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Low resolution crystal structure of hagfish insulin
J. Mol. Biol. (1974) 87, 23-30
Low Resolution Crystal Structure of Hagfish Insulin J. F. CUTFIELD, S. M. CUTFIELD, E. J. DODSON, G. G. DODSON AND M. N. SABESAN Laborntory of Molecular Biophysics Zoology Department South Parks Road Oxford OX1 3PS, England (Received 11 March 1974) Insulin from the Atlantic ha&h, Myxine glutino8a, crystallizes in space group P4,2,2 with a monomer in the asymmetric unit. The application of the Rossmann & Blow (1962) rotation function, utilizing the known 2-zinc pig insulin
crystal structure, has established the existence of an insulin climer containing a crystallographic
2-fold
axis. The position
of the hagfIsh
insulin
molecule
in the
unit cell has been determined and a set of calculated phases derived. These are compared to phases found from isomorphous replacement studies. A 6 A resolution electron density map has been calculated which shows the A and B chains are folded in a similar way to pig insulin and that the monomers are similarly organized into dimers.
1. Introduction Our attempts to relate the three-dimensional crystal structure of insulin to its biological properties have involved structural studies of different insulin species and crystalline forms. In most of those we have examined, the insulin occurs as a hexamer and the relations between the crystals can, to a first approximation, be defined by the use of the rotation function (Rossmann & Blow, 1962). Insulin from the hagfish, however, presented a different problem because in the crystal form obtained there is no hexameric aggregation. The hagfish together with the lamprey, are the sole extant representatives of the Agnatha (jawless fishes) and are generally referred to as cyclostomes. The insulinproducing apparatus of this primitive creature possesses characteristics of both vertebrates and invertebrates (Falkmer et al., 1973; Steiner et al., 1973). The biological activity of insulin from Myxine glutinosa (the Atlantic hagfish) is reported to be about 8% of mammalian insulin (Weitzel et al., 1967) and the amino acid sequence (Emdin, Peterson, Coulter, Ostberg, Falkmer & Steiner, 9th Internat. Gong. B&hem., Stockholm, l-7 July 1973) indicates changes in more than a third of the pig insulin molecule, though the residues believed to be essential for conserving the basic structure appear to be present. Significantly, one of these changes is the substitution of BlO histidine by aspartic acid which does not allow the formation of zinc-containing hexamers. Hagfish insdin CryetaIlizes in the absence of zinc as tetragonal bipyramids, space group P412,2 (or enantiomorph), with cell dimensions a = 38.4 A, c = 85.3 A, in agreement with Emdin et al. (9th Intern&. Gong. Biochem. Stockholm, 1973). If we assume a monomer (mol. wt approx. 6000) in the asymmetric 23
J. F. CUTFIELD
24
ET
AL.
unit the solvent content is found to be approximately 54%, dthin the range commonly observed for most proteins (Matthews, 1968). The crystal structure of rhombohedral a-zinc pig insulin has been analysed at high resolution (Blunclell et al., 19’71,1972) and a complete set of atomic co-ordinates derived. In this form the insulin is organized as s, compact hexameric unit made up of three equivalent dimers which co-ordinate through their BlO histidine residues to two zinc atoms. A non-crystallographic 2-fold axis perpendicular to the crystallographic 3-fold axis relates the two monomers in the climer (see Fig. l(a)). In hagfish insulin
b
(b)
of insulin molecules in the crystals of pig insulin (a) and FIG. 1. A schematic representation hagfish insulin (b). (a) Represents the 2-zinc pig insulin hexamer in space group R3, with the dashed lines representing non-crystallographic %-fold axes which relate monomers in the dimer. (Similar $-fold axes between the dimers are not shown.) (b) Shows part of the arrangement in the hagfIsh insulin crystal structure, space group P4i2i2 or P4,212. A crystallographio 2-fold axis relates two insulin molecules, which may or may not form a dimer. The Eulerian angle 0s refers to rotation ebout this axis.
the space group requires two monomers to be related by a crystallographic 2-fold axis (see Fig. l(b)) which offers the possibility that their organization with respect to this did is similar to that found in the pig insulin dimer. If this were the case then the known pig insulin monomer would have the same orientation as the hagfish monomer in its cell after rotations through 8, = - 104”, an arbitrary 8,, and 8, = 45”, where B,, 0,, d3 are Eulerian angles as defined by Rossmann $ Blow (1962).
TABLE
1
Prdimina y cystd Species
Spaoe group
Pig HugfIsh
R3 P412,2 or P422,2
Asymmetrio Dimer Monomer
da.ta unit
Aggregation
state
Hexamer Monomer or dimer
STRUCTURE
OF HAGFISH
INSULIN
“5
Table 1 summarizes crystal data for the two insulin species under comparison as known at the start of the investigation. We considered two separate approaches to the problem of determining the crystal structure of hagfish insulin. The Grst involved the application of the rotationtranslation functions using the pig insulin monomer and dimer, respectively, as search units. Here we used our knowledge of the crystal structure of rhombohedral pig insulin by calculating the appropriate subunit transforms from the atomic coordinates. The second approach was the conventional isomorphous heavy atom replacement method, treating hagfish insulin as an independent crystal study.
2. Rotation Function By effectively rotating the Patterson function of one unit on to the Patterson function of a structurally similar unit the rotation function determines the orientation which brings about maximum overlap. As pig and hagfish insulins have different aggregation properties, it was necessary to calculate Patterson functions for the isolated pig insulin search units. This was achieved by placing the pig insulin monomer and dimer, respectively, in suitable triclinic cells, then calculating their transforms. The atomic co-ordinates used were derived from a 2.8 A resolution analysis of rhombohedral 2-zinc pig insulin (Blundell et al., 1971). The backbone conformations at the terminal regions A21, B27-30 and Bl-5 were either poorly defined in the pig insulin structure or influenced by its hexameric aggregation. Thus, in considering 41 out of 51 residues in the pig insulin molecule, we used the co-ordinates of 208 atoms (out of a possible 403), comprising backbone, p-carbon and disulphide bridge atoms. No side-chain atoms were included because the hagfish insulin sequence was incomplete at the time of the investigation and their conformations anyway are uncertain given the different aggregation states. The set of atomic co-ordinates chosen was that of molecule 1 in the pig insulin dimer (Blundell et al., 1971) and for the dimer search unit an exact 2-fold axis was assumed, reproducing molecule 1. A set of native hagfish insulin data was collected to a resolution of 45 A from one small crystal (O-35 mm maximum dimension) using a Hilger Watt PDPS controlled four circle diffractometer. After the application of Lorentz-polarization and absorption (North et al., 1968) corrections 1301 measurements were combined giving 477 independent reflections with a merge index R, of 0.054, where
(a) Monomer search The “reduced” pig insulin monomer (defined above), an object approximately 30 A x 20 A x 15 A, was placed in a triclinic cell, 60 A x 40 A x 40 A (all angles 90”) and structure factors calculated. An overall temperature factor, B, of 12 A2 was assumed. We considered the rotation of the hagfish insulin Patterson function on to the Patterson function of this pig insulin monomer over a sphere of radius 15 A. The required asymmetric unit in Eulerian space of 0 < d1 < ~12, 0 < 62 _< 7r, 0 5 0, < r was sampled at 15” intervals. The peaks were further explored at 5” intervals. The triclinic unit cell dimensions and the radius of the sphere in the rotation function were chosen so that only intramolecular vectors would be present. In order
26
J. F. CUTFIELD
to save computing time only about 20% of the data, were The interpretation of the there were a large number strongest peaks corresponded
ET
AL.
the stronger reflections between 5 A and 10 A spacing, included in the calculations. rotation function results was not straightforward, as of peaks significantly above background. The three to the following orientations for hagfish insulin:
(i) 8, = 0” (ii) 13,= 50” (iii) e1 = 80”
O2= 160’ 8, = 155” 0, = 155”
8, = 90” e3 = 90” e3 = 120”
R = 430; R = 438; R = 428,
where the rotation function background level was R = 404+3. Using the equivalent position relationship R(8, Ba6,) = R(--8, 7~--t t$ t?,) (Tollin et al., 1966) and remembering that R(B, tY20,) is the reverse of R(--8, -0, -0,) we can see that solution (ii) is equivalent to a “pig on hagfish” rotation of 0, = -9O”, e2 = 25’, e3 = 50”. The simple case visualized from Figure 1 suggested 8, = -104”, 8, arbitrary, e3 = 45”. Thus, it seemed plausible that the local a-fold axis in the pig insulin dimer had indeed become a crystallographic 2-fold axis in hagfish insulin, with a rotation of 25” about this diad. For this reason, we decided to test for the existence of a hagfish insulin dimer by using the pig insulin dimer as search unit. (b) Dimer search A pig insulin dimer, as described above, was placed in a suitably large triclinic cell and the transform calculated. The spherical radius of the rotation function was set to 25 A. The requirement that the dimer axis should be along the 2-fold axis of P4,2,2 (or P4,2,2) means that e1 and t$ are fixed, so there is only one degree of freedom, i.e. rotation of t$, about the 2-fold axis. Results are shown in Figure 2, the dominant peak corresponding to &=30°, very close to the value of 25” given by the monomer solution.
ro
3603 0
40
SO %
120
160
FIG. 2. A plot of the dimer rotation fun&ion against & in degrees. The peak at & I mines the orient&ion of the dimer about the Z-fold axis.
313~deter-
3. Translation Function It now seemed likely that hagfish insulin formed a dimer similar to that of pig insulin. It remained to determine the one translation parameter which would define its centre along the crystallographic a-fold axis. This was achieved by a one-dimen-
STRUCTURE
OF HAGFISH
INSULIN
27
65-
FIG. 3. R-factor search of the correctly oriented pig insulin dimer along the crystallogrsphic ) and P43212 (----). Two data sets were used, co + 10 A 2-fold axis of P41212 ( (upper) and 10 +6 A (lower). The search need only be mede to the midpoint of the e-fold axis, a length of 27 A.
R-factor search, moving the centre of the now correctly oriented dimer along the S-fold axis in 2 il steps and calculating structure factors. The same set of atomic co-ordinates employed in the rotation function was used in these calculations. The search, which must be carried out in both P4,2,2 and P4,2,2 was done using two separate data sets, co -+ 10 a and lo+6 8. The results are shown in Figure 3. The minimum R factor (R = 21 l$‘ObSl- 1Ii’Coalol l/CIB’O,,Sl) corresponds to a translation of 5 A from the origin of the P4,2,2 unit cell along the 2-fold axis. The sharpness of the minimum provides satisfying confirmation of a pig insulin-like dimer and as well clearly resolves the space group ambiguity. We were thus reasonably confident that we had established the position of the hagfish insulin molecule in the tetragonal unit cell and felt that the best way to confirm these results would be by determining heavy atom positions in isomorphous derivatives using phases calculated from the appropriately transformed atomic coordinates of pig insulin. These could then be compared to positions found from the more conventional Patterson and “direct methods” approaches. sional
J. F. CUTFIELD
28
ET
AL.
4. Isomorphous Replacement Studies Heavy atom soaking experiments on a limited number of small crystals (0.3 mm maximum dimension) yielded good intensity changes for Pb’ + , AuCI, -, PtClk2 - and UOz2 + . We decided to collect diffraction data from the lead and uranyl derivatives as these showed minimal changes in cell constants. They were prepared under the following conditions : (i) 0.01 M-lead acetate, 0.05 M-sodium acetate buffer, pH 6.0, 24-hour soak; (ii) 09075
M-Uranyl
acetate, 0.05 M-sodium citrate buffer, pH 6.0, 20-hour soak.
Unfortunately, the data did not extend much beyond 6 A spacing and anomalous dispersion differences were considered too weak to be useful. Two symmetry related equivalents were measured for each derivative giving R, figures for the merged 6 A data sets (210 independent reflections) of O-068 (lead) and 0,059 (uranyl). In addition, centric data to 4.5 A spacing were collected for the lead derivative. Both isomorphous difference Patterson and AE syntheses (K. Wilson, personal communication; Germain et al., 1971) established major binding sites for the lead and uranyl ions in the two derivatives, though the correct hand of these solutions can not be distinguished by either method. Difference Fourier syntheses employing phases calculated from the transformed atomic co-ordinates of 2-zinc pig insulin showed the same major sites, and also resolved the problem of hand. Refinement was carried out using centric data (representing 60% of the 6 A sphere) and several minor sites were detected in the difference maps. However, they were barely significant after refinement. A set of “best” phases was calculated according to the method of Blow % Crick (1959), and a 6 A electron density map computed. The overall figure of merit was 0.73. It should be noted that the 101 reflection, which is the largest, had very small heavy atom contributions in both derivatives. This gave it a low figure of merit and a phase differing by m from the calculated value. Since the calculated amplitude was also very large for this reflection it seemed the isomorphous phase was probably wrong. Changing its sign and increasing its weight noticeably improved the appearance of the electron density map.
5. Discussion Figure 4 shows part of this isomorphously phased map with, superimposed, the pig insulin a-carbon skeleton positioned by the rotation-translation functions. As expected the overall organization of the dimer appears similar to that observed in a-zinc pig insulin but there are significant differences in regions involving both ends of the B chain. It is interesting to inspect the measure of agreement between the phases estimated from isomorphous replacement and those calculated from the pig monomer backbone. We considered centric and non-centric data separately and the results are summarized in Table 2. The number of reflections is rather small but, as expected, there is better agreement for those with high figure of merit or high Sim weight. A comparison of the three-dimensional crystal structures of hag&h and pig insulins may shed some light on the reason for their different biological activities. It may also allow us to decide why a local S-fold axis, as in pig insulin, becomes a crystallographic Z-fold axis in hagfish insulin. We would like to know if it can be
STRUCTUR.3.Z
OF HACFISH
INSULIN
29
FIG. 4. An 8 A thick slab of the 6 A isomorphously phased hagf%sh insulin eleotron density map viewed &long c. The projection is made up of sect,ions z = $2c/40 to -22c/40, The diagonal E-fold axis is contained in the z = 0 plane. Appxoprista a-carbon positions of the pig insulin molecule are suparimpoF,ed, namely BlZ-B26 (solid backbone) and AZO-A21 (hollow), Parts of nsighbouring molecules have not been drawn in for the sake of clarity.
TABLET Agreement un&?ysis of calcu&ed and experimental phases Centria
Non-cent&3 Mean discrepancy
Range
No. in zone
Fraction agreeing
No. in zone
4 sin2 S/h2 i: 0.02 > 0.02
86 34
0.69 0.66
60 40
73” 12”
0.0 0.2 0.4 04 0.8
< < < < <
7%5 o-2 7n < 0.4 ?n ( 0.6 m _< 0.8 m 5 1.0
I 12 6 14 81
0.71 O-42 0.67 O-67 o-73
12 22 8 30 18
109” 61” 91” 11” 47”
o,o 0.2 0.4 0.6 0.8
< < < < <
s s S S s
11 18 19 24 48
0.54 0.44 0.63 0.67 0.81
0 8 10 17 66
82” 71” 8””M 68”
120
0.68
90
73”
2 0.2 < 04 _< 0.6 I< 04 2 1-o
Tots1
Here rn is the figure of merit and S is a Sim-type (Sim, 1969) as I,(X),
where X = 21Fo\lF,l/ifa(i)
weight. and xf”(
The Sim weight
has been defined
z‘) 10 * p resents the scattering
contri-
bution from those atoms omitted from the a&&&ion. It ia well known that for low resolution protein data Wilson at&i&es are not obeyed and zf2 is not B good represent&ion of the observed scattering. We examined the distribution of
J. F. CUTFIELD
30
1T
AL.
explained, for example, in terms of sequence changes away from the monomermonomer interface region (there are very few amino acid substitutions in this region), as a result of being in a lower aggregation state caused by the absence of Zn2+, by crystal packing effects, or, perhaps we are seeing an average of two different orientations. These questions remain unanswered until we have a high resolution structure. We are indebted to Dr Stefan Emdin, Professor Sture Falkmer, Professor Don Steiner and colleagues for giving us a sample of crystalline hagfish insulin. Our grateful thanks are due to the Kristineberg Zoological Station, Fiskebilckskil, Sweden. This work was supported by the Royal Society, British Diabetes Association and Science Research Council. One of us (J. F. C.) was the recipient of an ICI Fellowship. We thank Professor D. C. Hodgkin of this paper.
for valuable
discussions during the preparation
REFERENCES Blow, D. M. & Crick, F. H. C. (1959). Acta Crysfollogr. 12, 794-802. Blundell, T. L., Cutfield, J. F., Cutfield, S. M., Dodson, E. J., Dodson, G. G., Hodgkin, D. C., Mercola, D. A. & Vijayan, M. (1971). Nature (London), 221, 506-511. Blundell, T., Dodson, G., Hodgkin, D. & Mercola, D. (1972). A&. Prot. Chem. 26, 279402.
Falkmer, S., Emdin, S., Havu, N., Lundgren, G., Marques, M., Ostberg, Y., Steiner, D. F. & Thomas, N. W. (1973). Amer. ZooE. 18, 626-638. Germain, G., Main, P. & Woolfson, M. M. (1971). Acta Cry&aUogr. ser. A, 27, 368-376. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. North, A. C. T., Phillips, D. C. & Mathews, F. S. (1968). Actu Crystdlogr. 8ep. A, 24, 351359. Rossmann, M. G. & Blow, D. M. (1962). Acta Crydullogr. 15, 24-31. Sim, G. A. (1969). Acta CrydaZZogr. 12, 813-816. Steiner, D. F., Peterson, J. D., Tager, H., Emdin, S., Ostberg, Y. & Falkmer, S. (1973). Amer. 2001. 13, 591-604. Tollin, P., Main, P. & Rossmann, M. G. (1966). Acta CryduZZogr. 20, 404-407. Weitzel, G., Stratling, W.-H., Hahn, J. & Martini, 0. (1967). Hoppe-SeyZer’s Zeit. Physiol. Chem. 348, 525-532.
Low Resolution Crystal Structure of Hagfish Insulin J. F. CUTFIELD, S. M. CUTFIELD, E. J. DODSON, G. G. DODSON AND M. N. SABESAN Laborntory of Molecular Biophysics Zoology Department South Parks Road Oxford OX1 3PS, England (Received 11 March 1974) Insulin from the Atlantic ha&h, Myxine glutino8a, crystallizes in space group P4,2,2 with a monomer in the asymmetric unit. The application of the Rossmann & Blow (1962) rotation function, utilizing the known 2-zinc pig insulin
crystal structure, has established the existence of an insulin climer containing a crystallographic
2-fold
axis. The position
of the hagfIsh
insulin
molecule
in the
unit cell has been determined and a set of calculated phases derived. These are compared to phases found from isomorphous replacement studies. A 6 A resolution electron density map has been calculated which shows the A and B chains are folded in a similar way to pig insulin and that the monomers are similarly organized into dimers.
1. Introduction Our attempts to relate the three-dimensional crystal structure of insulin to its biological properties have involved structural studies of different insulin species and crystalline forms. In most of those we have examined, the insulin occurs as a hexamer and the relations between the crystals can, to a first approximation, be defined by the use of the rotation function (Rossmann & Blow, 1962). Insulin from the hagfish, however, presented a different problem because in the crystal form obtained there is no hexameric aggregation. The hagfish together with the lamprey, are the sole extant representatives of the Agnatha (jawless fishes) and are generally referred to as cyclostomes. The insulinproducing apparatus of this primitive creature possesses characteristics of both vertebrates and invertebrates (Falkmer et al., 1973; Steiner et al., 1973). The biological activity of insulin from Myxine glutinosa (the Atlantic hagfish) is reported to be about 8% of mammalian insulin (Weitzel et al., 1967) and the amino acid sequence (Emdin, Peterson, Coulter, Ostberg, Falkmer & Steiner, 9th Internat. Gong. B&hem., Stockholm, l-7 July 1973) indicates changes in more than a third of the pig insulin molecule, though the residues believed to be essential for conserving the basic structure appear to be present. Significantly, one of these changes is the substitution of BlO histidine by aspartic acid which does not allow the formation of zinc-containing hexamers. Hagfish insdin CryetaIlizes in the absence of zinc as tetragonal bipyramids, space group P412,2 (or enantiomorph), with cell dimensions a = 38.4 A, c = 85.3 A, in agreement with Emdin et al. (9th Intern&. Gong. Biochem. Stockholm, 1973). If we assume a monomer (mol. wt approx. 6000) in the asymmetric 23
J. F. CUTFIELD
24
ET
AL.
unit the solvent content is found to be approximately 54%, dthin the range commonly observed for most proteins (Matthews, 1968). The crystal structure of rhombohedral a-zinc pig insulin has been analysed at high resolution (Blunclell et al., 19’71,1972) and a complete set of atomic co-ordinates derived. In this form the insulin is organized as s, compact hexameric unit made up of three equivalent dimers which co-ordinate through their BlO histidine residues to two zinc atoms. A non-crystallographic 2-fold axis perpendicular to the crystallographic 3-fold axis relates the two monomers in the climer (see Fig. l(a)). In hagfish insulin
b
(b)
of insulin molecules in the crystals of pig insulin (a) and FIG. 1. A schematic representation hagfish insulin (b). (a) Represents the 2-zinc pig insulin hexamer in space group R3, with the dashed lines representing non-crystallographic %-fold axes which relate monomers in the dimer. (Similar $-fold axes between the dimers are not shown.) (b) Shows part of the arrangement in the hagfIsh insulin crystal structure, space group P4i2i2 or P4,212. A crystallographio 2-fold axis relates two insulin molecules, which may or may not form a dimer. The Eulerian angle 0s refers to rotation ebout this axis.
the space group requires two monomers to be related by a crystallographic 2-fold axis (see Fig. l(b)) which offers the possibility that their organization with respect to this did is similar to that found in the pig insulin dimer. If this were the case then the known pig insulin monomer would have the same orientation as the hagfish monomer in its cell after rotations through 8, = - 104”, an arbitrary 8,, and 8, = 45”, where B,, 0,, d3 are Eulerian angles as defined by Rossmann $ Blow (1962).
TABLE
1
Prdimina y cystd Species
Spaoe group
Pig HugfIsh
R3 P412,2 or P422,2
Asymmetrio Dimer Monomer
da.ta unit
Aggregation
state
Hexamer Monomer or dimer
STRUCTURE
OF HAGFISH
INSULIN
“5
Table 1 summarizes crystal data for the two insulin species under comparison as known at the start of the investigation. We considered two separate approaches to the problem of determining the crystal structure of hagfish insulin. The Grst involved the application of the rotationtranslation functions using the pig insulin monomer and dimer, respectively, as search units. Here we used our knowledge of the crystal structure of rhombohedral pig insulin by calculating the appropriate subunit transforms from the atomic coordinates. The second approach was the conventional isomorphous heavy atom replacement method, treating hagfish insulin as an independent crystal study.
2. Rotation Function By effectively rotating the Patterson function of one unit on to the Patterson function of a structurally similar unit the rotation function determines the orientation which brings about maximum overlap. As pig and hagfish insulins have different aggregation properties, it was necessary to calculate Patterson functions for the isolated pig insulin search units. This was achieved by placing the pig insulin monomer and dimer, respectively, in suitable triclinic cells, then calculating their transforms. The atomic co-ordinates used were derived from a 2.8 A resolution analysis of rhombohedral 2-zinc pig insulin (Blundell et al., 1971). The backbone conformations at the terminal regions A21, B27-30 and Bl-5 were either poorly defined in the pig insulin structure or influenced by its hexameric aggregation. Thus, in considering 41 out of 51 residues in the pig insulin molecule, we used the co-ordinates of 208 atoms (out of a possible 403), comprising backbone, p-carbon and disulphide bridge atoms. No side-chain atoms were included because the hagfish insulin sequence was incomplete at the time of the investigation and their conformations anyway are uncertain given the different aggregation states. The set of atomic co-ordinates chosen was that of molecule 1 in the pig insulin dimer (Blundell et al., 1971) and for the dimer search unit an exact 2-fold axis was assumed, reproducing molecule 1. A set of native hagfish insulin data was collected to a resolution of 45 A from one small crystal (O-35 mm maximum dimension) using a Hilger Watt PDPS controlled four circle diffractometer. After the application of Lorentz-polarization and absorption (North et al., 1968) corrections 1301 measurements were combined giving 477 independent reflections with a merge index R, of 0.054, where
(a) Monomer search The “reduced” pig insulin monomer (defined above), an object approximately 30 A x 20 A x 15 A, was placed in a triclinic cell, 60 A x 40 A x 40 A (all angles 90”) and structure factors calculated. An overall temperature factor, B, of 12 A2 was assumed. We considered the rotation of the hagfish insulin Patterson function on to the Patterson function of this pig insulin monomer over a sphere of radius 15 A. The required asymmetric unit in Eulerian space of 0 < d1 < ~12, 0 < 62 _< 7r, 0 5 0, < r was sampled at 15” intervals. The peaks were further explored at 5” intervals. The triclinic unit cell dimensions and the radius of the sphere in the rotation function were chosen so that only intramolecular vectors would be present. In order
26
J. F. CUTFIELD
to save computing time only about 20% of the data, were The interpretation of the there were a large number strongest peaks corresponded
ET
AL.
the stronger reflections between 5 A and 10 A spacing, included in the calculations. rotation function results was not straightforward, as of peaks significantly above background. The three to the following orientations for hagfish insulin:
(i) 8, = 0” (ii) 13,= 50” (iii) e1 = 80”
O2= 160’ 8, = 155” 0, = 155”
8, = 90” e3 = 90” e3 = 120”
R = 430; R = 438; R = 428,
where the rotation function background level was R = 404+3. Using the equivalent position relationship R(8, Ba6,) = R(--8, 7~--t t$ t?,) (Tollin et al., 1966) and remembering that R(B, tY20,) is the reverse of R(--8, -0, -0,) we can see that solution (ii) is equivalent to a “pig on hagfish” rotation of 0, = -9O”, e2 = 25’, e3 = 50”. The simple case visualized from Figure 1 suggested 8, = -104”, 8, arbitrary, e3 = 45”. Thus, it seemed plausible that the local a-fold axis in the pig insulin dimer had indeed become a crystallographic 2-fold axis in hagfish insulin, with a rotation of 25” about this diad. For this reason, we decided to test for the existence of a hagfish insulin dimer by using the pig insulin dimer as search unit. (b) Dimer search A pig insulin dimer, as described above, was placed in a suitably large triclinic cell and the transform calculated. The spherical radius of the rotation function was set to 25 A. The requirement that the dimer axis should be along the 2-fold axis of P4,2,2 (or P4,2,2) means that e1 and t$ are fixed, so there is only one degree of freedom, i.e. rotation of t$, about the 2-fold axis. Results are shown in Figure 2, the dominant peak corresponding to &=30°, very close to the value of 25” given by the monomer solution.
ro
3603 0
40
SO %
120
160
FIG. 2. A plot of the dimer rotation fun&ion against & in degrees. The peak at & I mines the orient&ion of the dimer about the Z-fold axis.
313~deter-
3. Translation Function It now seemed likely that hagfish insulin formed a dimer similar to that of pig insulin. It remained to determine the one translation parameter which would define its centre along the crystallographic a-fold axis. This was achieved by a one-dimen-
STRUCTURE
OF HAGFISH
INSULIN
27
65-
FIG. 3. R-factor search of the correctly oriented pig insulin dimer along the crystallogrsphic ) and P43212 (----). Two data sets were used, co + 10 A 2-fold axis of P41212 ( (upper) and 10 +6 A (lower). The search need only be mede to the midpoint of the e-fold axis, a length of 27 A.
R-factor search, moving the centre of the now correctly oriented dimer along the S-fold axis in 2 il steps and calculating structure factors. The same set of atomic co-ordinates employed in the rotation function was used in these calculations. The search, which must be carried out in both P4,2,2 and P4,2,2 was done using two separate data sets, co -+ 10 a and lo+6 8. The results are shown in Figure 3. The minimum R factor (R = 21 l$‘ObSl- 1Ii’Coalol l/CIB’O,,Sl) corresponds to a translation of 5 A from the origin of the P4,2,2 unit cell along the 2-fold axis. The sharpness of the minimum provides satisfying confirmation of a pig insulin-like dimer and as well clearly resolves the space group ambiguity. We were thus reasonably confident that we had established the position of the hagfish insulin molecule in the tetragonal unit cell and felt that the best way to confirm these results would be by determining heavy atom positions in isomorphous derivatives using phases calculated from the appropriately transformed atomic coordinates of pig insulin. These could then be compared to positions found from the more conventional Patterson and “direct methods” approaches. sional
J. F. CUTFIELD
28
ET
AL.
4. Isomorphous Replacement Studies Heavy atom soaking experiments on a limited number of small crystals (0.3 mm maximum dimension) yielded good intensity changes for Pb’ + , AuCI, -, PtClk2 - and UOz2 + . We decided to collect diffraction data from the lead and uranyl derivatives as these showed minimal changes in cell constants. They were prepared under the following conditions : (i) 0.01 M-lead acetate, 0.05 M-sodium acetate buffer, pH 6.0, 24-hour soak; (ii) 09075
M-Uranyl
acetate, 0.05 M-sodium citrate buffer, pH 6.0, 20-hour soak.
Unfortunately, the data did not extend much beyond 6 A spacing and anomalous dispersion differences were considered too weak to be useful. Two symmetry related equivalents were measured for each derivative giving R, figures for the merged 6 A data sets (210 independent reflections) of O-068 (lead) and 0,059 (uranyl). In addition, centric data to 4.5 A spacing were collected for the lead derivative. Both isomorphous difference Patterson and AE syntheses (K. Wilson, personal communication; Germain et al., 1971) established major binding sites for the lead and uranyl ions in the two derivatives, though the correct hand of these solutions can not be distinguished by either method. Difference Fourier syntheses employing phases calculated from the transformed atomic co-ordinates of 2-zinc pig insulin showed the same major sites, and also resolved the problem of hand. Refinement was carried out using centric data (representing 60% of the 6 A sphere) and several minor sites were detected in the difference maps. However, they were barely significant after refinement. A set of “best” phases was calculated according to the method of Blow % Crick (1959), and a 6 A electron density map computed. The overall figure of merit was 0.73. It should be noted that the 101 reflection, which is the largest, had very small heavy atom contributions in both derivatives. This gave it a low figure of merit and a phase differing by m from the calculated value. Since the calculated amplitude was also very large for this reflection it seemed the isomorphous phase was probably wrong. Changing its sign and increasing its weight noticeably improved the appearance of the electron density map.
5. Discussion Figure 4 shows part of this isomorphously phased map with, superimposed, the pig insulin a-carbon skeleton positioned by the rotation-translation functions. As expected the overall organization of the dimer appears similar to that observed in a-zinc pig insulin but there are significant differences in regions involving both ends of the B chain. It is interesting to inspect the measure of agreement between the phases estimated from isomorphous replacement and those calculated from the pig monomer backbone. We considered centric and non-centric data separately and the results are summarized in Table 2. The number of reflections is rather small but, as expected, there is better agreement for those with high figure of merit or high Sim weight. A comparison of the three-dimensional crystal structures of hag&h and pig insulins may shed some light on the reason for their different biological activities. It may also allow us to decide why a local S-fold axis, as in pig insulin, becomes a crystallographic Z-fold axis in hagfish insulin. We would like to know if it can be
STRUCTUR.3.Z
OF HACFISH
INSULIN
29
FIG. 4. An 8 A thick slab of the 6 A isomorphously phased hagf%sh insulin eleotron density map viewed &long c. The projection is made up of sect,ions z = $2c/40 to -22c/40, The diagonal E-fold axis is contained in the z = 0 plane. Appxoprista a-carbon positions of the pig insulin molecule are suparimpoF,ed, namely BlZ-B26 (solid backbone) and AZO-A21 (hollow), Parts of nsighbouring molecules have not been drawn in for the sake of clarity.
TABLET Agreement un&?ysis of calcu&ed and experimental phases Centria
Non-cent&3 Mean discrepancy
Range
No. in zone
Fraction agreeing
No. in zone
4 sin2 S/h2 i: 0.02 > 0.02
86 34
0.69 0.66
60 40
73” 12”
0.0 0.2 0.4 04 0.8
< < < < <
7%5 o-2 7n < 0.4 ?n ( 0.6 m _< 0.8 m 5 1.0
I 12 6 14 81
0.71 O-42 0.67 O-67 o-73
12 22 8 30 18
109” 61” 91” 11” 47”
o,o 0.2 0.4 0.6 0.8
< < < < <
s s S S s
11 18 19 24 48
0.54 0.44 0.63 0.67 0.81
0 8 10 17 66
82” 71” 8””M 68”
120
0.68
90
73”
2 0.2 < 04 _< 0.6 I< 04 2 1-o
Tots1
Here rn is the figure of merit and S is a Sim-type (Sim, 1969) as I,(X),
where X = 21Fo\lF,l/ifa(i)
weight. and xf”(
The Sim weight
has been defined
z‘) 10 * p resents the scattering
contri-
bution from those atoms omitted from the a&&&ion. It ia well known that for low resolution protein data Wilson at&i&es are not obeyed and zf2 is not B good represent&ion of the observed scattering. We examined the distribution of
J. F. CUTFIELD
30
1T
AL.
explained, for example, in terms of sequence changes away from the monomermonomer interface region (there are very few amino acid substitutions in this region), as a result of being in a lower aggregation state caused by the absence of Zn2+, by crystal packing effects, or, perhaps we are seeing an average of two different orientations. These questions remain unanswered until we have a high resolution structure. We are indebted to Dr Stefan Emdin, Professor Sture Falkmer, Professor Don Steiner and colleagues for giving us a sample of crystalline hagfish insulin. Our grateful thanks are due to the Kristineberg Zoological Station, Fiskebilckskil, Sweden. This work was supported by the Royal Society, British Diabetes Association and Science Research Council. One of us (J. F. C.) was the recipient of an ICI Fellowship. We thank Professor D. C. Hodgkin of this paper.
for valuable
discussions during the preparation
REFERENCES Blow, D. M. & Crick, F. H. C. (1959). Acta Crysfollogr. 12, 794-802. Blundell, T. L., Cutfield, J. F., Cutfield, S. M., Dodson, E. J., Dodson, G. G., Hodgkin, D. C., Mercola, D. A. & Vijayan, M. (1971). Nature (London), 221, 506-511. Blundell, T., Dodson, G., Hodgkin, D. & Mercola, D. (1972). A&. Prot. Chem. 26, 279402.
Falkmer, S., Emdin, S., Havu, N., Lundgren, G., Marques, M., Ostberg, Y., Steiner, D. F. & Thomas, N. W. (1973). Amer. ZooE. 18, 626-638. Germain, G., Main, P. & Woolfson, M. M. (1971). Acta Cry&aUogr. ser. A, 27, 368-376. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. North, A. C. T., Phillips, D. C. & Mathews, F. S. (1968). Actu Crystdlogr. 8ep. A, 24, 351359. Rossmann, M. G. & Blow, D. M. (1962). Acta Crydullogr. 15, 24-31. Sim, G. A. (1969). Acta CrydaZZogr. 12, 813-816. Steiner, D. F., Peterson, J. D., Tager, H., Emdin, S., Ostberg, Y. & Falkmer, S. (1973). Amer. 2001. 13, 591-604. Tollin, P., Main, P. & Rossmann, M. G. (1966). Acta CryduZZogr. 20, 404-407. Weitzel, G., Stratling, W.-H., Hahn, J. & Martini, 0. (1967). Hoppe-SeyZer’s Zeit. Physiol. Chem. 348, 525-532.