THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 19M hy The American Society of Biological Chemists, Inc

Vol. 259, No. 20, Issue of October 25, pp. 12714-12717, 1964 Printed in U. S. A.

Preliminary X-ray Crystallographic Data on Mouse Submaxillary Gland Renin and Renin-Inhibitor Complexes*

(Received for publication, April 9, 1984)

Manuel A. Navis$, James P. Springer$, Martin Poe$, Joshua Bogert, and Karst HoogsteenS From the $Department of Biophysics, Merck Institute for Therapeutic Research, Merck, Sharp, and Dohme Research Laboratories, Rahway, New Jersey 07065 and the §Department of Medicinal Chemistry, Merck, Sharp, and Dohme Research Laboratories, West Point, Pennsylvania 19486

We have crystallized renin from the submaxillary gland of male mice, both in its native state, and in binary complex with transition-state analog inhibitors. The best of the many crystal forms examined consisted of tetragonal bipyramids with space group symmetry P41 Z1 2 (or itsFnantiomorph) and unit cell dimensions a = b = 91.4 A, c = 211.6 A; a = j3 = y = 90”. This tetragonal form was compatible with both inhibited and uninhibited renin. Two of the inhibitors used were synthesized as iodinated analogs; their binary com- plexes with renin may serve as single-site rational heavy atom derivatives. X-ray data beyond 2.8-A res- olution have been collected by oscillation photography using the Cornel1 High Energy Synchrotron Source in Ithaca, NY.

Hypertension is a leading public health problem in indus- trialized societies, contributing, among other things, to car- diovascular disease, stroke, and renal failure (1). Novel strat- egies for the treatment of hypertension have recently centered on the renin-angiotensin system through the inhibition of angiotensin I1 production (2). Angiotensin I1 is a potent octapeptide hormone which both constricts the vasculature, and induces fluid retention through the stimulated release of aldosterone (3). The additive effect of more fluid in less space leads to higher blood pressure. The hormone angiotensin I1 is produced from an inactive circulating protein prohormone, angiotensinogen, in two steps; a specific cleavage to the in- active NH,-terminal decapeptide angiotensin I by the action of renin, and the subsequent removal of the COOH-terminal dipeptide of angiotensin I by angiotensin-converting enzyme (4). Inhibitors of angiotensin-converting enzyme, a zinc di- carboxypeptidase, have already proven themselves clinically as safe and effective antihypertensives. I t is generally thought that angiotensin-converting enzyme inhibitors function by preventing angiotensin I1 formation, demonstrating the use- fulness of the renin-angiotensin system as a drug target (4, 5 ) , but it is possible that a more complicated set of interactions may be in operation involving inhibition of other enzymes (e.g. Ref. 6). Renin inhibitors would better define the clinical importance of angiotensin I1 by blocking the renin-angioten- sin system at the earlier step of angiotensin I production, by- passing angiotensin-converting enzyme-dependent effects al- together. The fact that renin is a more selective enzyme than angiotensin-converting enzyme should in principle provide the opportunity for more selective inhibition.

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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At present, renin inhibitors potent enough to lower blood pressure are peptidyl in nature and not orally active. This situation, strongly reminiscent of the early work on angioten- sin-converting enzyme inhibition (4), makes them difficult to work with in a clinical setting. One of the objectives of our renin structural program is to provide a basis for the design of non-peptidyl inhibitors with more favorable pharmaco- kinetic properties. To meet this objective, we have purified (7) and crystallized renin from the submaxillary gland of the male mouse (a rich source of the enzyme), both in its native state and in binary complex with a series of statine-containing peptidyl inhibitors synthesized at Merck (8).’

Mouse submaxillary gland renin is strongly homologous in sequence (9, 10) with human kidney renin (11, 12), the pre- sumed medicinal target enzyme. Nearly 70% of the amino acid residues of the two enzymes are identical, so that struc- tural insights from the one should be readily applicable to the other. Moreover, mouse submaxillary gland renin can be more readily obtained in quantity (7,13). Mouse submaxillary gland renin is a member of the class of aspartyl proteases which includes pepsin (14) and a number of microbial enzymes (15- 18), whose structures have been solved crystallographically. Comparisons between these molecules and mouse submaxil- lary gland renin should contribute to a better understanding of the mechanism of action of the aspartyl proteases, the high substrate selectivity of renin as compared to the rest of the class, and the anomalous pH optimum for renin activity (19).

EXPERIMENTAL PROCEDURES

Mouse submaxillary gland renin was isolated and purified as de- scribed by Poe et al. (7). In a typical crystallization experiment, a frozen 3-ml fraction of mouse submaxillary gland renin from the final purification step was thawed out which contained approximately 7 mg of the enzyme (as determined spectrophotometrically (20) assum- ing an absorption coefficient E;% nm = 10.0), in 50 mM sodium acetate, at pH 5.38, 200 mM sodium chloride, saturated in cholesterol. The solution was concentrated to a volume of approximately 0.5 ml by ultrafiltration over an Amicon YM-10 membrane. The retained so- lution was dialyzed overnight with stirring a t 4 “C versus 100 ml of 5 mM sodium acetate, 100 mM sodium chloride, pH 5.38, in a dialysis bag with a 12,000-dalton cut off. To prevent the growth of microor- ganisms all solutions were made in 0.01% sodium azide.

Renin inhibitors used are listed in Table I; they were synthesized as described in Ref. 8.’ Binary mouse submaxillary gland renin- inhibitor complexes were prepared by adding 1.2-1.8 eq of inhibitor complexes were prepared by adding 1.2-1.8 eq of inhibitor dissolved in 0.05 ml of N,N-dimethylformamide to the starting thawed fractions before proceeding as above. At these levels, N,N-dimethylformamide did not interfere with the enzymatic activity of mouse submaxillary gland renin. Excess inhibitor was lost in the concentration and dialysis steps, so that all starting solutions contained binary 1 to 1 complexes.

Crystallizations were performed using the hanging drop vapor

M. Poe and J. Boger, unpublished results.

12714

Crystallography of Renin and Renin-Inhibitor Complexes 12715

TABLE I Structure of statine-based transition state analog inhibitors used in

binary complex with mouse submaxillary gland renin for crystallization.

The abbreviations used are: Boc, tert-butyloxycarbonyl; Ibu, iso- butyryl (2-methylpropanoyl); Sta, statyl(3S,4S)-4-amino-3-hydroxy- 5-methyl-heptanoyl; IPh, p-iodophenylalanyl.

Inhibitor Structure K, Ref. nM

1 Boc-His-Pro-Phe-His- 4 . 9 8

2 Boc-His-Pro-Phe-IPh- 0.62 -'

3

Sta-Leu-Phe-NH,

Sta-Leu-Phe-NH,

Sta-Leu-Tyr-NH,

Sta-Leu-Phe-MI,

-Sta-Leu-Phe-NH,

Sta-Leu-IPh-NH2

Boc-His-Pro-Phe-His- 100.0 8

4 Boc-His-Pro-Phe-Phe- 5.0 -b

5 Ibu-His-Pro-Phe-His 15 .0 8

6 Boc-His-Pro-Phe-His- 11.0 -b

-, M. Poe, and J. Boger, unpublished results. -, this study.

diffusion method essentially as described (21). Crystallization condi- tions are summarized in Table I1 and include the starting protein concentration, the nature and concentration of precipitant and buffer in the reservoir solutions, the pH, crystallization time, and tempera- ture. Protein solutions were set up in bulk with a precipitant concen- tration 10-25% below that of the reservoir solutions. They were cleared by centrifugation at 30,000 X g for 1/2 h using a fixed angle head and large plastic centrifuge tubes. By this means, the superna- tant can move away from any debris pellet left at the end of a run. Individual 15-p1 droplets of protein solution were placed on hydro- phobic plastic coverslips which were inverted over 0.5-ml reservoirs in alternate wells of Linbro tissue culture plates. Using alternate wells prevents the coverslips from sliding underneath each other. Microscope immersion oil was used to seal the wells.

As indicated in Table 11, "macroseeding" (22-24) was required for the growth of very large crystals of form A, as shown in Fig. la. Small crystals (Fig. lb), which grew spontaneously to 0.1 mm in their largest dimension, were placed in a wash whose precipitant concentration was such that the crystals would dissolve overnight. Using a micro- pipette and working quickly, each seed crystal was serially transferred through 9-12 separate 100-pl aliquots of the wash before being introduced into the final crystallization droplet. In all transfers, a minimal volume (less than a microliter) was used to transport crys- tals. The crystals would, in any case, usually drop out of the pipettes on contacting liquid.

TABLE I1 Summary of crystal forms and crystallization conditions for mouse submaxillary gland renin and mouse

submaxillary gland renin-inhibitor complexes. PEG 6000 is polyethylene glycol with an average M, = 6000.

Form Complex Morphology Space group Cell parameters Zb Crystallization

conditions"

A None, 1, Tetragonal 2, 3

B 4

C 5

D 6

E

F

G

None

None

None, 1

H None

I None

bipyramids

Chunky rods

Plates

Thick plates

Irregular thick rods

Plates

Hexagonal (?)plates

Thin rods

Clusters of needles

15 mg/ml; 30% PEG 6000, 30 mM sodium acetate, pH 5.9; 1 week; 25°C; macro seeds needed.

15 mg/ml; 6% PEG 6000,6 mM sodium acetate, pH 5.9; 2 months; 25 "C

15 mg/ml; 8.3% PEG 6000, 100 mM sodium acetate, pH 5.9; 1 month; 25 "C

15 mg/ml; 12% PEG 6000, 30 mM sodium acetate, pH 5.9; 1 mM myristic acid; 2 weeks; 25 "C

15 mg/ml; 5% PEG 6000,20 mM sodium acetate, pH 7.3; 2-3 months; 4 "C, grows from precipite.

10 mg/ml; 20% PEG 6000, 25 mM sodium acetate, pH 5.2; 1-2 months; 4 "C

5 mg/ml; 7% PEG 6000, 25% methylpentanediol, 100 mM sodium acetate, pH 6.2; 1 month; 25 "C

15 mg/ml; 5% PEG 6000,6 mM sodium acetate, 80 mM sodium cacodylate, pH 5.9; 3 months; 4 "C

15 mg/rnl; 7.5% PEG 6000, 60 mM sodium acetate, 40

pH 5.9; 1 week; 25 "C mM sodium cacodylate,

P4]2]2 or is enantio- morph

P212121

Uncharac- terized

P21212 (mar- ginal)

Uncharac- terized

Uncharac- terized

Uncharac- terized

a = b = 91.4 A, c = 211.6 A, 2-3 a = p = y = g O ~

a = 94.7 A, b = 114.2 A, c = 2 148.6 A, 01 = 0 = y = 90"

a = 98.1 A, b = 130.2 A, c = 8+ 265.4 A, 01 = 0 = y = 90"

a = 67.1 A, b = 67.9 A, c = 2 100.8 A, 0 = 98.5', 01 = y = 90"

a = 94 A, b = 106 A, c = 4-6 188 A, a = /3 = y = 90'

Crystallization conditions include: the protein concentration, the precipitant and its concentration, the buffer and its concentration, any additives and their concentration, pH, crystallization time, crystallization temperature, and whether seeds were used.

b Z is molecules/asymmetric unit. Mornon et al. (27) have reported crystals in space group P2, with cell dimensions a = 96.4 A, b = 104.4 A, c = 77.4 A, 0 = 101.2", 01 = y = 90".

12716 Crystallography of Renin and Renin-Znhibitor Complexes

I!- - b F. a

FIG. 1. Crystals of form A of mouse submaxillary gland renin. The bar corresponds to 1 mm. a, data quality single crystal resulting from the seeding process. b, seed crystals grown spontaneously.

Large crystals suitable for x-ray diffraction were transferred into protein-free stabilizing solutions which were generally at IO-25% higher precipitant concentration than the reservoir solutions used in the crystallizations. These were subsequently mounted in glass cap- illaries for data collection. Precession photographs for surveys were obtained using conventional nickel-filtered copper K-radiation. Large scale data collection was by oscillation photography using focused, monochromatized 1.5-A synchrotron x-rays at the Cornell High En- ergy Synchrotron Source (CHESS) in Ithaca, NY.

RESULTS AND DISCUSSION

Mouse submaxillary gland renin crystals were first reported by Cohen et al. (251, but were not characterized crystallo- graphically. Recently, Mornon et al. (26) have described a mouse submaxillary gland renin crystal form with four inde- pendent molecules per asymmetric unit which diffracts to 5- A resolution. We have obtained multiple crystal forms of mouse submaxillary gland renin in its native state, and in binary complex with several inhibitors synthesized at Merck. As shown in Table 11, very small differences in crystallization conditions or in the nature of the inhibitors used can shift growth to one or another crystal form. This suggests that the region around the binding pocket is involved in crystal pack- ing. The best of these forms, A in Table 11, is tetragonal, in space group P41212 (or it; enantiomorph), with unit cell dimension!: a = b = 91.4 A, c = 211.6 A, and volume, V = 1,718,000 A3. Fig. 2 is an (Ok l ) precession photograph of our form A crystals taken at CHESS. Assuming M, = 36,500 (10) and two molecules per asymmetric unit, one can calculate the crystal volume/unit of protein molecular weight, V, = 3.026 A3 dalton" corresponding to 60% solvent content. This is within the range of values which have been observed with

FIG. 2. 12.5O ( O H ) precession photograph of form A crystal taken at CHESS.

protein crystals (27), although three molecules per asymme!- ric unit can also be accommodated, giving V,,, = 2.018 A3 dalton" (39% solyent). If one assumes M, = 38,000 (9, 261, then V,,, = 2.907 A ? dalton" (57% solvent), and V, = 1.938 A3 dalton" (36.5% solvent) for two and three molecules/ asymmetric unit, respectively.

Mouse submaxillary gland renin crystals are particularly difficult to obtain and show moderate radiation sensitivity. With high-intensity synchrotron radiation, we have observed

Crystallography of Renin and Renin-Inhibitor Complexes 12717

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with mouse submaxillary gland renin will be described else- (1979) Biochemistry 18, 1638-1640 where.’ A Patterson search with the related microbial aspartyl 19. Inagami, T., Chang, J. J., Dykes, C. W., Takii, Y., Kisaragi, M., protease from Rhizopus chinensis (15) as a structural prohe and Misono, K. S. (1983) Fed. P m . 42,2729-27.34

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_.. . . 5 1,283-308

Phurmac. 28,2867-2882

355-361

( L o n d . ) 303.81-84

FIG. 3. Oscillation photograph of form A crystals taken at Proc. Natl. Acad. Sci. U. S. A. 80,7405-7409

have now collected a complete set 0.f oscillation PhotoSaPhs 15. Bott, R. Subramanian, E., and Davies, D. R. (1982) Biochemistry 12,922-936

has been carried out using the iodine derivative structure 20. PW, M., and Liesch, J. M. (1983) J. Bbl. Chem. 258,9856-9860

tak, Wiley-Interscience, New York

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Fourme, R., Kahn, R., Gadet, A., Bartels, K. S., and Bartunik,

29. Reynolds, R. A., Remington, S. J., Weaver, L. H., Fisher, R. G., 1. Freis, E. D. (1979) Clin. Sci. 57,349s-353s Anderson, W. F., Ammon, H. L., and Matthews, B. W. (1984) 2. Ondetti, M. A., and Cushman, D. W. (1982) Annu. Rev. Biochem. Acta Crystallogr. A, in press

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