J. Mol. Biol. (1976) 100, 23-34

Structure of Methemerythrin at 5 .~ Resolution RONALD ]~. STENKAMP, LARRY C. SIEKER, LYLE H. J~.~SE~

Department of Chemistry and Department of Biological Structure University of Washington, Seattle, Wash. 98195, U.S.A. ~_WD J o ~ a ~ S. LO~.HR

Delaartment of Chemistry Portland State University, Portland, Ore. 97207, U.S.A. (Received 11 July 1975) Methemerythrin from Themiste dyscritum crystallizes in the tetragonal system with a ~ b = 86-58(4) A, c ---- 80.84(4} A, space group P4 and two octameric molecules of weight ~108,000 daltons per unit cell. Since the molecules were found to lie on the 4-fold axes, the asymmetric unit is composed of two subtmits from one of the molecules in the unit cell and two from the other one. The electron density map at 5/~ resolution was based on phases calculated from a single mercury iodide derivative. The molecules are seen to resemble square doughnuts of dimensions 60 ~- • 60 A • 40/~ with a square central hole 20/~ on a side and are made up of two layers of four subunits each. Each subunit is approximately 40/~ long and 20 A across with four prominent helical sections of chain extending in the long direction. The sites of the iron pairs have been identified as the regions of highest density in the subunits. 1. I n t r o d u c t i o n Hemerythrin is an oxygen transport protein-found in the erythrocytes of certain marine organisms. The molecule is an octamer of weight ~-~108,000 daltons, each subunit containing two iron atoms and binding one oxygen molecule (Klotz, 1971). Despite its name, hemerythrin has no heine groups, but it resembles hemoglobin in the sense t h a t it has oxy, deoxy, and met forms. Furthermore, a myohemerythrin corresponding to the subunit of hemerythrin is found in the muscle tissue of at least one sipunculid marine worm, Themiste pyroides (Klippenstein ct al., 1972). The structure of myohemerythrin from this organism has recently been reported at 5.5 A resolution (Hendrickson ct al., 1975). We report here the results of a 5 A resolution X - r a y study of the structure of hemerythrin from the sipunculid Themiste

dyscritum. 2. E x p e r i m e n t a l The method of crystallization and preliminary data for the native crystals have been reported (Loehr et al., 1975). A satisfactory isomorphous derivative was obtained by soaking crystals in 0.001 M-K2HgI4 for 24 days. Precession photographs of the derivative crystals showed relatively large intensity changes when compared to the corresponding

23

24

R.E.

STENKAMP ET AL.

TABLE 1

Crystal Data Native crystals a ( : b) c

86.58(4) A 80.84(4)

V

606,000 .~a

Space group

-P4

Derivative crystals 86.68(4) A 81.11(5) 609,400 A a P4

Cell parameters based on ~c~Ka = 1'5418 A.

photographs of the native crystals, indicating substantial binding of the h e a v y atoms to the protein. Crystal data for the native and derivative crystals appear in Table 1. Diffraction data to 5 /~ resolution were collected on a computer-controlled, 4-circle diffractometer. B o t h reflections of each Friedel pair were collected, 7467 reflections from a crystal of the native protein, including m a n y symmetry-related ones, and 6049 reflections from a derivative crystal. The method used was a 5-step scan bracketing the peak m ax i m a, each step being 0.1 ~ in 28 and counted for 4 s. Backgrotmds were assumed to be a function of 28 only and were measured at intervals between pairs of reciprocal lattice rows. B y this technique over 3000 reflections can be collected per day. Intensities were obtained by fitting the 5-step d a t a for each reflection with a Gaussian curve (Hanson e/al., 1973). T h e y were corrected for absorption and deterioration, and the usual Lorentz-polarization correction was applied. A modification of the m e t h o d of Singh & Ramaseshan (1966) was used to scale the derivative to the native data, the scale factor being 1.26 exp -- (27 sin28/a2). The relatively large positive B value indicates t h a t the derivative intensities fall off less rapidly than the native ones with increasing sin 0/h. F o r d a t a limited to 5 A resolution, however, B cannot be precisely determined, and it should be regarded only as an adjustable constant. Coefficients (f) representing the structure amplitudes of the h e a v y atoms were derived as suggested by Matthews (1966a). F r o m our data the value of k in his formulation was determined as 10.0 and w was arbitrarily taken as 1.0. Solution of the P at t er so n function calculated with these h e a v y - a t o m coefficients and subsequent difference maps led to a 6-site model which refined by least-squares to R = 0.31. R is defined by the expression ~ I f -- f e t / ~ f where f is the observed h e a v y - a t o m coefficient a n d f c is the calculated value. Refinement was based on 2439 of the 2993 possible coefficients; reflections with I < 2o(I) and sin 8/A < 0.0125 were given zero weight. I n the model for the h e a v y atoms, H g was assmned to represent the merem'y iodide complexes since the H g and I atoms separated by a distance of ~ 2 . 7 / ~ will not be resolved by 5 A data. Table 2 lists the parameters of the H g atoms. The sites are equally occupied and the large anisotropie " t h e r m a l " parameters can be ascribed to the use of H g atoms to fit w h a t appear to be HgIz or H g I a - groups. The h e a v y a t o m model was used with the Bi j v o et differences of the mercury iodide derivative to determine phases for n~tive structure factors (North, 1965; Matthews, 1966b). The root-mean-square isomorphous lack of closure and the root-mean-square anomalous lack of closure errors for the phase determination were 26-0 and 14.0, respectively. The average isomorphous and anomalous h e a v y a t o m signals were 192.6 and 39.0, respectively, on the same relative scale. The overall figure of merit for the 5 A resolution d a t a was 0-82. Two electron density maps were calculated corresponding to the two interpretations of the difference Patterson function (Blow & Rossmann, 1961), and the cmu'ect enantiomer was chosen as the one showing an essentially continuous polypeptide chain. A Fourier map with Bijvoet differences from the native crystal as coefficients and appropriately modified

STRUCTURE

OF METHEMERYTHRIN

25

phases was calculated, t h e 4 largest p o s i t i v e p e a k s coinciding w i t h 4 h i g h - d e n s i t y regions in t h e electron d e n s i t y m a p (Strahs & K r a u t , 1968).

3. Description o f the Structure The two molecules in the unit cell lie on the 4-fold axes in space group P4. One molecule is located at 0,0,z and designated I; the other is at 1/2, 1/2, z A- ~-~1/2 and designated II. In addition to the translation relating the two independent molecules,

f

FIG. 1. Schematic diagram of packing of the subunits in hemerythrin octamcr.

FIe. 2. View of balsa wood model of hemerythrin molecule showing the central cavity. Direction of view at a small angle to the 4-fold axis. Arrows show positions of non-crystallographic 2-fold axes through the sides and corners of the molecule.

~2

% 6

6 I

6 I

6 I

6

6

+

% I

I

I

I

+ o

6

6

o

6

I

6 I

+

I....1

.~

o

6

o

~

6

o

6

+ 6

+ 0

~• L~

II

STRUCTURE

OF

METHEMERYTHRIN

9.7

one is rotated by approximately 20 ~ about the 4-fold axis relative to the other. The arrangement of the molecules is roughly body-centered. The eight subunits in each molecule are related b y approximate s y m m e t r y D~ (422) (Figs 1, 2 and 3). The 4-fold axes of the molecules are exact, coinciding with the 4-fold axes Of the space group, but the 2-fold axes are non-crystallographic. Thus two subunits in each molecule are crystallographically independent, and the asymmetric unit consists of four subunits, two from each molecule. The molecules are made up of two layers, each formed b y four subunits related by the 4-fold axes. Figure 4 shows stereo diagrams of the electron densities in the two layers of molecule I. Figure 4(a) shows subunit I a with its 4-fold related subunits forming the layer with greater z co-ordinates (the top of the molecule when viewed down the z axis). Snbunit I b and its symmetry-related subunits form the lower layer of this molecule (Fig. 4). Figure 5 shows similar diagrams for molecule I I ~ith subunits I I a and IIb. The subunits in each layer arrange themselves to form a square hole ~ 2 0 A on a side. Superposing the layer in Figure 4(a) on that in Figure 4(b) (and likewise for the layers in Fig. 5(a) and (b)) shows the molecule to resemble a square doughnut. The four subunits in one layer of a molecule are inverted with respect to the four subunits in the other layer of the same molecule, being related b y the non-crystallographic 2-fold axes. I f this were not so, formation of higher oligomers would be possible.

:Fra. 3. View of the balsa wood model of the hemerythrin molecule with one subunit removed exposing heavy atom sites within the central cavity. Three independent heavy-atom sites on one oetamer are indicated: 2 on the wall of the inner cavity by (. . . . . >}; one on noncrystallographic 2-fold axis by (........ > ). Sites of iron pairs visible in this view are indicated by ( >).

28

R. E. S T E N K A M P E T A L .

(0)

X

(b)

x.-..,,.

.,,-w-

Fie. 4. (a) Stereo view of electron density in subunits of layer Ia; sections in z from 52/50 to 67/50, x and y from --1/2 to 1/2. (b) Stereo view of layer Ib; sections in z from 38/50 to 51/50, x and y from --1/2 to 1/2.

Inspecting the superposed layers of subunits in Figures 4 and 5 reveals the relation among the subunits in the octamer, l~or a n y one subunit there are three different types of possible interaction regions with other subunits, one with subunits in the same layer and two where the subunit overlaps two others in the adjacent layer. The n u m b e r of different types of such regions corresponds to the square antiprism arrangement described b y Klotz for packing spheres (Klotz, 1971). The subunits are not spherical, however, and the description of their arrangement in terms of a cube or antiprism will depend on the point of reference in the subunit. The contours in the m a p appear to be more dense toward one end of the subunits, and the midpoints of these dense regions approximate the vertices of a cube as deduced b y North & Stubbs (1974) from the diffraction p a t t e r n of crystals of hemerythrin from Golfingia gouldii. The subunits are roughly cylindrical in shape, ~ 2 0 / ~ in diameter and ~ 4 0 / ~ long. The four independent subunits appear to be identical at the present resolution,

STRUCTURE

OF METHEMERYTHRIN

29

t (I))

X~

T-1

t (b)

x --~

F r o . 5. (a) Stereo v i e w o f e l e c t r o n d e n s i t y in s u b u n i t s of l a y e r I I a ; s e c t i o n s in z f r o m 26/50 to 41/50, x a n d y f r o m 0 to 1. (b) Stereo view of l a y e r I I b ; s e c t i o n s in z f r o m 14/50 to 26/50, x a n d yfrom0t0 1.

and a composite view is similar to the myohemerythrin structure (Hendrickson et al., 1975). Figure 6 shows a stereo view of the four independent subunits. The quality of the electron density map and the repetitive views of the subunits are sufficient to enable one to follow the course of the main chain. Four prominent columns of electron density in each subunit correspond in diameter to ~-helices. The four helical sections are roughly parallel, extending in the long direction of the subunit. Measurement of the length of the helical sections in one of the subunits corresponds roughly to 80 amino acids indicating --~71% helix, if one assumes 113 amino acids in the subunit. This compares well with the value of 75% found b y Klotz (1971). In addition to sections of chain connecting the helices, a piece of electron density corresponding to ~-~20 amino acids lies on the external side of the subunits opposite the 4-fold axis. In subunits Ia, Ib, and IIb this piece appears to be disconnected from

(0)

(b)

(c)

.IO

(d)

F~a. 6. ((a) to (d)). Stereo views of subunits in layers Ia, Ib, I I a a n d I I b , respectively. Contour levels a t a r b i t r a r y equal intervals of electron density. Arrows indicate the sites of iron pairs in each subunit. Highest contours have been omitted from the map.

STRUCTURE OF METHEMERYTHRIN

31

FIo. 7, Drawing of h e m c r y t h r i n subunit.

the rest of the electron density, but in IIa it is weakly connected to a helix giving a structure similar to that found for myohemerythrin (Hendrickson et al., 1975). Accordingly, the notation of regions in that molecule, namely N-terminal peptide, A helix, A-B turn, B helix, B-C turn, C helix, C-D turn, X) helix will be adopted. Figure 7 is a drawing showing the current interpretation of the electron density in the subunit of hemerythrin. One feature of the biochemistry of hemerythrin best observed in an electron density map is the nature of the region of possible contact between the subunits within the octamer, but only preliminary results can be deduced at the present resolution. Within each layer, weak electron density connects the ends of one subunit and the side of an adjacent one. The major connections between layers are between the N-terminal peptide and the A and B helices in one layer with the same areas related by the non-crystallographic 2-fold axes. Figure 8 is a schematic diagram of the criss-cross arrangement of the A and B helices in adjacent layers. The resolution of the present map is too low, however,

~

lii~

'

~~

ilix\'

Nt(~r~~i-nol pep/ido 2- fold oxis F1G 8. Schematic diagram showing one region of c o n t a c t between subunits. The non-crystallographic 2-fold axis is t h e diagonal one t h r o u g h t h e corners of t h e molecule.

32

R. E. STENKAMP

ET

AL.

to identify the specific residues involved in the binding. No commctions between the layers are seen in the region of the other non-crystallographic 2-fold axes. The site of the two iron atoms in each subunit has been confirmed by a Bijvoetdifference Fourier map (Strahs & Kraut, 1968). The four largest peaks in the map corresponding to the four pairs of Fe atoms in the four independent subunits and coinciding with the highest peaks in the electron density map are indicated by arrows in Figure 6. Although the electron density in the native protein map is somewhat greater at the iron sites than in the helical regions, it was less than certain that these peaks represented the iron pairs in view of the limited resolution of the map. Thus the Bijvoet map was important in confirming these peaks as the iron sites. The iron peaks are slightly elongated in the z direction, suggesting this as the orientation of the F e - F e vectors. The iron pairs are located at the corners of a square antiprism, the distances between adjacent pairs in any given layer of subunits being ,~30 •, very nearly the same as the distances between closest pairs in different layers of the same molecule. Table 3 lists the co-ordinates of the iron pairs. Several openings can be found leading from the iron pairs to the solvent. Some of these openings m a y be filled by amino acid side-chains which are not seen at this resolution. In myohemerythrin the iron ligands have been tentatively identified as His25, His54, Tyr67, His73, Hisl06, and T y r l l 4 (Hendrickson et al., 1975). Connections between the iron pairs and the helices in our map (Table 4) correspond to some of TABLE 3

Co-ordinates of iron pairs

Fe Fe Fe Fe

1 2 3 4

x

y

z

Subunit

0"04 0-21 0.26 0-34

0.24 0.12 0-45 0-31

0.20 0-92 0.69 0.41

]a ]b IIa lib

TABLE 4

Protein-iron pair connections Subunit

h ' o n pair

la

Fe 1

Ib

Fe 2

IIa

Fe 3

IIb

Fe 4

D i s t a n c e fl,o m amino-terminal e n d of helix

Helix

1/4

A

3/5

B

1/2 3/4 I/2 1/4 2/3 I/2 1/4

C D B C D B C

2/3

D

1/2

B

]13 112

D

c'

STRUCTURE

OF METHEMERYTHRIN

33

these assignments, but we do not see electron density connections for all of t h e m at the present contour levels.

4. The H e a v y A t o m s The locations of the h e a v y atoms used to determine the structure confirm the reality of the hole in the octamer and t h a t it is accessible to the solvent and not an artifact of the phasing process. Figures 2 and 3 show the location of the h e a v y - a t o m sites. Four of the six independent h e a v y atoms are bound to the four independent subunits on the sides forming the walls of the hole. The 4-fold axes operating on these four sites, labeled Hg 1, 2, 4 and 5 in Table 2, generate 16 of the 24 sites in the unit cell. The remaining atoms, Hg 3 and 6 are located at the interface region between the subunit layers, one on each molecule. They are found on the outside corners of the molecules and lie on the 2-fold axes passing through the corner edges of the molecule. The 4-fold axes operating on these sites generate the remaining eight sites in the unit cell. Figure 9 is an O R T E P plot (Johnson, 1965) of the " t h e r m a l " ellipsoids of the h e a v y atoms. Presumably the anisotropy is caused b y fitting H g atoms to what are in reality mercury iodide complexes. I t is interesting t h a t H g 1, 2, 4 and 5, the sites bound to similar regions of the subunits, are all prolate ellipsoids, suggesting those sites to be HgI2; and the oblate ellipsoids of H g 3 and 6 suggest planar H g I 3-, but these interpretations are highly uncertain in view of the low resolution of the data. I t is also interesting t h a t the 20 ~ rotation of molecule I relative to molecule I I can be deduced from the orientations of Hg 1, 2, 4 and 5. These h e a v y atoms, which are located on the walls of the inner cavity, are midway along the B helix. The other h e a v y - a t o m sites are located between the two N-terminal peptides in the two layers of each molecule. D a t a for the native and derivative crystals have been collected to 2.8 A resolution and a more detailed analysis will be undertaken. The sequence determination for T. dyscritum is in progress and this will greatly facilitate the interpretation of the electron density m a p at higher resolution.

Fro. 9. "Thermal" ellipsoid plot of Hg atoms. Direction of view along --z.

3

34

R.E.

STENKAMP ET AL.

This work was supported under U n i t e d States Public H e a l t h Service g r a n t AM-3288 from the National Institutes of H e a l t h and is in partial fulfillment for the Ph.D. degree of one of the authors (R.E.S.). REFERENCES Blow, D. M. & Rossmann, M. G. (1961). Acta Crystallogr. 14, 1195-1202. Hanson, J. C., W a t e n p a u g h , K. D., Sieker, L. C. & Jensen, L. H. (1973). Amer. Crystallogr. Assn. Abstr. p. 55, University of Florida, Gainesville. Hendrickson, W. A., Klippenstein, G. L. & W a r d , K. B. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 2160-2164. Johnson, K. C. (1965). Oak Ridge National L a b o r a t o r y R e p o r t ORNL-3794. Klippenstein, G. L., van Riper, D. A. & Oosterom, E. A. (1972). J. Biol. Chem. 27, 59595963. Klotz, I. M. (1971). Biol. Macromolecules (Timasheff, S. N. & F a s m a n , G. D., eds), voL 5, pp. 55-103, M. Dekker, Inc., New York. Loehr, J. S., Meyerhoff, K. N., Sieker, L. C. & Jensen, L. H. (1975). J. -~Iol. Biol. 91, 521-522. Matthews, B. W. (1966a). Acta Crystallogr. 2@, 230-239. Matthews, B. W. (1966b). Acta Crystallogr. 20, 82-86. North, A. C. T. (1965). Acta CrystaUogr. 18, 212-216. North, A. C. T. & Stubbs, G. J. (1974). J. Mol. Biol. 88, 125-131. Singh, A. K. & Ramaseshan, S. (1966). Acta Grystallogr. 21, 279-280. Strahs, G. & K r a u t , J. (1968). J. Mol. Biol. 35, 503-512.