Notes to the Editor

Preparation and X-ray studies of crystals of anhydroelastase

Sean Murphy and G. Michael Blackburn Department of Chemistry, The Unil'ersity, She[liehl $3 7HF, UK

and Lindsay Sawyer and Herman C. Watson Department of Biochemistry, The University, Bristol BS8 ITD, U K (Received 10 July 1981, revised 17 February 1982)

The preparation, purification and crystallization of porcine anhydroelastase using affinity chromatography with turkey- ovomucoid inhibitor is described. The structure of the active site at 3.5A (0.35 nm) resolution is compared with that of the natice enzyme. An attempt is made to locate the substrate (A lah-Lys-Phe bound to anhydroelastase.

Keywords: Enzyme; enzyme activity; crystal soakiml; ,';uhslrale binding: electron density

Introduction

Substrate binding studies have shown that elastase (EC 3.4.21.11) has an extended specificity site l, or series of subsites, which compensates for the relatively weak interaction between the residue adjacent to the cleaved bond and the enzyme. Shotton et al. 2 have attempted to investigate the interaction of substrates with elastase using crystals of the native and tosylated forms of the enzyme soaked in various peptide solutions. Because the lifetime of a complex between elastase and a true substrate at physiological temperatures is very much shorter than the time required to collect X-ray diffraction data these studies were confined to the binding of competitive inhibitors which in themselves were the products of the hydrolysis of good substrates.

N-Acetyl-Pro-Ala-Pro-Ala is an excellent inhibitor of elastase ~ and is ideal for crystal soaking experiments because the presence of the proline residues prevents hydrolysis under conditions where most N-acetyl tetrapeptides would be broken down. The crystallographic study of the resulting enzyme inhibitor complex revealed a major peptide binding site at the active centre of elastase which appeared to satisfy the subsite specificity for residues on the amino terminal side of the scissile bond as previously reported by Atlas et a l ) The corresponding study using the dipeptide Lys-Phe, which is one of the products of the good hexapeptide substrate, was less successful in demonstrating the position on the enzyme at which it would bind as part of the true substrate. As a direct result of this inability to define the position of that part of the substrate which is C- terminal to the point of proteolytic cleavage, the studies reported here on the preparation and crystallization of anhydroelastase were undertaken. Anhydroelastase is a modified form of the enzyme in which the active centre serine hydroxyl is removed to prevent proteolytic cleavage.

Experimental

Anhydroelastase was prepared from once crystallized elastase by treatment with [~-14C]-toluenesulphonyl fluoride followed by aqueous potassium hydroxide in a

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250 Int. J. Biol. Macromol., 1982, Vol 4, June

similar way to that described by Feinstein and Feeney s for chymotrypsin (EC 3.4.21.1). The resulting anhydroelastase/elastase mixture (70 mg) was treated with diisopropyl fluorophosphate (DIFP), dissolved in Tris buffer solution (2 ml, pH 8.1) and applied to a Sepharose turkey ovomucoid inhibitor column 6 and washed with Tris buffer. The anhydroelastase was eluted from the column (2 x 5 x 7 cm, 0.67 mg ml- ~ capacity) with aqueous acetic acid (0.1 M, pH 3.0). This method of purification of anhydroelastase proved efficient if the elution rate was high enough to effect rapid dilution of the eluate into buffer at pH 5.

For crystallization a small volume of acetate buffer (0.01 M, pH 5.0) was added to the desalted, freeze-dried material giving a final protein concentration of 5.3 mg ml 1. Cautious addition of sodium sulphate solution (1 M) to a final concentration of 0.02 M produced small, high quality crystals. These crystals were found to be isomorphous with crystals of native and tosyl (toluene sulphonyl) elastase v both before and after soaking in solutions containing substrate. Even though the crystals were judged to be better than 90% inactive they were further soaked in salt solution containing 1 mM tosyl fluoride for 48 h to ensure that cleavage of the added substrate would not occur. The crystals were then back soaked against a salt buffer solution for 24 h followed by a seven day soak against saturated (~ 10 mM) solution of Ala-Ala-Ala-Ala-Lys-Phe. The crystals were mounted in the usual way and a computer controlled four circle diffractometer used to collect Friedel pairs of reflections (for a full description of the measurement procedure see Watson et al. 8) within the reciprocal lattice sphere corresponding to 0.35 nm resolution.

Results and discussion

Preparation of anhydroelastase was accomplished by specific tosylation of serine-195, with p-toluenesulphonyl fluoride, followed by mild alkaline treatment of the modified enzyme to effect the elimination of the tosyloxy function which was monitored by carbon-14 labelling of the tosyl group. After incubation with turkey ovomucoid inhibitor followed by the addition of native elastase and assaying for activity using p-nitrophenyl-N-t-butoxy carbonylalaninate, an equivalence point was found of 1.4 mmol anhydroelastase for each mmol turkey ovomucoid inhibitor. This compares favourably with the equivalence of 3.5:1 determined for anhydrochymotrypsin using the same inhibitor s. Although detailed kinetic analysis of the binding of anhydroelastase and turkey ovomucoid inhibitor was not attempted, the data suggest that binding of the protein inhibitor to the enzyme's active site cannot be dependent on covalent interaction of the active centre serine hydroxyl with the amide carbonyl of the inhibitor as has been suggested by Finkenstadt and Laskowski ~.

Apart from the use of somewhat smaller crystals the X- ray procedures adopted were the same as in earlier successful substrate binding studies 2. Figure la shows the difference electron density map calculated with the coefficients:

(IFlanhydro--IF]~,,~y0 exp i~o~y,

For comparison purposes Figure lh shows the same region of the native-tosyl difference map. The principal feature common to both maps is the removal of the tosyl

group which allows His-57 to take up a position hydrogen bonded to Asp-102. In the native enzyme ~° a large positive peak corresponding to a sulphate ion (SU-2) appears 0.34 nm from O7'-195 and 0.40 nm from N~'2-57, a feature also found in crystals of ~-chymotrypsin ~ ~ and in crystals of benzamidine-inhibited trypsin 12. In the anhydroelastase difference map (F igure la) no large 'sulphate' peak appears showing if not the absence of the solvent ion, then an increase in its thermal motion due to the loss of the hydrogen bond to O ~' of Ser-195. At 0.35 nm resolution it is not possible to give precise values for the changes in the main-chain conformation associated with the formation of the double bond between C ~ and C ~ of residue 195 in the anhydro form of the enzyme. The indications available from the anhydroelastase difference map would suggest that these changes are minimal. Indeed, Huber et al. ~3 reported a movement of only 0.1 nm for C t~ from its position in the structure of native trypsin (EC 3.4.21.4) and changes of 2 '~, 9 ~, 11 ° in ~0, ~9, ¢,~ respectively in their careful study of anhydrotrypsin concluded some time after these experiments with anhydroelastase were completed. Although these changes are small they exist and must have an effect on the positions of Asp-194 and Gly-196 thus modifying, albeit slightly, the binding of substrates.

~, ~. ~'~ .2

~ . . ~ ,~, " "~ "

,

N o t e s to the E d i t o r

Although the difference Fourier calculations using the anhydro and tosyl elastase diffraction data show conclusively that the active centre serine has been successfully dehydrated, the experiments using the substrate-soaked crystal failed to reveal any appreciable binding. Since a hexapeptide concentration some 20 times that of the K~ 4 was used in the soaking experiment, we conclude that the peptide binding in the crystal, with its concentrated salt environment, must be considerably weaker than in free solution. Unfortunately, the lack of good quality crystals meant that further experiments to optimize soaking conditions with this and other substrates could not be undertaken.

The possibility that the substrate was hydrolysed early in the course of the crystal experiment by the presence of active elastase is considered unlikely in view of the precautions taken to eliminate this particular problem. Also, had the hexapeptide been hydrolysed, the experiments with product inhibitors 2 would indicate that binding of di- or tri- alanine would have been observed. Without further soaking experiments using a variety of different substrates to ascertain the general applicability of the method to elastase, it would be premature to say that this particular approach to the complete mapping of the enzyme's specificity site is invalid. Whilst Alber et al. t 5

b

t

i~r~1- "11

Figure 1 (a) Stereo drawing showing the difference in electron density in the active site region between anhydro and tosyl elastase. Positive electron density is shown with full lines; negative electron density is indicated by dashed contours. The position of the tosyl group linked to serine 195 is shown as is the side chain of histidine 57 both in its position in the native (see positive peak) and tosyl (lower negative peak) elastase structures. The atomic coordinates used for this computer drawing were taken from Sawyer et al.10. The contour level was set at 1/5th of the maximum density and only the first positive and negative contours have been drawn on each of 12 sections. (b) A similar drawing to (a) but showing the difference in electron density between native and tosyl elastase. The large positive peak adjacent to the tosyl 'ring' represents a sulphate ion which binds to the native but not to the tosylated or dehydrated (anhydro) foetus of the enzyme

Int. J. Biol. Macromol., 1982, Vol 4, June 251

Notes to the Editor

have shown that the low temperature trapping of substrates in the active site of an enzyme is feasible, doubt cast by the recent work of Hassall, Johnson and Roberts ~ ~' about the mode of substrate binding proposed by Shotton 2 makcs these physiological temperature studics essential.

References

1 Gertler, A. and Hofmann, "f. Can. J. Biochem. 1970, 48, 384 2 Shotton, D. M., White, N. J. and Watson, H. C. Cohl Spring

ttarhor Syrup. Quant. Biol. 1971, 36, 91 3 Thompson, R. C. and Blout, E. R. Proc. Natl. Acad. Sci. 197(I, 67,

1734 4 Atlas. D., Legit, S.. Schec/cr, I. and Berger, A. FEBS Left. 1970,

II, 281

5 Feinstein, G. and Feeney, R. J. Biol. Chem. 1966, 241, 5183 6 Feinslem, (]. I.EBS Lett. 1970, 7, 353 7 Shotton, D. M. Methods Enzymol. 1970, 19, 113 8 Watson, H. C., Shotton, D. M., Cox, J. M. and Muirhead, H.

Nature 1970, 225, 806 9 |-inkensladl, W. R. and Laskowski, M..I. Biol. ('hem. 1965. 240,

962 l0 Sawyer, L., Shouon, D. M., Campbell, J. W., Wendell, P. L.,

Muirhead, H., Watson, H. C., Diamond. R. and Ladncr, R. C. J. Mol. Biol. 1978, 118, 137

11 Tulinsky, A. and Wright, L. H. J. Mol. Biol. 1973, 81, 47 12 Bode, W. and Schwager, P. J. Mol. Biol. 1975, 98, 693 13 Huber, R., Bode, W., Kukla, D., Kohe, U. and Ryan, C. A.

Biophys. Struct. Mech. 1975, I, 189 14 Atlas, D. J. Mol. Biol. 1975, 93, 39 15 Alber, T., Petsko, G. A. and Tsernoglou, D. Nature 1976, 263, 297 16 Hassall, C. H., Johnson, W. H. and Roberts, N. A. Bioorg. Chem.

1979, 8, 299

Studies on actin fragments obtained by digestion with thrombin, BNPS-skatole and nitrothiocyanobenzoic acid Peter Johnson and Verna B. Stoekmal Department of Chemistry and Colle#e of Osteopathic Medicine, Ohio Unirersity, Athens, Ohio 45701, USA (Receiced 14 October 1981: recised 8 December 19811

1+~, hate isolat ed fi'agments of actin prepared by thrombic digestion (residues 40 374 [1], and 114 374 [II]1, BN PS-skatole cleat,age (residues 87 339 [III]i and nitrothiocyanobenzoic acid treatment (residues 10 2]6 [II/]) using preparatit:e electrophoretic aml chromatographic techniques. Analysis of the filament-lbrming and tropomyosin-hinding properties of demonstrably homo~jeneous

.li'a~lment.s recealed that only .l)'agmellt,s l and II could .lorm l:-aclin-like fihmwnts qlier otlempted remllurali+m, and thal only

fi'agment I couht hind to tropomyosin. These resuhs ill addition to our precious studies on actin.lJ'agments and chemically-modilied intact actin suqgesf that residues 1 86 and 340 374 are not required fi," F-actin.lilamenl .lbrmation, whereas residues 70 86 and/or 340 374 are essential lbr tropomyosin-binding acticity.

Kcvu'ord& /~lllscl¢': actill," tl'Ol~OttO'ositl. thrombin. nilrothiocyanohen=oi~ acid; BNPS-skatole

Introduction

The actin molecule is now recognized as being one of the most widely distributed proteins in eukaryotic cell types, and considerable interest centres on its detailed molecular structure because of its apparent involvement in a variety of biological processes such as muscular contraction, protoplasmic streaming and cell division 1'2

Although the primary structure of rabbit skeletal muscle actin has been known for some time a and the sequences of other actins have recently become available 4's, relatively little is still known about the detailed tertiary structure of actins, with the latest reported X-ray crystallographic map being down to a resolution of 6 A and showing only generalized poly- peptide folding features 6. In the absence of a detailed tertiary structure for actin, chemical modification and polypeptide fragmentation studies have been used in attempts to identify the involvements of various residues and polypeptide regions in the various biological

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activities of actin v t.s so that these results may be correlated with an eventual solution of the tertiary structure of actin in order to establish the location of the different types of protein, nucleotide and cation binding sites that exist on the molecule.

In earlier studies la, we reported on the renaturation and interaction characteristics of fragments of actin obtained by enzymatic digestion, and concluded that residues 1 68 of rabbit skeletal muscle actin were not essential for filament formation, t ropomyosin and myosin binding, whereas residues 208 374 were essential for these properties. In this report, we extend these findings from analysis of the properties of fragments of actin obtained by different fragmentation procedures and conclude that whereas residues i-86 and 34(>374 are not required for filament formation, residues 7(> 86 and/or 340 374 are essential for tropomyosin-binding activity.

Experimental

Actin and tropomyosin were prepared from rabbit skeletal muscle as described elsewhere 10,17. The commercially-obtained reagents used in digestion studies were thrombin (Grade 1, Sigma Chemical Co.), BNPS- skatole* [Pierce Chemical Co.) and NTCB (Eastman Chemicals).

Thrombic digestion of actin was performed according to the protocol of Muszbek et al. is except that the concentration of G-actin in the digestion was 2 mg ml t, 10 NIH units of thrombin were used per mg of actin, and the digestion was performed for 48 h at 2Y'C. After termination of the digestion by incubation for 1 h at 23 C in the presence of I mM DFP, the solution was lyophilized and then redissolved at a concentration of 10 nag ml 1 in 6 M urea, 2?; SDS, 1% /#mercaptoethanol, 50 mM phos- phate, pH 7.0. Samples (40 mg actin) were then subjected to preparative-scale electrophoresis using the apparatus of Koziarz eta/. 19 using a 5.9 x 12 cm column containing a 7.8'!; acrylamide, 0.2°; bisacrylamide gel with a running buffer of 0.2% SDS, 2 mM EDTA, 20 mM sodium acetate, 20 mM thioglycollic acid, 40 mM Tris acetate, pH 9.0. Elution of undigested actin and actin fragments was

* Abbreviations: BN PS-skatole, 2-{2-nitrophenylsulphenyl)-3- methyl-3-bromoindolenine: DFP, diisopropyl fluorophosphate; EDTA, ethylenediamine tetraacetic acid: M,, molecular weight ratio; NTCB, 2- nitro-5-thiocyanobenzoic acid: SDS, sodium dodecyl sulphate; SDS- PAGE, electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate.