0 1988 by The American Society for Biochemistry and Molecular Biology, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 263, No. 8, Issue of March 15, pp. 3960-3962, 1988

Printed in U.S.A.

Crystallization and Preliminary Analysis of Crystals of Apolipophorin I11 Isolated from Locusta migratoria”

(Received for publication, November 18, 1987)

Hazel M. Holden, Michael R. Kanost, John H. Law, Ivan RaymentS, and Michael A. Wells From the Department of Biochemistry, University of Arizona, Tucson, Arizona 85721

Crystals of apolipophorin 111, isolated from the locust Locusta migratoria, have been reproducibly grown from ammonium sulfate solutions and are well suited for an x-ray crystallographic analysis. Locust apoli- pophorin 111 is a glycosylated protein with a molecular weight of 19,100 and interacts with lipophorin, the major lipoprotein complex in insects. The crystals be- long to the space group P6+?2 or P6a22 owwith unit cell dimensions of a = b = 67.5 A, c 7 155.6 A and diffract to a nominal resolution of 2.5 A. They are physically robust and are stable in the x-ray beam for over a week. A complete native x-Tay data set has been col- lected and processed to 3.0-A resolution.

In some insect orders such as Lepidoptera and Orthoptera P-oxidation of fatty acids serves as the source of fuel for prolonged flight (Beenakkers et al., 1985). During flight tri- acylglycerols normally stored in the fat body cells are con- verted to diacylglycerols and released into the hemolymph (Shapiro et al., 1988). Once in the hemolymph these lipids are associated with lipophorin and transported to the rapidly metabolizing flight muscles.

Lipophorin is the major lipoprotein complex found in in- sects (Chino et al., 1981) and is typically comprised of 60% protein and 40% lipid. The protein portion of the lipophorin particle consists of two apolipoproteins referred to as apoli- pophorin I (apoLp-I)’ and apolipophorin I1 (apoLp-11). Both apoLp-I and apoLp-I1 are integral constituents of lipophorin and have molecular weights of approximately 250,000 and 80,000, respectively (Ryan et al., 1984; Shapiro et al., 1984). In some insects there exists another apolipoprotein designated as apoLp-111. This apolipoprotein has a molecular weight of approximately 20,000 and exists as a soluble monomer in the hemolymph. During flight, however, when lipophorin is loaded with diacylglycerol, apoLp-I11 associates with the lip- oprotein particle, and it has been suggested that this smaller apoprotein serves to stabilize the lipid-enriched complex (Ka- wooya et al., 1984; Kawooya et al., 1986; Wells et al., 1987).

ApoLp-I11 has been isolated from a variety of insects in- cluding the sphinx moth Manduca sexta (Kawooya et al., 1984), the mesquite bug T h u s acutangulatus (Wells et al., 1985), the grasshopper Gastrimargus africanus (Haunerland

Health Grants AM GM 351865 and BRSG 829023 (to I. R.), Grant *This research was supported in part by National Institutes of

HL 39116 (to M. A. W.), and Grant GM 29238 (to J. H. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “udvertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Established investigator of the American Heart Association. ’ The abbreviations used are: apoLp, apolipophorin; Hepes, 4-(2-

hydroxyethy1)-1-piperazineethanesulfonic acid.

et al., 1986) and the locust Locusta migratoria (Van der Horst et al., 1984; Chino and Yazawa, 1986). In recent years the protein from the sphinx moth has been the most extensively characterized (Kawooya et al., 1984; Wells et al., 1985; Ka- wooya et al., 1986; Wells et al., 1987). It has been suggested that this apoLp-I11 is a prolate ellipsoid with an axial ratio of approximately 3 based on its hydrodynamic properties and its behavior during gel permeation chromatography (Kawooya et al., 1986). Furthermore, the circular dichroic spectrum of apoLp-I11 indicates that the protein contains about 50% a- helix. The amino acid sequence of the sphinx moth apoLp- 111, deduced from the cDNA sequence, shows similarity to several mammalian apolipoproteins and is composed of re- peated sequences with amphiphilic helical potential (Cole et al., 1987). In an effort to understand, on a three-dimensional level, the role of apoLp-I11 in the stabilization of the lipid- enriched lipophorin complex we initiated experiments de- signed to grow large single crystals of the protein isolated from M . sexta. All attempts to prepare such crystals failed. However, the apolipoprotein isolated from the locust L. migratoria crystallized readily and in a form suitable for a high resolution structural analysis.

Locust apoLp-111 was first studied by Van der Horst et al. (1984) and subsequently purified to homogeneity by Chino and Yazawa (1986). The protein is glycosylated with a total sugar content of approximately 12%. A cDNA coding for locust apoLp-I11 has recently been cloned, and the amino acid sequence has been determined based on the cDNA sequence.’ Locust apoLp-111 contains 161 amino acids with no methio- nine, tyrosine, or cysteine residues.

In this report we describe the crystallization and prelimi- nary analysis of crystals of locust apoLp-111. The three- dimensional structure determination of this protein is of considerable interest since it will not only provide a better understanding of insect apolipoproteins but may also yield valuable insight into the mammalian lipid transport systems.

EXPERIMENTAL PROCEDURES

Locust apoLp-I11 was purified according to the method of Kanost et al. (1987). This purification procedure takes advantage of the exceptional thermal stability of apoLp-111. Nearly all of the hemo- lymph proteins, with the exception of apoLp-111, precipitate when the hemolymph is heated to 100 “C. Minor contaminants are then re- moved by gel filtration and reversed phase high performance liquid chromatography. ApoLp-111 is stored as a lyophilized powder.

For crystallization experiments the protein was dissolved in a buffer solution of 10 mM Hepes, 5 mM sodium azide, pH 7.0, to a concentration of 30 mg/ml. A survey for potential crystallization conditions was conducted at room temperature using the hanging drop method of vapor diffusion. Once promising conditions were identified, the sitting drop method was employed in order to grow

* M. R. Kanost, M. S. Boguski, M. Freeman, J. I. Gordon, G. R. Wyatt, and M. A. Wells, manuscript in preparation.

3960

Crystallization of Apolipophorin III 3961

larger crystals (for a review of crystallization techniques, see Mc- Pherson, 1982).

X-ray diffraction photographs were recorded using nickel-filtered copper Kcu radiation from an Elliot GX20 rotating anode x-ray generator operated a t 35 kV and 40 mA with a 200-pm focal cup. Because the apoLp-111 crystals tended to slip in the quartz capillary tubes, a small volume of a 0.2% solution of poly(vinylforma1) in 1,2- dichloroethane was drawn over each crystal and allowed to dry. This formed a very thin plastic film around the crystal and was exceedingly helpful in preventing such problematic slippage (Rayment et al., 1977).

Precession photographs were typically recorded using a precession angle of IO", a crystal-to-film distance of 100 mm, and exposure times of about 17-20 h. The three-dimensional native data set was collected by the method of oscillation photography using an Arndt-Wonacott camera. An oscillation angle of Z.O"/film pack was used with an exposure time of 6 h and a crystal-to-film distance of 100 mm. The crystal was rotated about the c axis through a net rotation of 30".

The x-ray films were digitized with an Optronics PI000 film scanner and subsequently processed using a set of programs originally developed by Dr. Michael Rossmann and modified by Dr. Brian Matthew's laboratory (Rossmann, 1979; Schmid et al., 1981).

To ensure that we had crystallized the complete apoLp-111 mole- cule, the crystal used for data collection was subsequently dissolved in 10 mM Hepes, pH 7.0, and subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis. The gel clearly showed that the protein in the crystal was intact apoLp-111.

RESULTS AND DISCUSSION

Crystals of apoLp-111 were first obtained using 3.0 M am- monium sulfate as the precipitant and a protein concentration of 30 mg/ml. The ammonium sulfate solution was buffered with 50 mM potassium/sodium phosphate, pH 7.4. The crys- tals grew as a cluster of small needles. Subsequently, single hexagonal rods with dimensions of 0.2 X 0.2 X 1.0 mm were produced in about 2 weeks by lowering the protein concentra- tion to 10 mg/ml and the ammonium sulfate concentration to 2.8 M. There was, however, some difficulty in growing large crystals reproducibly. Indeed, many experiments failed to produce crystals a t all.

In an effort to overcome this nucleation problem we used the technique of "macro-seeding" which proved extremely successful in other crystallization studies (Thaller et al., 1981; Thaller et al., 1985). For such experiments, small apoLp-I11 crystals, grown from 2.8 M ammonium sulfate, were first washed in 2.4 M ammonium sulfate, 50 mM phosphate, pH 7.4, for approximately 15 min and then transferred to 50-pl

. .

. .

FIG. 2. IO" precession photograph. The precession photograph shown is of the h0l zone and was recorded using a crystal-to-film distance of 100 mm and an exposure time of 19 h a t 35 kV and 40 mA. Diffraction maxima a t the edge of the precession circle corre- spond to a resolution of 4.4 A.

. .

. .

. - . .

. . .

FIG. 1. Crystal of locust apoLp-111. The crystal shown was grown from 2.8 M ammonium sulfate, 50 mM phosphate, pH 7.4. The thick bar corresponds to a length of 0.5 mm. By using the technique of "macro-seeding'' crystals with a length of 4.0 mm can be readily grown.

FIG. 3. Typical oscillation photograph. The photograph shown here was recorded using an oscillation angle of 2.0" and an exposure time of 6 h. The crystal-to-film distance was 100 mm. Diffraction maxima at the edge of the film correspond to a resolution of 3.0 A.

droplets containing 12 mg/ml protein and 2.4 M ammonium sulfate, 50 mM phosphate, pH 7.4. The droplets were then equilibrated by vapor diffusion against 2.8 M ammonium sulfate, 50 mM phosphate, pH 7.4. The seed crystals increased in size over a period of 2 weeks with some achieving dimen- sions of 0.4 X 0.4 X 4.0 mm. With this technique large single crystals can be obtained from at least 80% of the crystalliza- tion experiments. A photomicrograph of a typical crystal is shown in Fig. 1.

3962 Crystallization of Apolipophorin 111

TABLE I Data processing statistics for the native crystals Number of film packs 17 Average Rama (%) 4.8 Average Re." (%) 3.8

Total reflections measured 13,992 Independent reflections 4,261 Resolution (A) 3.0

RmergeO (%) 6.7

" R = II - TI/CI. R,, measures the agreement between sym- metry-related reflections on the same film, R,, measures the agree- ment between reflections recorded on successive films in a given film pack, and Rmew gives the overall agreement between intensities measured on different films.

ApoLp-I11 crystals belong to the hexagonal space group P6122 or P6522 with unit cell dimensions of a = b = 67.5 A and c = 155.6 A. A typical lo" precession photograph is shown in Fig. 2. Assuming one molecule/asymmetric unit and a molecular weight of 19,100, the volume/unit molecular weight in the unit cell or V, is 2.7 A3/dalton. Most globular proteins lie within the range of 1.68-3.53 A3/dalton (Matthews, 1974).

Tbe crystals are easily manipulated and diffract strongly to 3.0-A resolution using a conventional x-ray source. A consid- erable number of weaker diffraction maxima can be observed to 2.5-A resolution, and it should be possible to collect this higher resolution data using synchrotron radiation. At pres- ent, a three-dimensional na$ve x-ray data set has been col- lected and processed to 3.0-A resolution. A typical oscillation photograph is shown in Fig. 3, and relevant data processing statistics are given in Table I. Since the crystals are long rods it was possible to collect the entire data set from one crystal by making appropriate translations along its length. Data collection and processing took 1 week. A search for heavy atom derivatives is in progress.

In recent years there have been many investigations di- rected toward understanding those structural elements of the apolipoproteins that allow them to interact with lipid surfaces. It has been suggested that amphiphilic helices may play a key role in such protein:lipid interactions (Segrest et al., 1974). These helices are thought to contain polar and nonpolar amino acid residues distributed in such a manner that the nonpolar residues penetrate into the lipid milieu while the charged and polar residues interact with the aqueous environ- ment. Evidence for the amphiphilic helix comes from a variety of studies including protein sequence comparisons, peptide syntheses, and numerous physiochemical investigations (for a review see Gotto et al., 1986). Furthermore, Eisenberg et al. (1982, 1984) have formulated a mathematical model to quan- tify the amphiphilicity of a helical region, and this theoretical approach has been recently applied to various mammalian apolipoproteins (De Loof et al., 1987). Analysis of the amino acid sequence of locust apoLp-111 also suggests the presence of amphiphilic regions.' To date, however, there is a lack of three-dimensional structural information concerning apoli-

poproteins and the putative amphiphilic helices. The crystals of apoLp-I11 described here present a unique opportunity to examine, on a structural basis, the current hypotheses con- cerning the interactions between apolipoproteins and lipid surfaces.

Acknowledgment-We thank Dr. G. R. Wyatt for providing locust hemolymph.

REFERENCES Beenakkers, A. M. T., Van der Horst, D. J., and Van Marrewijk, W.

Chino, H., and Yazawa, M. (1986) J. Lipid Res. 2 7 , 377-385 Chino, H., Downer, R. G. H., Wyatt, G. R., and Gilbert, L. I. (1981)

insect Biochem. 11,491 Cole, K. D., Fernando-Warnakulasuriya, G. J. P., Boguski, M. S.,

Freeman, M., Gordon, J. I., Clark, W. A., Law, J. H., and Wells, M. A. (1987) J. Biol. Chem. 262,11794-11800

De Loof, H., Rosseneu, M., Brasseur, R., and Ruysschaert, J. M. (1987) Biochim. Biophys. Acta 911.45-52

Eisenberg, D., White, R. M., and Terwilliger, T. C. (1982) Nature

Eisenberg, D., Weiss, R. M., and Terwilliger, T. C. (1984) Proc. Natl. Acad. Sci. U. S . A. 8 1 , 140-144

Gotto, A. M., Jr., Pownall, H. J., and Havel, R. J . (1986) Methods Enzymol. 128 , 3-41

Haunerland, N. H., Ryan, R. O., Law, J. H., and Bowers, W. S. (1986) Insect Biochem. 16,797-802

Kanost, M. R., McDonald, H. L., Bradfeld, J. Y., Locke, J., and Wyatt, G. R. (1987) in Molecular Entomology (Law, J , ed) pp. 275- 283, Alan R. Liss, Inc., New York

Kawooya, J. K., Keim, P. S., Ryan, R. O., Shapiro, J. P., Samaraweera, P., and Law, J. H. (1984) J. Biol. Chem. 259,10733-10737

Kawooya, J. K., Meredith, S. C., Wells, M. A., Kezdy, F. J., and Law, J. H. (1986) J. Biol. Chem. 261 , 13588-13591

Matthews, B. W. (1974) J. Mol. Biol. 8 2 , 513-526 McPherson, A. (1982) Preparation and Analysis of Protein Crystals,

Rayment, I., Johnson, J. E., and Suck, D. (1977) J. Appl. Crystallogr.

Rossmann, M. G. (1979) J. Appl. Crystallogr. 12, 225-238 Ryan, R. O., Schmidt, J. O., and Law, J. H. (1984) Arch. Insect

Biochem. Physiol. 1, 375-383 Schmid, M. F., Weaver, L. H., Holmes, M. A., Grutter, M. G.,

Ohlendorf, D. H., Reynolds, R. A., Remington, S. J., and Matthews, B. W. (1981) Acta Crystallogr. A37,701-710

Segrest, J. P., Jackson, R. L., Morrisett, J. D., and Gotto, A. M., Jr.

Shapiro, J. P., Keim, P. S., and Law, J. H. (1984) J. Biol. Chem. 259 ,

Shapiro, J. P., Law, J. H., and Wells, M. A. (1988) Annu. Reu.

Thaller, C., Weaver, L. H., Eichele, G., Wilson, E., Karlsson, R., and

Thaller, C., Eichele, G., Weaver, L. H., Wilson, E., Karlsson, R., and

Van der Horst, D. J., Van Doom, J. M., and Beenakkers, A. M. T.

Wells, M. A., Ryan, R. O., Prasad, S. V., and Law, J. H. (1985) Insect

Wells, M. A., Ryan, R. O., Kawooya, J. K., and Law, J. H. (1987) J.

J. A. (1985) Prog. Lipid Res. 2 4 , 19-67

299,371-374

Wiley Interscience, New York

10,365

(1974) FEBS Lett. 38 , 247-253

3680-3685

Entomol. 33,297-318

Jansonius, J. N. (1981) J. Mol. Biol. 147,465-469

Jansonius, J. N. (1985) Methods Enzymol. 114 , 132-135

(1984) Insect Biochem. 14,495-504

Biochem. 15,565-571

Biol. Chem. 262,4172-4176