (C ) B. Interconversion of hydrogen bonds at the a1b2 interface Absent in human but present in M. griseus Hb CO form a1G1Aspb (Od2) a1HC3Argb (NZ2) Deoxy form a1G1Aspb (Od2) a1G3Gln (Ne2) a1G3Gln (Oe1) Absent in M. griseus but present in human Hb CO form a1C6Thrc (Og1) a1FG4Argc (NZ2) Deoxy form a1C5Lysb (Nz) a1FG3Leub (O) a1FG3Leub (O)

Ê) Distance (A 1.76(2.00a) 2.53 (3.22) 1.38 (1.75) 2.90 (1.78)

b2C7Tyrc (OZ) b2B16Valb (O) b2G4Serc (Og) b2G6His (Ne2) b2G3Glyc (O)

2.63 2.89 2.91 2.72 2.79

b2C6Argb (NZ2) b2C2Prob (O) b2HC3Hisb (O) b2C6Argb (NZ2) b2C6Argb (Ne)

2.61 3.05 2.52 3.14 2.85

The values in parentheses are those of human Hb. Distance between a1E7His (Ne) and a1CO (O) in human Hb. b A common amino acid for human and M. griseus Hb. c Amino acids mentioned in Table 4. a

the D helix. In M. griseus Hb, which has no b D helix, this residue is a Lys, which has a long enough side-chain to form the new contact. The preservation of the three-dimensional structure of the a1b1 interface is probably a key requirement of the tetrameric conformation of vertebrate Hbs. Comparison of Hbs from several species and abnormal human Hbs shows this interface allows various mutations without affecting oxygen af®nity.23 As described above, it is possible to substitute or delete residues near the interface, altering the conformation of residues and contacts across the interface, but preserving its overall structure. a 1b b2 interface If the a1b1 interfaces of human deoxy and CO Hb are superimposed, the overall movement of the a2b2 dimer relative to a1b1 is a rotation of about 15  . The a1b2 interface, mainly the C and G helices and the FG corners, slides during the quaternary structure change that occurs on ligation of Hb. The contact between the a1FG corner and the b2C helix, near the rotation center, does not move signi®cantly and is called the joint region. In contrast, the switch region, the contact between the a1C helix and the b2FG corner away from the rotation center, shows large deviations.22 Conformational changes in the switch region of M. griseus Hb are very similar to that of human Hb. When the a1C helices of human and M. griseus Hb are superimposed, the b2FG corner in deoxy and CO form Hbs align closely even though the residues are not preserved in this region. The replacements of aC3Thr by Gly and aC6Thr by Ser do not in¯uence the quaternary structure, but replacing aC6 with Ser in an abnormal human Hb (Hb Miyano) increases the oxygen af®nity.24 The

different a1b2 contacts seen in the M. griseus Hb show that, compared to human Hb, the substitution at position aC6 loses one hydrogen bond in both quaternary states, to b2C6Arg in the CO form and to a1C3 Gly carbonyl group in the deoxy form. At the joint region, overlapping the BGH frames of deoxy human and M. griseus Hb it can be seen Ê (Figure 3(a)). This the a1FG corner shifts about 2 A shift is much smaller if the b2C helices are superimposed. The alteration of aFG4Arg to Leu results in a small displacement, but the basic mechanism of the quaternary structural change is common to human and M. griseus Hb. The same substitution is found in Hb Chesapeake, a human abnormal Hb that has high oxygen af®nity and little cooperativity.14 Despite strong preservation (71 % identity in the deoxy structure) of the sequence at the a1b2 interface, Table 3B shows many different combinations of hydrogen bonds. The total area of the a1b2Ê 2) is Ê 2; deoxy form, 628 A interface (CO form, 527 A not so different from that of human Hb however Ê 2). The same Ê 2; deoxy form, 681 A (CO form, 528 A number of hydrogen bonds are formed across the a1b2 interface of M. griseus Hb as that of human Hb (Table 3B) in both CO and deoxy forms. Gel®ltration of CO M. griseus Hb tetramer, however, shows that the protein dimerises much less readily than human Hb. Despite having diverged from other a2b2-type Hbs soon after tetrameric Hb appeared, the shark protein is clearly relatively stable. One notable mutation of the a1b2 interface is the replacement of bG4Asn by Ser (Table 4). In human Hb this residue stabilizes only the CO form but in the M. griseus Hb it forms a hydrogen bond across the a1b2 interface in both the CO and deoxy forms (Table 3B). Although the a1b2 interface

Structural Comparison of Vertebrate Hbs

265

Figure 4. A stereoscopic comparison of heme pockets in the heme frame. Black, human deoxy Hb; gray, deoxy M. griseus Hb. (a) a heme pocket. The position of a water molecule that plays an important role for ligand binding in other deoxy Hbs is different in M. griseus Hb. (b) b heme pocket. A thin black line shows the CO molecule of CO human Hb in the heme frame. Signi®cant differences are seen in distal residues, especially at the position of deoxy E11Val, disturbing ligand binding in human Hb but not in M. griseus same as in D. akajei Hb.12

shows better-than-average sequence conservation and the mechanism of the quaternary change is preserved, the conformations of interface residues and the contacts between them vary considerably. The Bohr effect M. griseus Hb shows only a small Bohr effect (Table 1) despite preservation of the residues responsible for the Bohr effect of human Hb. In deoxy human Hb, the imidazole group of the bCterminal His forms a salt-bridge with bFG1Asp, raising its pK value. The carboxyl group of the same His contacts aC5Lys across the a1b2 interface in deoxy Hb but not in CO Hb. Additional Bohr protons come from the N termini of the a subunits. The Cl ÿ binding between the guanidinium group of a1HC3Arg and the a2N terminus increases the pK value of the N-terminal amino group of deoxy human Hb.25 Although these amino acid residues are all the same in human and M. griseus Hb, the three-dimensional structure shows they have quite different conformations. The differences in the Cterminal structure of the a and b chains, and the probability of acetylation of the aN-terminal amino group (see Materials and Methods) can explain the

small Bohr effect in M. griseus Hb. Figure 5 shows bHC3His and its contacts of deoxy M. griseus Hb. The salt-bridge with aC5Lys is formed, but the salt-bridge with bFG1Asp is not, even though it could be, simply by reorienting the side-chain of bHC3His. In contrast, in D. akajei Hb, because bFG1Asp and bF9Cys are substituted by Glu and Gln, respectively, the hydrogen bond between bHC3His and bFG1Asp is disturbed and a different bond between bHC3His and bHC1Asn is found in the deoxy form.12 The structure of the bC terminus seen in Pagothenia bernacchii Hb (which has the Root effect) is like that of M. griseus Hb.8 The conformational change upon ligation of aHC3Arg is also different from that of human Hb. In both human Hb and D. akajei Hb, the guanidinium group of a1HC3Arg forms a hydrogen bond to the carbonyl group of b2B16Val and the C-terminal carboxyl group forms a salt-bridge with a2H10Lys in the deoxy form, which are broken in the CO form.12,26 The retention of these bonds in the CO structure of M. griseus Hb may partly explain the weakened Bohr effect, since they help maintain the same conformation around aHC3Arg in both quaternary states.

266

Structural Comparison of Vertebrate Hbs

Table 4. Amino acid residues of M. griseus Hb found in the substitutions of abnormal Hbs with altered oxygen equilibrium function and stability

a

b

Residue number

Amino acid substitution

Hb name

Abnormal property

C6 F9 FG4 H18 B2 C7 F6 G3

Thr!Ser Ala!Gly Arg!Leu Val!Glu Val!Met Phe!Tyr Glu!Lys Glu!Gly

Miyano Valparaiso Chesapeake Pavie Olympia Mequon Agenogi Alberta

G4 G10 G15 H17

Asn!Ser Asn!Asp Val!Glu Asn!Asp

Beth Israel Yoshizuka New York Geelong

Increased affinity Increased affinity; more stable Increased affinity; decreased cooperativity; unstable Decreased affinity Increased affinity; unstable Unstable Decreased affinity; unstable Increased affinity; decreased BoÈhr effect and cooperativity Decreased affinity; decreased cooperativity; unstable Decreased affinity Decreased affinity; decreased BoÈhr effect; unstable Unstable

The similarities of sequence and quaternary structure seem to show that the structural basis of a large Bohr effect is present but some minor changes weaken the effect. This suggests that protein functions can be ®ne-tuned by mutations distant from the principal residues involved directly in an interaction. Organic phosphate-binding site Organic phosphates stabilize the T-state structure and lower the oxygen af®nity of Hbs. The binding site of DPG and IHP is at the entrance to the central cavity in human Hb.27,28 bNA1Val, bNA2His, bEF6Lys, and bH21His interact with DPG or IHP. In the model of ATP binding in bony ®sh Hbs proposed by Perutz,5 the same residues are used but bNA2His is substituted by Glu or Asp. The carboxylate group of either Glu or Asp at this position is proposed to accept a hydrogen bond from the N-6 amino group of adenine, so that ATP lowers oxygen af®nity more than DPG. In M. griseus Hb both IHP and ATP work as allosteric effectors (Table 1). The residues interacting with DPG or IHP in human Hb are the same,

except bH21His, which is replaced by Lys, keeping a positive charge at the entrance of the central cavity. Consistent with the Perutz model, bNA2 is histidine, and IHP is found to bind more tightly than ATP. On the other hand, D. akajei Hb has lost positive charges at bEF6 and bH21, but still binds ATP.12 A candidate for a novel organic phosphate binding site was proposed for D. akajei Hb, which was composed of two Lys (bG6) and two Arg (bH13) along the molecular dyad axis just inside the DPG-binding site.12 In M. griseus Hb, however, this site cannot bind ATP, because bH13 is Glu. The mechanism by which organic phosphates reduce oxygen af®nity may not have been preserved. As binding sites for allosteric effectors are lost from some parts of the protein, new ones may evolve elsewhere, as shown by the bicarbonate effect of crocodile Hb.7 Mutations affect distant parts of the structure Of 507 variant Hbs with an amino acid substitution in either subunit listed in ``Human Hemoglobin Variants'',23 as many as 310 Hbs show some functional abnormalities (mainly concerning oxy-

Figure 5. A stereo view of deoxy M. griseus (gray) b C-terminal region superimposed on the BGH frame of deoxy human Hb (black). The b C-terminal His (HC3His) does not form a salt-bridge with bFG1Asp but forms contacts with bHC1Gln instead. Other residues have very similar conformations to those of human Hb.

267

Structural Comparison of Vertebrate Hbs

gen equilibrium property and stability). There are 21 variant Hbs with amino acid replacements found in M. griseus Hb. Of these 21, 12 variants are found to show abnormalities in their functions (Table 4). Table 3 presents some of the amino acids, preserved as well as mutated, whose deviations between human and M. griseus Hb appear to be functionally signi®cant. Some of the residues in Tables 3 and 4 have been described earlier. The same tables for the ray Hb (not shown) are very similar. There is a large number of functionally signi®cant alterations, which makes it extremely dif®cult to integrate all the effects in a predictive way. Moreover, the amino acid substitutions that cause signi®cant atomic shifts relative to human Hb and apparently silent or ``quiet'' ones are likely to have an important functional effect. The displacement of bE11Val of cartilaginous ®sh Hbs compared to human HbA must be caused by such quiet substitutions, because the heme contact residues are essentially preserved. In the case of oxygen equilibrium, they appear to compensate each other to give a similar af®nity. Destabilising mutations can also be compensated by changes elsewhere in the molecule. For example, only distal histidine and valine residues are mentioned in Table 3A among many heme contact residues, but there are signi®cant deviations for all the heme contact residues between the two Hbs except the proximal His, and each of them may exert some effect on function. The residues around the heme are good examples of highly conserved amino acids that show altered contacts and alterations in the three-dimensional structure. Although M. griseus and human Hb are functionally similar, it would be dif®cult to ``evolve'' one into the other by the accumulation of neutral mutations, given the high proportion of substitutions that are known to affect human Hb signi®cantly. Table 4 shows 12 such mutations. Clearly, substitutions at a distance can compensate for some of these effects, and in the case of the ten residue deletion in the b chain, the unfolding of the ®rst two residues of the E helix is needed to place CD7 Phe at the position occupied by CD4 Phe in most Hbs.

Conclusions Overall, the globin fold of the subunits, the alteration of the geometry of the heme and the proximal His (F8His) upon ligand binding, the structure of the a1b1 interface and the role of the switch and joint regions in the a1b2 interface upon quaternary structure change are very similar among the ®ve diverse a2b2-type vertebrate Hbs examined. However, the direct stereochemical mechanisms that control the binding of heme ligands on the distal side may be altered by the mutations outside the heme contact region. The stereochemical mechanisms that control the binding of heterotropic ligands (Bohr protons and organic phosphates) are sometimes altered or lost

by mutations in the binding sites, and by the conformational changes induced by the mutations of other parts of the protein. The preservation of the important amino acid residues does not always indicate the preservation of the function, and similarly the preservation of the function does not always mean that the same mechanism prevails. Therefore, the sequence of a novel Hb alone may have little predictive value. There are many functionally important amino acid replacements between cartilaginous ®sh and human Hb, and wider substitutions than usually considered as neutral have been accepted in the course of Hb evolution. Thus, adaptive molecular evolution is feasible as a result of both functionally signi®cant mutations and displacements of preserved amino acid residues by other amino acid substitutions.

Materials and Methods Hemoglobin purification Living sharks (M. griseus) were obtained from Taniume, a ®sh dealer of Minoshima port in Wakayama Prefecture, Japan. Blood was collected and lysed as described by Chong et al.12 The hemolysate was equilibrated with 2 mM Tris and then deionized by passage through a column of Amberlite MB-3 (Rohm & Hass). Deionized hemolysate was equilibrated with 5 mM TrisHCl buffer (pH 7.2) containing 5 mM 2-mercaptoethanol to reduce oxidized SH groups and loaded onto a CM-32 cellulose column equilibrated with the same buffer. It was eluted with a linear gradient to 0.2 M NaCl to give a single band. The protein was then passed through a Sephadex G-25 column to remove NaCl and 2-mercaptoethanol, and used for the crystallization. The deionized hemolysate showed a single band on starch gel electrophoresis and we could detect only one cDNA for each of a and b chains, but it contained nearly 10 % metHb. Therefore, for the measurement of oxygen equilibrium curves we used fresh hemolysate that was previously reduced by 10 mM sodium dithionite and passed through an anaerobic Sephadex G-25 column. The sample thus obtained was found to have a small amount of Hb with molecular mass higher than tetramer by gel-®ltration, probably because of some intermolecular disul®de bridge. In an oxygen equilibrium measurement at pH 7.4 we reduced half of the sample with dithiothreitol just before the measurement and carried out the oxygen equilibrium measurement and the gel®ltration. While the higher molecular mass component was signi®cantly reduced by the dithiothreitol reduction, the oxygen equilibrium curve did not change. All the manipulations were carried out as CO forms at 4  C or on ice, and CO was removed just before use when necessary. cDNA sequence analysis cDNA sequence was analysed as described by Chong et al.12 except that the N-terminal amino acid sequence for the a chain could not be determined by the automated protein sequencer, probably because of N-terminal acetylation. (Because of the ¯exibility of the a N-terminal regions, we could not con®rm the acetylation in the crystal structures.) We used a mix primer corresponding to the N-terminal sequence for the b

268

Structural Comparison of Vertebrate Hbs

chain, and for the a chain we adopted a mix primer corresponding to a comparatively preserved amino acid sequence among cartilaginous ®sh Hbs from E5 (usually Lys) instead of a primer corresponding to N-terminal sequence. Oxygen equilibrium measurement Oxygen equilibrium curves were measured as described by Imai et al.29 Measurement conditions were 60 mM protein concentration in 50 mM Bis-Tris and 50 mM Tris buffer containing 100 mM Clÿ at 25  C. The pH value was adjusted with concentrated NaOH: 2 mM ATP and 2 mM IHP were used as organic phosphate effectors. To minimize the autoxidation of Hb during measurements, catalase and superoxide dismutase were added to each sample.30,31 Deoxygenation curves were used to determine the p50 (partial pressure of oxygen at half saturation), Hill coef®cient, n, (the maximum slope of the Hill plots of oxygen equilibrium curves), K1 and K4 (the ®rst and the fourth stepwise Adair constant). Crystallization and data collection Oxy Hb solution was degassed in sealed tubes and ¯ushed with nitrogen or carbon monoxide before precipitant and sodium dithionite were added. Deoxy form crystals were obtained from about 1.7 M ammonium sulfate, 400 mM ammonium phosphate (pH 6.5), and 2.1 % protein. The dataset was obtained using synchrotron radiation source at RIKEN beam line-2 (BL44B2) station

of SPring-8, Harima, Japan.32 Intensity data were collected with a MAR CCD detector, which was mounted on a Huber alignment table, and processed with MOSFLM33 and scaled with SCALA.34 The CO form crystals were grown from 8 % (w/v) PEG2000, 50 mM sodium phosphate (pH 7.0) and about 2 % protein. Data were collected from three CO-form crystals at the Photon Factory, Tsukuba, Japan using the Weissenberg camera and Imaging Plate. The data were processed with DENZO and scaled with SCALEPACK.35 Structure determination and refinement Both structures were solved by molecular replacement using X-PLOR.36 The initial model for the deoxy structure was deoxy human Hb (PDB code, 2hhb) mutated to the shark Hb amino acid sequence. The re®ned deoxy tetramer was used as a search model for the CO structure. Re®nement was carried out using X-PLOR. The initial models were re®ned with the standard simulatedannealing protocol37 and bulk solvent correction38 using Ê resolution (CO form) or data between 30.0 and 2.0 A Ê (deoxy form). In each case, 5 % of 10.0 and 2.0 A randomly selected re¯ections were used as a test set to calculate free R.39 After each cycle of re®nement, the models were adjusted manually using the program FRODO.40 Isolated electron density greater than 3s in Fo ÿ Fc maps and 1.3s in 2Fo ÿ Fc maps was designated as water molecules if the locations were sterically reasonable. Crystallographic and re®nement data are summarized in Table 5. In the bCD corner (residues 44-47) and

Table 5. Crystallographic and re®nement data for the CO and deoxy M. griseus Hb A. Crystal data Space group Ê , deg.) Unit cell (A

Ê) Resolution range (A Observed reflections Independent reflections Completeness (%) Ramerge Asymmetric unit B. Refinement parameters Ê) Resolution (A Reflections (F>0s) Work set Test set R factor (%) Rbfree Rccryst Deviation from ideality Ê) Bonds (A Angles (deg.) Ramachandran plot (%) Most-favoured Additional allowed Generously allowed Disallowed

CO form

Deoxy form

P212121 a ˆ 66.70, b ˆ 80.45, c ˆ 114.83; a ˆ b ˆ g ˆ 90.00 100-2.0 260,718 39,951 91.5 7.5 a2b2 tetramer

P21 a ˆ 57.68, b ˆ 90.55, c ˆ 61.45; a ˆ g ˆ 90.00, b ˆ 98.45 30-2.0 181,593 41,763 98.8 4.4 a2b2 tetramer

20.0-2.0

10.0-2.0

37,342 1963

39,340 2078

25.5 19.9

22.3 18.1

0.008 1.086

0.008 1.156

91.2 0.9 0.9 0.0

92.1 0.2 0.2 0.2

a Rmerge ˆ h ijI(h,i) ÿ hI (h,i)ij/h  ijI (h,i)j, where I(h,i) is the intensity value of the ith measurement of h and hI(h)i is the corresponding mean value of I(h,i) for all i measurements; the summation is over the re¯ections with I/s(I) > 1.0 (CO form) and 0.0 (deoxy form). b Rfree is the R factor calculated for 5 % of re¯ections that were randomly selected and were excluded from the X-PLOR re®nement. c Rcryst ˆ jjFoj ÿ jFcjj/jFoj, where Fo is the observed structure factor and Fc is that calculated from the model.

269

Structural Comparison of Vertebrate Hbs bC-terminal regions (residues 133-136) of the CO form the electron density is broken at the main-chain level and these residues are omitted from the model.

3.

Comparison of various Hbs The coordinates of various Hbs were obtained from the PDB; human deoxy Hb (2hhb), deoxy P. bernacchii Hb (1hbh), CO P. bernacchii Hb (1pbx), deoxy Trout Hb (1out), CO Trout Hb (1ouu), deoxy D. akajei Hb (1cg5), and CO D. akajei Hb (1cg8). The model of CO human Hb has been re®ned to very high resolution by Park and coworkers (unpublished results). The comparison of three-dimensional structures was done with CCP4.34 The buried area between subunit interfaces was calculated from the difference in solventaccessibility between the subunits and dimer. The solvent-accessibility calculation was carried out in Ê . In order to AREAIMOL41 using a probe radius of 1.4 A compare the several Hbs, the coordinates were superimposed on the BGH frame, heme frame, or the C helix with LSQKAB.42 The BGH frame was originally de®ned by Baldwin and Chothia22 but differs slightly here, and is de®ned here to be B8 to B18, G8 to G19, and H5 to H17 in the a subunit and B8 to B16, G6 to G19, and H1 to H10 in the b subunit. These residues have relatively small rmsd values between human and M. griseus Hbs in both the CO and deoxy forms, and between the CO and deoxy forms of each Hb. The heme frame contains 28 atoms, four nitrogen and 20 carbon atoms of the porphyrin ring, and four methyl groups. The evaluation of hydrogen bonds across the subunit interfaces was done using with CONTACT in CCP434 in which the de®nition of the cut-off distance between the acceptor and the Ê , the minimum caluclated angle donor atoms was 3.3 A of O-H-N was 120  , and the minimum angle of a donor atom-O-C was 90  .

4. 5. 6.

7.

8.

9. 10. 11.

12.

Data Bank accession numbers The nucleotide sequence data reported in this paper has been deposited to the DDBJ/EMBL/Genbank nucleotide sequence databases with the accession numbers AB023800 (a-chain) and AB023801 (b-chain). The Protein Data Bank (PDB) accession numbers of the CO and deoxy M. griseus Hb are 1gcw and 1gcv, respectively.

13.

14. 15. 16.

Acknowledgments The authors thank Drs Y. Kawasaki and T. Suzuki of the Department of Biology, Faculty of Science, Kochi University for help with N-terminal sequencing and cloning. Data collection at Photon Factory was performed under the approval of the Photon Factory Advisory Committee (Proposal No. 97G354). H.M. is a member of the TARA project of Tsukuba University.

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Edited by K. Nagai (Received 11 July 2000; received in revised form 10 December 2000; accepted 4 January 2001)