J. Mol. Biol. (1992) 228, 1269-1270

Crystallization

and Preliminary X-ray Analysis of Human Angiogenin

K. Ravi Acharya-f-, Vasanta

University

Robert

Subramanian

Department of Biochemistry of Bath, Claverton Down, Bath BA2

Shapiro,

James F. Riordan

7A Y, U.K.

and Bert L. Vallee

Center for Biochemical and Riophysical Sciences and Medicine and Department of Pathology Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115, U.S.A. (Received 6 August

1992; accepted 9 September 1992)

Crystals of recombinant human angiogenin have been grown from solutions containing sodium potassium tartrate and polyethylene glycol as precipitants. They belong to the space group C222, (a = 83.36 A, b = 120.64 A, c = 37.72 A) and contain a single molecule in the asymmetric unit. The crystals diffract to at least 2.3 A resolution and are suitable for threedimensional X-ray structural analysis.

Keywords: human

angiogenin;

Angiogenin, an extracellular protein, is a potent inducer of the formation of new blood vessels (Fett et al., 1985). This process, known as angiogenesis, is highly regulated in normal growth, embryonic development and wound healing, and during the menstrual cycle. However, progression of many diseases involves its degradation, as for example in arthritis, diabetes and tumour growth (Vallee et al., 1985; Folkman & Cotran, 1976). A number of molecules have been isolated and characterized which are involved in angiogenesis (Folkman & Shing, 1992) but in no case has a mode of action been established. Thus, an understanding of the physiological role and molecular mechanism of an angiogenin might have practical benefit for the treatment of a variety of disorders in which either enhanced blood supply or inhibition of vascular growth is desired. Human angiogenin is a single chain polypeptide (J& 14,124) initially isolated from tumour cell conditioned medium on the basis of its capacity to induce neovascularization on the chick chorioallantoic membrane (Fett et al., 1985). It was demonstrated subsequently to be a normal component of serum (Shapiro et al., 1987) and milk (Maes et al., 1988). Angiogenin has about 35% amino acid sequence ribonuclease (RNase) pancreatic identity to t Author to whom correspondence should be addressed.

crystallization;

X-ray

et al., 1985; Kurachi et al., 1985) amd it (Strydom also exhibits ribonucleolytic activity albeit markedly different in both magnitude and specificity (Shapiro et al., 1986; Rybak & Vallee, 11988). A considerable body of evidence now indicates that the enzymatic and biological activities of angiogenin are interrelated (Shapiro et al., 1986, 1989; Shapiro & Vallee, 1987, 1989). Based on the amino acid sequence homology between angiogenin and bovine pancreatic FLNase A, a preliminary three-dimensional structure of angiogenin was proposed by Palmer et al. (1986). This predicted structure was used to identify similarities and differences among amino acid residues known to be crucial for the enzymatic activity of RNase and also present in the active site of angiogenin. Conversely, because RNase is not angiogenic, positions in angiogenin that differ in RNase must contribute to the angiogenic activity of angiogenin. Thus, a major focus of current angiogenin research has been on the elucidation of structural features important for its ribonucleolytic and angiogenic activities, in particular; by examining the relationship between the in vitro activities of angiogenin and angiogenesis using protein engineering techniques. Here we report the crystallization of human angiogenin and its initial crystallographic analysis. Met-(-l) angiogenin was obtained from a recombinant expression system in Escherichia coli (Slhapiro et al., 1988). This extension derivative is func1269

0022%2836/92/241269-02

$08.00/O

0 1992 Academic Press Limited

1270

K.

R. Acharya

et al. References Fet,t,

Figure

1. Crystals

of human

angiogenin

tionally indistinguishable from the native < Glu- 1 protein. It was purified by Mono S cation-exchange and Cl8 high pressure liquid chromatography, then dialysed versus water and finally concentrated in an

Amicon Centricon 10 ultrafiltration device. Crystals of human angiogenin were grown at room temperat,ure using the hanging-drop method (McPherson, 1982). Droplets of 8 ~1 initial volume on siliconized glass coverslips were prepared suspended over reservoirs containing 1.0 ml of 62 Msodium potassium tartrate, 20 to 25% polyethylene glycol 6000, 0.5% /?-octyl glucoside in 0.01 Msodium citrate buffer (pH 5.2). Each droplet consisted of 4 ~1 of the reservoir plus 4 ,ul of a protein solution containing 20 mg/ml human angiogenin in 0.01 M-sodium citrate buffer. Rectangular shaped crystals of maximum dimensions 0.25 mm x 62 mm x 61 mm appeared after two days and grew to their maximum size in one week (Fig. 1). A single crystal of these was mounted in a thin

walled,

glass

capillary

tube

and

data

were

collected using a Siemens area detector mounted on a Rigaku rotating anode X-ray source using CuKor radiation (50 kV, 80 mA). The crystals are ordered and diffract to at least 2.3 A resolution (1 A = 0.1 nm). They belong to space group C222, (a = 83.36 8: b = 12664 A, c = 37.72 A). Assuming one molecule per asymmetric unit’, volume of the protein is 3.36 a3/Da,

the specific which falls

within the range normally observed for globular proteins, and corresponds to a 63% (v/v) solvent content (Matthews, 1968). Due to high solvent content, the crystals are sensitive to X-rays during the diffraction experiments. A preliminary 2.5 A data

set

has

been

colle&ed

on

the

Siemens

area

detector. We would like to thank Sir David Phillips and Professor A. R. Rees for their support and encouragement and Dr David Stuart for helpful discussion regarding the space group. K.R.A. would like to acknowledge the financial support by the Royal Society and the Medical Research Council, U.K.

Edited

5. W.. Strydom, D. J., Lobb, R. it., .Alderman, E. M., Bethune? J. L.: Riordan, J. F. & Vailee. B. L. (1985). Isolation and characterization of angiogenin, an angiogenic prot,ein from human carcinoma cells. Riochemnistry, 24, 5480-5486. Folkman, J. & Cotran, R. S. (1976). Relation of vascular proliferation to tumor growth. Int. Rev. Expt. Path. 16, 207-248. Polkman: J. 8r Shing, U. (1992). Sngiogenesis. J. Biol. Chem. 267, 10931-10934. Kurachi, K., Davie, E. W., Strydom, D. J., Riordan, J. F. & Vallee, B. L. (1985). Sequence of the cDN.4 and gene for angiogenin, a human angiogenesis factor. Biochemistry, 24; 5494-5499. Maes, P., Damart.? D., Rommens, C.; Montreuil: J., Spik, G. & Tartar, A. (1988). The complete amino acid sequence of bovine milk angiogenin. FEBS Letters, 241,41-45. Nlatthews, B. W, (1968). Solvent content of protein crystals. J. 1MoZ. Biol. 33; 491-497. McPherson, A. (1982). The Preparation and Analysis of Protein Cry&a& pp. 82-159: John Wiley C Sons: New York, U.S.A. Palmer, K. A., Scheraga, H. A.; Riordan, J. F. & Vallee, B. L. (1986). A preliminary 3-dimensional structure of angiogenin. PTOC. Xat. Acad. fki., C.8.A. 83. 1965-1969. Rybak, S. N. & Vallee, B. L. (1988). Base cleavage specificity of angiogenin with Sacchnromyees cereaisiae and E. coli 5 S RNAs. Bioch.emistry, 27, 228%2294. Shapiro, R. & Vallee, B. L. (1987). Human placental ribonuclease inhibitor abolishes both angiogenic and ribonucleolytic activities of angiogenin. Proe. Sat. dead. Sci., U.S.A. 84, 2238-2241. Shapiro; R. & Vallee, B. L. (1989). Site-directed mut,agenesis of histidine-13 and histidine-114 of human angiogenin. alanine derivatives inhibit angiogenininduced angiogenesis. Biochemistry, 28, 7401-7408. Shapiro, R.; R,iordan, J. F. & Vallee, B. L. (1986). Characteristic ribonucleolytic activity of human angiogenin. Biochemistry, 25, 3627-3532. Shapiro, R., Strydom, D. J., Olson, K. A. & Vallee; B. L. (1987). Isolation of angiogenin from normal human plasma. Biochemistry, 26. 5141-5146. Shapiro, R., Harper, J. W, Fox, E. A., ,Jansen, H.; Hein, F. & Uhlmann, E. (1988). Expression of %Iet-(-1) angiogenin in Escher&in coli: conversion to the authentic