J. Mol. Biol.

(1990) 214, 7-14

Structure of Deoxy-quaternary Haemoglobin Liganded p Subunits

with

Ben Luisi Department of Molecular Biophysics and Biochemistry Yale University, 260 Whitney Avenue New Haven, CT 06511, U.S.A.

Bob Liddington Department of Biochemistry and Molecular Biology Harvard University, 7 Divinity Avenue Cambridge, MA 02138, U.S.A

Giulio Fermi MRC Laboratory of Molecular Biology Hills Road, Cambridge CB2 2&H, England

and Naoya Shibayama Jichi

Medical

(Received

Department of Physics School, Minamikawachi-machi, Tochigi-Ken 329-04, Japan

17 November

Kawachi-Gun

1989; accepted 23 February

1990)

We have determined the structure of a T-state haemoglobin in which the haem groups of the /I subunits have carbon monoxide bound, and the a subunits have nickel replacing the haem iron and are ligand-free. The structural adjustments on binding ligand in the T state are in as those associated with the quaternary transition, and a translational the same direction shift of the haem is severly restricted. We explain how these observations may account for the low ligand affinity of the /? haem of T-state haemoglobin.

et al., 1987). We are now in a position

to analyse the /I subunits. The hybrid haemoglobin (~Ni(u))2(/IFe(1’))2 was et al. (1986b). prepared as described by Shibayama The Ni haem groups of this hybrid do not coordinate CO. Crystals were grown under a, CO atmosphere from 16.9% (w/w) polyethylene glycol 1000, 5.5 mM-potassium phosphate (pH 6+3), 1 mMinositol hexaphosphate (IHPT), and 0.5 mM of the hybrid haemoglobin. The crystals belong to space group P2, and have cell dimensions a = 63.18 A, b = 82,26 A, c=5506it (1 A = 0.1 nm),

The liganded B. T-state haemoglobin presented here completes the spectrum of unliganded and symmetrically liganded mammalian haemoglobins in both the low and high-affinity quaternary conformations (T and R states, respectively) whose crystal structures have been solved and refined. With these structures we can compare the tertiary changes that occur with ligand binding in the R quaternary state (R-liganded versm R-deoxy) with those occurring in the T quaternary state (T-liganded versus T-deoxy) and so identify the restraints that are responsible for the low ligand affinity in the T state. Such an analysis has already been performed for the CI subunits (Arnone et al., 1986; Liddington et al., 1988; Luisi & Shibayama, 1989; for a review, see Perutz 0022-2836/~/l

30007O8

$03.00/0

t Abbreviations used: IHP, inositol hexaphosphate; r.m.s. root-mean-square; Hb, haemoglobin.

7

0

1990 Academic

Press Limited

B. Luisi et al. olphal-beta2

interface

L befaFG

alohal-beta2

alnh;l-beta2

Interface

Interface

R

hraFG4

CP

(b)

alohal-beta2

alohai-beta2

lnterfsce

Fig. 1.

Interfecs

o n

9

Communications alphal-beta2

alphal-beta2

interface

lnterfoce

HlsFGl

HlsFGl

alpha \

PheC7b

-

ValFGS

-

(dl

Figure 1. (a) A stereoscopic view of 1 half of the al/pz interface: the packing of the C helix of a1 and the FG corner of flz shown for deoxyhaemoglobin in the T state (open bonds) and (aNi)z(flFeCO), (filled bonds). The aI C helices have been superimposed with a root-mean-square fit of peptide backbones of 920 A. The lower case a denotes residues of the a subunit. With the quaternary transition, the /3FG corner jumps along 1 notch of the a C helix, in the manner of a ratchet (see (c) for comparison with the R state). The deoxy T-state haemoglobin (human) co-ordinates are from the 1.74 A model (Fermi et al., 1984). (b) The 2nd half of the al//?* interface: the packing of the C helix of & and the FG corner of aI for deoxy (open bonds) and (LX~‘)~(~~~“CO),(filled bonds). The flz C helices have been superimposed, with r.m.s. fit of 919 A. The lower case b denotes residues of the fl subunit. This interface acts as the swivel point for the rotation of the subunits (see (d) for cornwith the R state). (c) Th e same interface as shown in (a), comparing the oxy R state (open bonds) and (aNi)&? ‘CO), (filled bonds). r.m.s. fit of C helices 929 A. The oxyhaemoglobin (human) co-ordinates are from the 2.1 A model (Shaanan, 1983). (d) The same interface as shown in (b), here comparing oxy (open bonds) and (aNi)z(fiF’CO), (filled bonds). The r.m.s. fit of C helices is 032 A.

p = k3.42”. The asymmetric unit holds one tetramer, and the molecular dyad does not lie on a crystallographic symmetry axis. A complete data set of diffraction intensities, including measurements from Friedel symmetry-related reflections, was collected from one crystal to 2.6 A resolution with a Watts-Hilger four-circle diffractometer at room temperature. Reference reflections in the highresolution range were measured periodically, and the maximum extent of change 20%. A linear, time-dependent

in intensity correction

was was

applied to the data to compensate for this intensity decay. A total of 34,793 Iobs were merged to yield 17,218 unique I,,, with an R-factor of 58%. The structure was solved by molecular replacement (Dodson, 1982), using deoxyhaemoglobin (Fermi et al., 1984) as the search model, and was refined by conventional least-squares techniques (Jack & Levitt, 1978; Konnert & Hendrickson, 1980) to an R-factor of 21.4%. The restraints used in the refinement were varied between cycles to maintain the root-mean-square (r.m.s.) deviation of C-C bonds from the ideal value (1.52 A) at 603 A. Fifty-two water and one IHP molecules were included in the final model. The IHP was poorly ordered but occurs in the same place in the central

cavity as it does in deoxy-Hb (Arnone & Peurtz, 1974). The al/fil and az//.lz subunits, which are related by non-crystallographic symmetry, were refined independently. The root-mean-square fit of the main-chain atoms of the a1 and a2 subunits is 630 A and of the fil and /I2 subunits is 0*36 A. The positional uncertainty of the atoms in the wellordered parts of the molecule is about 63 A and for the Fe atoms it is about @l 8. The atomic temperature factors of the CO molecules were refined with occupancy fixed at lOOo/o, since refinement with both these parameters is impractical, owing to the high correlation between them. The values of the refined CO temperature factors were between 19 and 22 A2, which is in the same range as the refined parameters of neighbouring atoms (8 to 54 A2 for the Fe and ring atoms of His F8 and E7/?). This suggests that the CO must in fact have an occupancy close to lOO%, since otherwise its temperature factor would have refined to a much larger value than that of the neighbouring atoms. In agreement with spectroscopic studies (Shibayama et al., 1986a, 1987), we find that the bond between the Ni and the proximal histidine, His F8, is broken. The co-ordinating N” of His F8 and the Ni atom are separated by 3.2 A. This

B. Luisi et al. beta:

T-CO

and

T-deoxy

beta:

T-CO

and

R-oxy

beta:

T-CO

beta:

TACO

and

T-deoxy

and

R-oxy

H,st,

VaiEll

P

LeuFG3

‘i ti 1 SF8

(b)

Figure 2. (a) A stereoscopic comparison of the haem pocket of the fi subunits of deoxyhaemoglobin in t,he T state (open bonds) and (aN’)2(flF”CO), (filled bonds). The haem centres of the respective models have been superimposed. The view is from the interior of the haem pocket, looking towards the solvent side (into which the propionic groups project). (b) The haem pockets of the /I subunits of (aN’)2(/IF”CO), (filled bonds) and oxyhaemoglobin (R-state) (open bonds). (c) The haem pockets of the /I subunits of (LY~‘)~(/?““CO), (filled bonds) and met-T haemoglobin (open bonds). co-ordinates are from a 2.1 a model (Liddington et al., 1988; Liddington. 1986). (d) Ligand binding in R (open bonds) and oxy-R haemoglobin (filled bonds). The deoxy-R co-ordinates (horse) are from a 1986; Perutz et al., 1987). The deoxy-R intermediate was prepared by crystallizing the met, (Fe3’) haemoglobin, haems.

which

had been treated

with

broken bond results in small structural tions in the CI haem pocket, and it may constraints on globin movement that ligation in the normal T state. However, is functionally a model of the low-affinity (p(W )2(jFe(“))2 binds oxygen (at the Fe non-co-operatively in solution and with

the cross-linking

perturbareduce the accompany this hybrid state: for atoms only) roughly the

agent

bis-maleimidoethyl

ether,

The met (human) the R state: deoxr2.0 A model (L&i.

derivative of horse and then reducing the

same affinity as that of the T state of native haemoglobin (Shibayama et al., 19863). Although (LX~‘)~(/I~“CO), crystallizes in a difierent space group from that of deoxyhaemoglobin crystals grown under similar conditions (Ward et al., is nonetheless in the 7 1975)> the molecule quaternary state, as defined by the packing of i he

Communications beta:

T-CO

and

beta:

T-met

T-CO

and

T-met

,

beta:

R-deoxy

and

beta:

R-oxy

R-deoxy

and

R-oxy

(d)

Fig. 2.

a,//Iz and az//I1 interfaces (Fig. l(a) to (d)). It has been proposed that the b chains do not bind ligand in the T state (DiCera et al., 1987a,b), but the structures of (aNi)z(/3F”CO), and of (c?‘“)~(~“‘CO), (Amone et al., 1988) show that they do. Th,e role of the distal residues In Figure Z(a) to (d), we compare the /I haem pockets of T-deoxy, T-P-CO, T-met, R-deoxy and R-oxy haemoglobins by overlapping the haem atoms. The Val El1 side-chain projects over the haem and occupies the ligand binding site in

T-deoxy and, to a lesser extent, R-deoxy haemoglobin. The arrival of oxygen in the R state leads to structural adjustments of the haem pocket such that Val El1 moves well clear of the ligand (Fig. 2(d)). In the T state, these adjustments cannot occur to the same extent (Fig. 2(b)), and the ligand’s fit in the T-state haem pocket is very tight, with close contacts to both Val El1 and the distal His E7 (the distance between His E7 N” and CO is 3-O A, and Val El1 Cy’ to CO is 3.2 A; see Table 1). Because it protrudes from an a-helix, the Val side-chain is restricted from rotating about the C”-C? bond, and it can only be displaced with a

12

B. Luisi et al.

Table 1 Distances (8) between atoms of the distal residues and the ligand in the /I subunit R HbCOt R HbOJ T Hbb-CO His E7 r-0, His E7 N’-C or 0, Val El1 CY-0

Val El1 CY2-Cbr0 2

330 345 328 373

30 31 30 3.2

32 35 36 34

The errors in the distances are approximately t Derewenda et ~2. (1999). $ Shaanan (1983).

&@l A.

concerted shift of the entire E helix. Relative to the haem of deoxy-haemoglobin, the E helix of is displaced to accommodate the t~NiMBFecoh ligand and the distal histidine, His E7, rotates about the C”-CB bond. With ligand binding in the R-state, the E helix undergoes a concerted shift and the structural change here is not localized to the distal residues. Probably the E helix cannot be displaced much further in the T state. For example, if a ligand smaller than CO, such as a water molecule, is bound, the displacement of the E helix is of similar magnitude. However, the larger ligand is accommodated by a greater rotation of His E7 (Fig. 2(c)). The bulkier CO results in a O-9 A greater shift of the His E7 side-chain relative to the orientation found with bound water in T-state methaemoglobin. As the distal histidine must move to accommodate the bulkier ligands, it is possible that the steric bulk of the residue may also restrict T state ligand binding. The position of the E-helix relative to the fl haem group in the intermediate states, ligated T and unligated R, is intermediate between the positions in the end states, unligated T and ligated R states

Table 2 Stereochemistry of the B subunit haem groups Molecule T-state R-state T-state R-state T-state R-state R-state

Fe-N” deoxy deoxy met met CO HbCO HbO,

217 2.14 2.17 2.15 2.23(5) 220 2.07

Fe-P,?

Fe-P

@36(5) 0.27 009(5) @lo @15(5) -@IO -@11(5)

050(3) 0.45 026 019 @27(5) @00(8)

Reference 1 2 3 4 5 6

References: 1, Fermi et al. (1984); 2, Perutz et al. (1987); 3, Liddington et aE. (1988); 4, Luisi (1986); Perutz et al. (1987); 5, Derewenda et al. (1990); 6, Shaanan (1983). t Fe-P, is the separation of the Fe and the mean plane of the pyrrole nitrogens; Fe-P is the separation from the mean plane of the porphyrin N and C atoms, including the first atoms of each side-chain. Fe-N” is the separation of the Fe and the N” of His FS, the proximal histidine. Negative values indicate that the Fe is on the distal side of the haem plane. All values tabulated are the mean of the /fl and j& subunits, except for R-state HbCO and HbO,, for which there is a dimer in the asymmetric unit. For T-state CO, the values in parenthesis are the estimated standard deviation of the mean.

oxy-Hb 4.7 I

F I

I

boxy-Hb 3.7 J

Figure 3. A comparison of the distance between Val CY2 and the haem centres in the /I subunit of intermediate and end-state haemoglobins.

(see Fig. 3). Consequently, the shift of the E helix that accompanies ligation within either quaternary state is in the same direction as that which accompanies the T to R transition. This is as would be expected if, as has been proposed, the dependence of oxygen affinity of the /? haem group on quaternary structure is mediated through the steric blockage, by Val El1 in going from T to R (Perutz, 1970). This proposal is also consistent with the results of functional studies of mutant haemoglobins prepared by site-directed mutagenesis (Nagai et al., 1987; Olsen et al.? 1988).

Structural changes on the proximal

side of the haem

Ligand binding at the T-state J haem results in a shift of the Fe atom from its position on the proximal side of the haem by 021 8; about half way to its position found in R-state carbonmonoxy haemoglobin (Table 2). We do not know whether this intermediate geometry is strained. The hydrogen-bonding network of the F helix is not affected by the movement of the Fe atom. In the a subunits, the hydrogen bond between His F8 Ng and Leu F4 C=O is stretched in T-state a-liganded haemoglobin (Liddington et al., 1988). Movement of the /I haem is constrained in the T state Our analysis has thus far revealed how the direct interactions between the ligand, the haem and the globin might regulate affinity. We now turn to the relationship between the restrictions to tertiary structural adjustments and the quarternary state. The interface between the a1 and fll subunits is relatively invariant in the two quaternary structures and provides a suitable point of reference for examining global tertiary structural changes (Baldwin & Chothia, 1979). If we compare deoxy-T and deoxy-R, we find that the position of the b haem differs principally by a translation (@9 A) toward the interior of the globin (Fig. 4(a)). (In contrast, the a haem does not move significantly with the allosteric transition.) Ligand binding in both the T (Fig. 4(b)) and R states (not shown) results in a rotation of the haem of 7 to 8”. The haem translates roughly @4 A with ligand binding in the T state. Further translation of the haem in the T state would bring the ligand away from the distal residues, and so restrictions on haem translation may decrease the ligand affinity by enforcing close steric contacts with the distal residues. Our results support the proposed role of Val E 11

Communications

(b)

Figure 4. A stereoscopic comparison of the tertiary structural changes in the environment of the /l haem pocket with the quaternary transition. Deoxy-T (filled bonds) and deoxy-R state (open bonds) molecules have been superimposed on the main-chain atoms of the a,/fil interface (a residues 20-36, 94-112, 118-138; fl residues l&-33,98-116, 122-140). The r.m.s. fit is @51 A. The propionic groups have been removed from the haem groups for clarity. (b) The tertiary structural changes upon ligand binding at the T-state fl haem. Deoxy-T is shown by filled bonds and CO-T by open bonds. As in (a), the residues of the al/j1 interface have been superimposed. The r.m.s. fit is 024 A. The propionic groups have been removed from the haem groups for clarity.

in regulation of the T state ligand affinity of the fi haem. They also point to other possible sources of the low affinity; namely, the steric bulk of the distal

generated by T-state ligand binding with the drive for the quaternary transition.

histidine and the restricted movement of the Fe. The relief of steric hindrance is prevented by restrictions on haem translation, which is determined by the quaternary state. In the reference frame of the al/P1 interface, the FG and CD corners undergo little structural change in the /I-liganded T state (Fig. 4(b)). Residues of these corners make contacts on the distal (Phe CDl) and proximal (Val FG5 and Leu FG3) sides of the haem. As these contacts are part of the al//l2 interface and are dependent on the quaternary state, they may serve to link the strain

We thank the MRC and the Usher Foundation for financial support, and Eleanor Dodson and Zygmunt Derewenda for help in finding the molecular replacement solution. We are grateful to Joyce Baldwin, Cyrus Chothia, Guy Dodson, Hideki Morimoto, Kiyoshi Nagai, Max Perutz and Boaz Shaanan for many stimulating discussions. We dedicate this work to Max Perutz on the occasion of the 20th anniversary of his mechanistic model of haemoglobin allostery. The co-ordinates of (aNi(‘*))2(~Fe(“)CO)Z have been deposited with the Brookhaven protein structure data bank; the deposit code is 1NIH.

14

B. hisi

References Amone, A. & Perutz, M. F. (1974). Nature (London), 249, 34-36. Amone, A., Rogers, P., Blough, N., McGourty, 331L336. Shibayama. N;., Morimoto. H. & Miyazaki. (:. (1986/j). J. Mol. Biol. 192 123-329. Ward, K. B., Wishnerl B. C.. Lattman, E. E:. B Love. W. E. (1975).