J. Mol. Biol. (1975) 98, 179-194

Crystal Structure of Sickle-cell Deoxyhemoglobin at 5 .~ Resolution B. C. WISm~R, K. B. WAI~D~, E. E. I ~ T T ~ $

AND W. E. LOVE

Thomaz C. Jenldns Department of Biophysics Johns Hopkins University Baltimore, Md 21218, U.S.A. (l~eceived 17 March 1975) Crystals of sickle-cell deoxyhemoglobin were grown from solutions containing polyethylene glycol and citrate--phosphate buffer at a pH between 5 and 6. The crystals have the symmetry of the monoclinic space group -P21, with a ~ 63.28 A, b ~ 184.19 A, c ~ 52.84/~, and fl ~ 92-67~ The structure was determined by rotational and translational search procedures. Structure amplitudes and phases were calculated from the atomic co-ordinatss of deoxy Hbw A molecules appropriately positioned in the unit cell of the deoxy Hb S crystal. An $'o -- -~o difference Fourier for the Hb S crystal was computed at 5 .~ resolution. Portions of the Hb S molecules near the Val6fl residues do not appear to be significantly different from the same portions ofdeoxyHb A molecules crystallized in polyethylene glycol solutions at p H 7. I n the Hb S crystal the molecular ~ axes enclose angles of less than 10~ with the crystallographic a axis. The molecules are arranged in pairs of interlocking strands aligned with the a axis. The two strands in each pair are related appro~dmately by a 2-fold screw axis ~mn~ng between them longitudinally. Intsrmolecular contacts within each pair of strands involve Val6fl and other residues that are believed to affect sieldlug interactions. Double strands, slm~lar to those found in the Hb S crystal, can be incorporated into a fiber model that is consistent with available information on the structure of deoxy Hb S fibers i~ v/vo. 1. I n t r o d u c t i o n Sickle-cell hemoglobin, Hbw S, differs from normal h u m a n hemoglobin, H b A, b y a single amino acid substitution at the sixth position from the N-terminus of each fl chain (Ingram, 1957). The glutamate residues normally present at these positions are replaced b y valine residues in H b S. The 6fl residues, in H b A, are the third residues in the A helices of the fl chains and lie on the outside surface of the molecule ('Fermi, personal communication). Erythrocytes from individuals with sickle-cell anemia tend to assume elongated, sickle shapes under reduced oxygen pressure. Pau]ing e~ al. (1949) proposed t h a t siclding might be caused b y the aggregation of deoxy H b S molecules inside ery~hrocytes. Subsequently, Perutz & Mitchison (1950) found t h a t the solubility of deoxy H b S is only 0.02 t h a t of deoxy H b A. Present address: Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, D.C. 20375, U.S.A. :~Present address: Structural Biology Laboratery, Rosenstiel Basic Medical Sciences Researeh Center, Brandeis University, Waltham, Mass. 02154, U.S.A. wAbbreviation used: Hb, hemoglobin. 179

180

B.C. WISHNER ET AL.

Investigations with the electron microscope have shown that deoxy Hb S, both inside erythroeytes and in ceU-free solution, forms rods or tubules with outer diameters between 140 and 170 A (Stetson, 1966; Murayama, 1966; White & Heagan, 1970). Finch et aJ. (1973) lysed sickled erythrocytes directly on electron microscope grids. Their micrographs show what appear to be tubules, each composed of six filaments. The filaments are strings of hemoglobin tetramers with repeat spacings of about 62 A. Each filament is wound around the surface of the tubule with a helical pitch of about 3000 A, and molecules in adjacent filaments are in longitudinal register, so that the tubule may also be viewed as a stack of fiat hexagonal rings. A different model was proposed by Edelstein d al. (1973) who studied deoxy Hb S gels forced through syringe needles into solutions of g]utaraldehyde. Their model is also a tubule composed of six filaments, having a periodicity of about 65 A. Edelstein et a~., however, suggest that the filaments are wound about the tubule with a helical pitch of 2000 ~, and that adjacent filaments are out of register by an axial translation of about 20 A. Hofrichter d a~. (1973) have established, by measurements on the absorption of polarized light by sickled cells, that the angle enclosed between the fiber axis and the molecular z axis (the pseudo-dyad relating ~1 to ill) is less than 23 ~ Although X-ray diffraction patterns have been obtained from oriented gels of deoxy Hb S (Magdoff-Fairchild e~ aL, 1972), they have not yielded additional information on the structure of the Hb S fiber. Solutions of deoxy Hb S, at concentrations above 24 g/100 ml, form viscous gels at 37~ (Bookchin & Nagel, 1971). These gels melt as the temperature is lowered to 0~ Such temperature dependence is consistent with the hypothesis that gelation depends, in part, on hydrophobic bonding, presumably involving Val6~ (Scheraga, 1963; Murayama, 1966). Investigations of the gelation of mixtures of deoxy Hb S with other mutant human hemoglobins have shown that, in addition to Val6~, residues Asp73fl, Glul21~ and Glu23~ might be located in regions of intermolecular contact in Hb S fibers. However, the molecular interactions involved in sickllng have not yet been defined. X-ray dit~action analysis of deoxy Hb S crystals could reveal structural changes caused in deoxy Hb S by the amino acid substitution at position 68, and might suggest possible interactions between molecules in the Hb S fibers. Perutz et al. (1951) crystallized met Hb S by dialyzing the hemoglobin against 2-8 M-phosphate buffer, at pH 6.8, and found those crystals to be isomorphous with crystals of Hb A grown in the same manner. The diffraction patterns obtained from the two crystals were indistinguishable, implying that there is little conformational difference between Hb S and Hb A in the liganded state. Stetson (1966) reported growing needle-shaped crystals of deoxy Hb S in phosphate buffer, but these crystals were too small for X-ray diffraction analysis. 2. Materials a n d M e t h o d s

(a) Hemoglobinlrreparation Blood obtained from patients homozygous for Hb S was supplied by Dr S. Charache (Johns HoptdnR University, School of Medicine). The red blood cells were washed three times with 0.9% I~TaC1,packed by centrifugation, and lysed by the addition of 1 vol. distilled water and 0.4 vol. toluene (Drabkin, 1946). After standing at 3~ overnight, the m~Tture was centrifuged to remove cell debris. The hemoglobin was dialyzed against

DEOXYHE~IOGLOBIN

S CRYSTALS

181

2.0% D r a b k i n ' s (1946) buffer (full strength D r a b k i n ' s buffer contains 283.1 g K 2 H P O 4 a n d 160 g KI~=PO4 per 1), then concentrated b y pressure dialysis to a b o u t 12 g/100 mi, a n d finally reduced with sodium dithionJte (Na=S=O~) a d d e d to t h e H b S solution in 10 mM concentration. The d e o x y H b S solution was stored under nitrogen. (b) GryataZl~z~on Crystals of d e o x y H b S were grown from solutions containing polyethylene glycol of average molecular weight 6000 (VClshner, 1974). A stock solution of polyethylene glycol was p r e p a r e d b y dissolving 50 g polyethylene glycol 6000 (J. T. B a k e r Chemical Co.) in 100 mi water. Crystals were grown in small nitrogen filled tubes, each containing 0.1 ml d e o x y H b S solution, 0.05 mi of 0.2 M-citrate buffer, with a p H between 4 a n d 5, a n d a volume of polyethylene glycol solution t h a t generally comprised between 15 a n d 35% of t h e t o t a l volume of m i x t u r e in t h e tube. All of t h e crystals examlned were twinned, a n d a n y a t t e m p t to cut t h e m resulted in their cracking a n d fragmentation. W~nen soaked in glutaraldehyde solutions, however, t h e crystals became strengthened sufficlently to be cut with a razor blade into large fragments suitable for X - r a y diffraction analysis. Before being crosslln]ced, the crystals were washed in a miYture of 35~o (v/v) polyethylene glycol stock solution, 20~o (v/v) 0.2 ~r-citrate buffer (pH 5.6) a n d 450/0 (v/v) of 2"0~o D r a b k l n ' s buffer. Sodium dithionite was added, in 10 m~-concentration, a n d the solution was k e p t under nitrogen. The measured p H of this solution was 5.9. A f t e r t h e crystals h a d soaked for one day, the t e m p e r a t u r e of the solution was lowered to 3~ a n d g l u t a r a l d e h y d e solution (50% w/w, B a k e r Chemicals) was added, amounting to 2~o of the t o t a l final volume. The crystals were soaked in this miYture a t 3~ overnight. L a t t i c e constants of the d e o x y H b S crystals before a n d after t h e ghitaraldehyde t r e a t m e n t are shown in Table 1. The crystals usually grew in t h e form of oblong plates, u p to 0.5 m m thick. T h e length a n d w i d t h of t h e crystals varied, extending in some cases u p to several rnm. The direction of most extensive crystal growth t e n d e d to be along the a axis of t h e u n i t cell; t h e direction of least growth was along the b axis. (c) X - r a y dam collection and proceaei~g Reflections from g l u t a r a l d e h y d e - t r e a t e d H b S crystals e x t e n d e d to a spacing of a b o u t 3 A. X - r a y diffraction intensities from these crystals were measured out to 6 A resolution on a S y n t e x P21 a u t o m a t e d r ] i ~ a c t o m e t e r , with r a d i a t i o n from a fine-focus Cu tube. A graphite m o n o c h r o m a t o r was used to select Cu K = radiation. Lorentz a n d polarization factors were applied to the intensities. A b s o r p t i o n corrections were m a d e b y t h e empirical m e t h o d of N o r t h e~ o~. (1968). The positions a n d orientations of the molecules in the unit cell of the H b S crystal were d e t e r m i n e d b y the r o t a t i o n a l a n d translational search procedures discussed in t h e preceding p a p e r ( W a r d e~ ~ . , 1975)..& horse deoxyhemoglobin t e t r a m e r was used as t h e t e s t molecule. The H b S r o t a t i o n function was sampled in the angular range 0+ ~ 0 to 180 ~ 0= ---- 90 to 270 ~ a n d 0_ ---- 0 to 360 ~ ( L a t t m a n , 1972). This range covers a n a s y m m e t r l c unit of the H b S r o t a t i o n space group, -PSa2 (Tollln e~ a~., 1966). On a scale from 0 to 100, t h e two largest peaks in t h e m a p h a d values of 100. The n e x t highest p e a k h a d a value of 89, a n d ~there were several other peaks w i t h values below 85. The two highest peaks were accepted as representing the correct alignment of the t e x t molecule with each of t h e two H b S molecules in the asymmetric u n i t of the crystal. Three translation functions were c o m p u t e d to find the vectors from one molecule to each of t h e other three molecules in t h e unit cell. E a c h translation function contained one large peak. Procedures described b y W a r d et ~ . (1975) were used to refine the molecular orientations a n d positions determined from the r o t a t i o n a n d translation functions. A t this p o i n t t h e atomic co-ordinates of h u m a n d e o x y H b A were received from P e r u t z ' s l a b o r a t o r y (Fermi, personal communication). T h e procedure described b y W a r d et a~. (1975) was used to replace t h e horse H b molecules in t h e d e o x y H b S crystal with those of d e o x y H b A. The atomic co-ordinates of t h e deoxy H b A molecules a p p r o p r i a t e l y positioned in t h e H b S unit cell were used to compute structure amplitudes a n d phases to 5 A resolution. A n overall t e m p e r a t u r e factor of 20 A = was used. The calculated intensities, I o, were

L~

c~

,.0

oO

A

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