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Volume 331. number 1,2, 134-140 FEBS 13024 September 1993 0 1993 Federation of European Biochemical Societies 00145793/93/$6 00

Astacins, , and matrix exhibit identical -binding environments (HEXXHXXGXXH and Met-turn) and topologies and should be grouped into a common family, the ‘metzincins’

Wolfram Bode”,*, Franz-Xaver Gomis-Riithb, Walter Stiickler”

“Mas-Planck-Institut fir Biochemie. Ahteihtng ji’ir Strukturforschung, 82152 Martinsried (be1 Miinchen ), Germaq blnstitut de Biologia Fonamental, Utmersitat Autdnoma de Barcelona, 08193 Belluterra, Barcelona, Spain ‘Zoologisches Institut der C&ziversitiitHeidelberg, Fachrichtwzg Physiologic, 69120 Heidelberg, German?>

Received 9 August 1993

The X-ray crystal structures of two zmc endopeptldases. from crayfish. and adamalysm II from snake venom, reveal a strong overall lopologlcal equivalence and virtually Identical extended HEXXHXXGXXH zinc-binding segments, but m addition a methlomne-containing turn of similar conformatlon (the ‘Met-turn’). which forms a hydrophobic basis for the zmc ion and the three ligandmg hlstidine residues. These two features are also present in a similar arrangement m the matrix metalloproteinases (matrixins) and in the large bacterial Serratra proteinase-like peptidases (serralysins). We suggest that these four proteinases represent members of distinct subfamilies which can be grouped together in a family, for which we propose the designation, metzmcins.

Astacm; Snake venom ; Matrix metalloprotemase; Thermolysm; Zmc family: crystal structure

1. INTRODUCTION ered prototypical representatives of two of these fami- lies [12,13,16]. These structures reveal that both pro- The majority of zinc exhibit a charac- teinases exhibit (in spite of low overall sequence similar- teristic HEXXH sequence integrated into an ‘active-site ity) significant topological equivalence and especially a helix’. The two residues serve as zinc ligands, virtually identical zinc environment, including a com- and the glutamic acid residue polarizes a water molecule mon ‘Met-turn’. A new structure analysis of the alkaline involved in nucleophilic attack at the scissile metalloproteinase isolated from Pseudomonas aerugi- bond. These features were first examined in the struc- nosa [23], belonging to the serralysins, shows that this ture of [1,2]. In this proteinase and in some proteinase shares these features. The matrixins also con- other closely related bacterial of the thermoly- tain the extended zinc-binding signature and, in addi- sin family [3,4], another glutamic acid which is found 20 tion, conserved sequence motifs. Structure and se- residues after the second histidine forms the fourth zinc quence comparisons of these four metalloproteinase ligand; this glutamic acid is likewise part of a conserved families (serralysins, , , and matrix- NEXXSD segment [5]. ins) give evidence that they can be grouped together into More recently, a considerable number of zinc en- a common family which we suggest be called ‘metz- dopeptidases have been sequenced where the zinc bind- incins’ due to the common zinc-binding region and ing motif is extended to HEXXHXXGXXH [6-S]. methionine turn. These peptidases include the astacins [9-131, the snake venom zinc endopeptidases [14,15] now called the 2. MATERIALS AND METHODS ‘adamalysins’ [ 161, the large bacterial S’erratia - like extracellular metalloproteinases (serralysins; [l7- The topological similarities of the crystal structures of astacin. 19]), and the matrixins (matrix metalloproteinases, II and thermolysm have been investigated by visual in- spection and using the program OVRLAP [24] which minimizes spa- mammalian [20&22]). tial distances as well as deviations of segmental orientations. Quite We have solved the catalytic domain crystal struc- generous equivalence probability weights are chosen for the spatial tures of astacin and adamalysin II which can be consid- and the orientational part (E, = Ez = 4.0 A). Sequence alignments were performed using the program CLUS- TAL Implemented m the software package HUSAR at the Deutsches Krebsforschungszentrum (Heidelberg) as described m detail elsewhere [IO]. These alignments were modified according to topological equiva- *Corresponding author. Fax: (49) (89) 8578 3516 lences obtained by OVRLAP. The pre-ahgned sequences were then

134 Published by Ekrvler Scrence Pubhshers B L/: Volume 331, number 1,2 FEBSLETTERS September 1993 analyzed with the program PAUP version 3.0s for relattonshtps and (proforms) presumably adopt quite different (and possi- evolutionary divergence of the involved [25]. bly inactive) conformations. This is clearly different from the situation in adamalysin II, where the N-termi- nus is freely exposed, allowing covalent coupling to 3. RESULTS AND DISCUSSION other preceeding domains. Astacin [12.13] and adamalysin II [16] are topologi- Both enzymes exibit virtually identical active-site en- tally similar molecules (Fig. la and b). Both consist of vironments (Fig. la; Fig. lb; Fig. 2a). Adamalysin II two domains separated by a long active-site cleft with and astacin have an ‘ helix’ comprising the the catalytic zinc ion at its basement. In both molecules, HEXXH zinc-binding consensus sequence, in common the ‘upper’ domain, essentially comprising the N-termi- with thermolysin. In contrast to the bacterial , nal chain parts is organized in relatively regular second- however, this helix terminates abruptly at a glycine res- ary structure elements, whereas the ‘lower’ domain idue (Gly-99 in astacin. Gly-149 in adamalysin II, see formed by the C-terminal segment is organized in a Table I) three residues after the second histidine residue, more irregular manner, with both chains, however, end- and the peptide chain turns sharply away from the helix ing in a C-terminal helix and an extended segment axis towards the third zinc-liganding histidine of the clamped to the upper domain. Furthermore, both upper ‘signature’ sequence (His-102 in astacin, His-152 in domains consist essentially of a central highly twisted adamalysin II). The following residue of astacin, Glu- five-stranded P-pleated sheet of identical connectivities 103, forms the aforementioned salt bridge with the N- flanked by two long helices on its concave side. The terminus, thereby locking in the active site conforma- adamalysin II sheet is, however, covered on its convex tion: the corresponding Asp-153 of adamalysin II is in side by two additional helices and by longer multiple strong hydrogen bond contact with the active site base- turn structures, thus giving rise to a shift of the zinc- ment (Met-turn, see below). binding residues to higher sequence numbers in adama- The peptide chains following these signature seg- lysin II (His-142, His-146, His-152) compared with as- ments in both metalloproteinases comprise the much tacin (His-92, His-96, His-102; see Table I). more irregularly folded lower domains. After a loop of Both polypeptide chains start in the lower domain. 10 residues interconnected by a disulphide bridge, the However, the N-terminus of astacin is completely bur- adamalysin II chain turns back to the active site and ied in the lower domain, with its ammonium group forms a 14 tight turn comprising Cys-164Ile-1655 involved in a solvent-mediated salt bridge formed with Met-166-Arg-167, with the methionine side chain pro- Glu-103 (the residue immediately following the third viding a hydrophobic base beneath the three zinc-li- zinc-liganding histidine). As a consequence, the (active) ganding histidine side chains (see Fig. 2b). On the other astacin conformation seems to require a precisely proc- hand, the astacin chain, only after passing a multiple essed N-terminus; N-terminally elongated molecules turn structure of 41 residues, enters a Ser-1455Ile-146-

Table I Sequence ahgnment of three representattves of each subfamily of the metzincins

Zinc-binding Signature Met-turn

Astacm TIIHELMHAIGFYHEHT(33) EDYQYYSIMHYGKYSF mouse TIEHEILHALGFFHEQS(35) TPYDYESLMHYGPFSF BMPl” IVVHELGHVVGFWHEHT(35) ETYDFDSIMHYARIJTF

Serralysin TFTHEIGHALGLSHPGD(l6) EDTRQFSLMSYWSETN Pseudomonas alkb TLTHEIGHTLGLSHPGD(16) EDTRAYSVMSYWEEQN Erwina b TLTHEIGHALGLNHPGD(16) EDTRQFSIMSYWSEKN

Collagenase f VAAHELGHSLGLSHSTD -mIGALMYPSYTFS n VAAHEFGHSLGLAHSSD -mPGALMYPNYAFR Matrilysin AATHELGHSLGMGHSSD --PNAVMYPTYGNG

Adamalysin II TMAHELGHNLGMEHDGK(2) RGASL-CIMRPGLTPG Atrolysin TMAHELGHNLGMEHDGK(2) RGASL-CIMRPGLTKG Trimerelysm TMTHELGHNLGMEHDDK(3) CEA--mCIMSDVISDK

Only the secttons containing the zinc-binding histidine residues and the Met-loop are shown. The histidme zinc-hgands. the catalytically active glutamic acid residue, the residue directly following the thud His ligand, the conserved Met residue, and the two positions after the Met are in bold face. The numbers in brackets indicate the intervening residues. a Human bone morphogenettc protein I and a homologue from the nematide worm, C aenorhabitrs ekgans. ‘Pseudomonas aeruginosa alkaline proteinase. ’ Erwinia chrysanthemi protease b.

135 Volume 331, number 1.2 FEBS LETTERS September 1993

a

b

Fig. 1. Ribbon stereo plot of (a) astacin and (b) adamalysin II using RIBBON [30] (as modified by A. Karshikoff). Both molecules are shown in then ‘standard orientation’ [12]. i.e. with the central active site cleft in the front running from left to right The znc ion and a few residues which are particularly addressed In the text are shown in full and are extra labelled.

Met-147--His-148 tight turn of identical topology com- ion, the corresponding Pro-l 68 of adamalysin II leaves pared with the Met-turn of adamalysin II (Fig. 2a and the fifth zinc position freely accessible (Fig. 2a). Table I). A structure comparison with the program OVRLAP Subsequent to this Met-turn, the astacin and the and visual inspection reveal that about 106 of the 202 adamalysin II chains deviate again. Whereas in the as- adamalysin II residues can be considered as topologi- tacin chain Tyr-149 (the fifth zinc ligand) protrudes tally equivalent with astacin. with the relatively high from the floor of the active site cleft towards the zinc rms deviations of 3.2 A for their CI carbon atoms. Of

136 Volume 331. number 1,2 FEBSLETTERS September 1993 a

I b l . . l --..I.

Fig. 2. (a) Stereoplot displaying the superimposed structures of adamalysin II (bold line) and astacin (dashed line) around their zinc-binding sites (using MAIN [31]). Only a few residues of adamalysin 11 are labelled. (b) Schematic representation of the zinc environment in the metzincins. Identical structures (zinc signature. Met-turn) are shown by a continuous line, variable features by a dashed chain trace. Standard orientation as in Fig. 1. these equivalent residues, however, only 15 are identi- glycine residue, and (iii) the Met-turn comprising an cal, 7 of which are located in the extended zinc-binding SVMS sequence (see Table I). Due to the absence of consensus region and 3 in the Met-turn segment (Table coordinates, however, a quantitative comparison with I). The a-carbons of the 17 residues forming the ‘zinc astacin and adamalysin II is not yet possible. Here also, signature’ and the Met-turn fit with rms deviations of about half of the catalytic domain residues seem topol- only 0.35 A. ogically equivalent, while only a few are identical. The structure of the Pseudomonas aeruginosa alkaline The availability of three spatial structures of zinc- proteinase [23], which is a representative of the serraly- endopeptidases representing distinct subfamilies pro- sins, displays all structural elements characteristic for vide excellent tools for deciphering the structural rela- astacin and adamalysin II: (i) the five-stranded tionships among these enzymes. We have in a first step p-pleated sheet and the two adjacent large ol-helices, (ii) aligned the corresponding sequences using the extended zinc-binding consensus sequence contain- the program CLUSTAL. We have then utilized the in- ing the three histidine residues and the intervening formation obtained with OVRLAP by superimposing

137 Volume 331, number I,2 FEBS LETTERS September 1993 the available three-dimensional structures for detection than originally expected (26% identity within the topol- of topologically equivalent segments. These segments ogically equivalent regions, compare Table II). Further- were arranged by hand and fixed. The intervening se- more. astacin is also close to (22%), but most quence regions were again aligned with CLUSTAL. Al- distant from human collagenase (10%). The though there is no crystal structure available yet for the distance between adamalysin II and serralysin is like- matrixin subfamily, we have also included this group of wise large (11%). The percentages of identical residues enzymes in our alignment, since they all contain the shared by adamalysin II with both astacin and collage- extended zinc-binding motif and share a conserved nase are about 15%. methionine residue seven positions downstream of the In all four subfamilies the zinc-binding segment proposed third histidine zinc ligand. In the absence of follows the common sequence pattern such tertiary structure information, additional second- HEBXHXBGBXHZ. The astacin and the adamalysin ary structure predictions have been taken into account II structures offer an explanation for the conservation in order to achieve optimal alignment. of these bulky. hydrophobic ‘B’ residues due to burying The alignment included 22 representatives belonging of their side chains in the hydrophobic cores of both to the astacin-. adamalysin-, serralysin- and matrixin- molecules. The last residue in this extended signature, subfamilies. A reduced section of the resulting align- Z. which immediately follows the third histidine zinc ment comprising the signature- and the Met-turn re- ligand, differs in all 4 subfamilies, but seems to be gions is displayed in Table I. All residues in a single row strictly conserved within each subfamily (Glu in astac- are considered to be topologically equivalent (either ins, Asp in adamalysins, Ser in matrixins, and Pro in based on crystal structures or solely on sequence align- serralysins). Hence, as pointed out by Jiang and Bond ments and secondary structure predictions). Table II [27], this residue may serve as a label to differentiate gives the lengths of the experimental or assumed topol- between the subfamilies (see Fig. 3). There are presum- ogically equivalent segments used for the alignment, the ably functional reasons for the conservation of these Z identical residues, and the percentage of identical resi- residues within a given subfamily. In both the astacin dues within these topologically equivalent regions for and the adamalysin II structure this residue seems to be four selected representatives as deduced from pairwise involved in active site stabilization; in astacin it is, in alignments. The overall sequence similarity is quite addition. presumably linked to activation. weak, compared with the sequence identities within the As shown in Table I. the matrixins also exhibit a subfamilies that are generally above 30% and where highly conserved Met-turn sequence, XBMX (almost very few single residue insertions and deletions are re- exclusively confined to ALMY). five residues behind the quired for optimal assignment. In contrast, the percent- third histidine. Simple model-building experiments age scores between the families lie in the region which show that if this connecting matrixin segment would run Doolittle [26] has called the ‘twilight zone’ (between along the adamalysin II chain, the adjacent XBMX 15% and 25% sequence identity). However. by taking segment could be placed exactly as observed for astac- into account not only primary structure information, ins, adamalysins, and serralysins (Fig. 2b). but also knowledge about zinc-binding environments Thus, the unifying feature of the four subfamilies (HEXXHXXGXXH and Met-turn), topologies from (besides the common zinc-binding signature) seems to three-dimensional structures, and secondary structure be the Met-turn: this turn, together with the side chains predictions, these percentages (albeit low) are more of the residues at turn positions 2 (Ile, Leu or Val) and meaningful. Surprisingly, there seems to be a much 3 (Met). obviously plays an important role in the struc- closer relationship between serralysins and matrixins tural integrity of the active site of these enzymes. The side chain of the following residue (at position 4 of the turn), in contrast, is, in all three known tertiary struc- Table II tures, directed away from the active site towards the Percentage similarity scores between the different subfamtlies of the bulk solvent. It varies not only between the 4 subfami- metzmcins calculated for four selected representatives lies, but also within 2 of the subfamilies (see Table I), AST COL SERR ADA which indicates its relative unimportance for the struc- tural integrity. AST X 118 (12) 165 (37) 139 (20) The residue immediately following this 1.4-tight turn COL 10.2 X 166 (44) 133 (21) is found to be strictly conserved in 3 of these 4 subfam- SERR 23.4 ‘6.5 X 158 (18) ADA 14.3 15.8 11.3 X ilies ( in all astacins and in all serralysins, but in all matrixins known so far), and less con- AST stands for astacm, COL for human fibroblast collagenase, SERR served (mostly proline) in the adamalysins (Table I). In for serralysm, and ADA for adamalysin II. The values m the upper the astacins, and very likely in the serralysins, this tyro- triangle Indicate the number of topologically and/or sequentially equivalent sequence positions and (in brackets) the identical restdues sine represents the fifth zinc ligand (which in the struc- within these eqmvalent stretches; those in the lower triangle indicate ture of the present serralysin structure [23] seems to be the percentage identity. slightly displaced by a bound peptide compared with its

138 Volume 33 1, number 1.2 FEBS LETTERS September 1993

tharmolysms Short Zn-bIndIng cO”se”S”S sequences HEXXH collagenaset and NEXXSD collagenase” d pump1

astac1n

meprin a mouse meprl” a rat PPH meprm R rat astacms Met-turn SBMH-Y sequence BMPI man Z=E HEXBHXBGXHZ tolloid Met-turn BMPl worm BP10

SPAN

HCE LCE

uvs.2 43adamalysins Fig. 3. Structural relattonships between the zincins (HEXXH zinc-endopeptidases) and the metzincins (HEXBHXGBGXHZIMet-turn metallopro- teinases). Left panel: Phylogram of 24 zmc-endopeptidases as obtamed by a maximum parsimony analysts of pre-aligned amino acid sequences using the program PAUP 3.0s [25]. The options employed were: branch and bound search, sequence addition: closest. The results of bootstrap analysis (100 replications) are depicted. The length of the horizontal lines reflect distance scores, t.e the number of ammo acid substttutions. Right panel: Topological and sequential features typical for the zincms and metzincms. B stands for bulky, apolar residues; Z represents the residue following the thud histidine zinc ligand. . Pseudomonas aerugrnosa elastase: thermolysm t, thermolysin from Bucrllus thermoprofeolyt- ICUS; thermolysin s. thermolysin from Buczllus stearothermophilus, collagenase f, human fibroblast collagenase; collagenase n. human collagenase, pumpl, matrilysin-‘punctuated’ or -putative metalloprotemase; serralysin, Serratla proteinase; erwima b, Erwinirr chrysanthemz protease b; erwima c, Erwzma chrysanthemi protease c; PPH, human AbzOH-peptide ; BMPl, human bone morphogenettc protein 1 and a homologue from the nematode worm, Cuenorhabitls elegans, BP10 and SPAN, blastula protein from sea urchin: HCE and LCE. fish embryonic hatching enzymes; UVS.2, frog embryonat protein: trimerelysm II, proteinase from TrznlrreszrrztsJlclvortdu. For references, see [10,12].

position in astacin). In adamalysin II, and very proba- active site helix extends far beyond the short HEXXH bly also in the matrixins, the equivalent main chain zinc-binding signature. Its polypeptide chain returns to position is occupied by a proline, which does not extend the active site only after running through a much longer into the zinc coordination sphere. loop to donate the third zinc-liganding residue (Glu- Thus, focussing on conserved tyrosine residues alone 166). The rest of the lower thermolysin domain is quite [27] may lead to incorrect alignments. Interestingly. the a-helical and bears no relation at all to any of the lower presence of a fifth tyrosine zinc ligand correlates with metzincin domains. Notably, in thermolysin the region the absence of a ‘cysteine switch region’ [28,29] in the approximately corresponding to the methionine sites of proparts of the corresponding metalloproteinases, pre- astacin and adamalysin II is occupied by the serine sumably reflecting unwanted interference of this tyro- (Ser-169) of the consensus NEXXSD sequence which is sine residue with the mercapto-group of a cysteine pep- otherwise structurally unrelated to the Met-turn. tide. A total of 78 adamalysin II and 62 astacin residues In comparing the metzincins with the archetypical are topologically equivalent to thermolysin, with rms zinc-endopeptidase thermolysin, it becomes apparent deviations of 3.5 A and 2.8 A. respectively, for the that there are some important similarities despite the corresponding a-carbon atoms; 12 and 5, respectively, overall structural variation. The helical part of the zinc of these equivalent residues are chemically identical (the binding signature of the metzincins is very similar to the higher numbers of equivalent residues in the astacin corresponding XXHEXXHX segment in thermolysin. thermolysin superposition as compared to our previous Furthermore, the upper domain of thermolysin con- paper [ 131 results from less rigorous equivalence para- tains a b-pleated sheet, the first four cleft-sided strands meters). of which are topologically identical to their metzincin ln summary this means that the metzincins are struc- conterparts. However, the upper domain of thermolysin turally related to the thermolysin-like enzymes forming is much larger and has N-terminal appendices, and the an HEXXH zinc endopeptidase superfamily, for which

139 Volume 33 1, number 1.2 FEBSLETTERS September 1993 we suggest the name ‘zincins’. This relatedness has been [5] Titan]. K., Hermodson, M.A.. Encsson, L.H., Walsh. K.A. and corroborated by including the thermolysin family into Neurath. H. (1972) Nature New Biol. 238. 35-37. the structure based alignment mentioned above. This [6] Stdcker, W., Ng. M. and Auld. D.S. (1990) Blochenustry 29, 10418-10425. alignment has then been used to analyze the relation- [7] Murphy. G.J.P., Murphy, G. and Reynolds. J.J. (1991) FEBS ships between a total of 24 different zinc-endopeptidases Lett. 289. 47. using the maximum parsimony method [25]. As already [8] Rawlings. N.D and Barrett. A.J. (1993) Blochem. J. 290, 205- anticipated, the enzymes form clusters corresponding to 218. [9] Stdcker, W., Sauer, B. and Zwilhng. R. (1991) Blol. Chem. the different subfamilies. Also by this analysis the ser- Hoppe-Seyler 372, 385-392. ralysins appear rather closely related to the matrixins, [lo] Stdcker, W.. Gomis-Riith. F.X.. Bode, W. and Zwilhng. R and the serralysin-matrixin-subcluster stands with (1993) Eur J Biochem. 214. 215-231. equal rights with the astacin- and the adamalysin sub- [I l] Dumermuth. E.. Sterclu. E.E . Jiang. W.. Wolz. R.L.. Bond. J.S.. clusters. These three subclusters are, however, more dis- Flannery, A. and Beynon. R J (1990) J. Biol. Chem. 266.21381- 21385. tantly related to the thermolysin-like proteinases (see [12] Bode, W.. Corms-Riith. F.X , Huber, R , Zwilling, R. and Fig. 3). StGcker. W. (1992) Nature 358. 164-167. The zincin superfamily comprises at the moment the [13] Corms-Ruth, FX., StGcker. W., Huber, R., Zwilling, R. and thermolysins and the metzincins and is characterized by Bode, W. (1993) J. Mol. Biol. 229. 945-968. [14] Shannon, J.D.. Baramova. E N . BJarnason. J.B. and Fox. J.W. the short zinc-binding sequence. HEXXH. The met- (1989) J. BIoI. Chem. 264, 11575-l 1583. zincin family, in turn, is formed by the 4 subfamilies [15] Kini, R.M. and Evans. H.J. (1992) Toxlcon 30, 265-293. mentioned, i.e. the astacins, adamalysins, serralysins [16] Gomis-Rhth, F.X.. Kress. L.F. and Bode, W. (1993) EMBO J. and matrixins, and has additionally in common the ex- (m press). tension of the zinc-binding signature and the Met-turn. [17] Nakahama, K.. Yoshimura. K., Marumoto, R.. Kikuchl. M., Lee, 1,s.. Hase. T and Matsubara, H. (1986) Nucleic Acids Res. Prominent features, by which these subfamilies can be 14, 5843-5855. distinguished from one another, are the Z residue fol- 1181 Delepelaire. P and Wandersman. C. (1989) J. Biol. Chem. 264. lowing the ‘third’ histidine zinc ligand, and the residue 9083-9089. following the 1,4-tight Met-turn (see Fig. 3). [19] Duong, F., Lazdunski. A.. Caml, B. and Murgler. M. (1992) 121. 47-54. [20] Matrisian. L.M. (1990) Trends Genet. 6. 121-125. Acknowledgernenis~ We thank Prof. Dr. Robert Huber, Prof. Dr. [21] Woessner, Jr. J.F. (1991) FASEB J. 5, 2145-2154 Robert Zwdling, and Prof. Dr. Francesc Xavier Avll&s for then con- [22] Nagase, H.. Barrett. A.J and Woessner, Jr. J F. (1992) Matrix tinuous encouragement, Dr. Lawrence Kress and Dr Ulrlch Bau- (Suppl. No. 1). 321323. mann for helpful comments. FXGR would like to acknowledge the [23] Baumann. U.. Wu, S . Flaherty, K M and McKay, D.B. (1993) help provided by a EMBO-long term fellowslup. The financial support EMBO J. (m press) by the Sonderforschungsbereich 207 der Umversitat Mhnchen (WB) [24] Rossmann. M.G. and Argos. P. (1975) J. I3101 Chem. 250, 7525- and the Deutsche Forschungsgemeinschaft (Sto 185/l-3) IS gratefully 7532. acknowledged. [25] Swafford, D.L. (1991) PAP: Phylogenetic analysis usmg parsi- mony: version 3.0s. Champaign, Illinois Natural HIstory Survey [26] Doolittle, R.F. (1985) Sci. Am 253, 74-83 REFERENCES [27] Jiang, W. and Bond. J.S. (1992) FEBS Lett 312, 110-114. [28] Springman. E.B., Angleton. E.L.. Birkedal-Hansen, H. and Van Matthews, B.W.. Jansonius. J.N., Colman, PM., Schoeborn. Wart, H.E. (1990) Proc. Natl. Acad. Sci. USA 87, 364-368. B.P. and Dupourge. D. (1972) Nature New Biol. 238, 3741. [29] Hate, L.A.. Fox, J.W and BJarnason. J.B. (1992) Biol. Chem. Matthews. B W. (1988) Accts. Chem. Res. 11, 333-340 Hoppe-Seyler 373, 381-385 Thayer, M.M., Flaherty, K.M. and McKay. D.B. (1991) J. Mol. [30] Pnestle, J.P (1988) J. Appl. Chrystallogr. 21. 573-576. Biol. 266, 2864-2871. [31] Turk. D. (1992) Ph.D. Thesis, Umversity of Munich, Mumch. Pauptlt, R.A.. Karlsson. R., Picot, D.. Jenkins, J.A., Niklaus- Reamer, A.S. and Jansonius, J.N. (1988) J. Mol. Biol. 199, 525$ 537.

NOTE ADDED IN PROOF

Our newly determined catalytic domain structure of a human collagenase now confirms that the collagenases share the characteristic overall topology and identical zinc environment with the other metzincins, and exhibit an active-site geometry very similar to the adamalysins.

140