1
Substrate specificity of a metalloprotease of the pappalysin family revealed by
an inhibitor and a product complex.
Raquel García-Castellanos a,c, Cynthia Tallant a,c, Aniebrys Marrero a, Maria Solà a,
Ulrich Baumann a,b,* & F.Xavier Gomis-Rüth a,*
of the
Departament de Biologia Estructural; Institut de Biologia Molecular de Barcelona, C.S.I.C.;
c/ Jordi Girona, 18 – 26; 08034 Barcelona and Parc Científic de Barcelona; c/ Josep Samitier, 1 – 5; 08028
Barcelona (Spain).
a
b
On sabbatical leave from the Department für Chemie und Biochemie, Universität Bern, Freiestrasse 3; CH-3012
Bern (Switzerland).
c
These two authors contributed equally to this work and share first authorship.
* Corresponding authors. UB: Tel. +41 31 631 4320/4324; Fax +41 31 631 4887; e-mail:
ulrich.baumann@ibc.unibe.ch. FXGR: Tel. +34 934 006 144; Fax. +34 932 045 904; e-mail: xgrcri@ibmb.csic.es.
Short title : Substrate specificity of ulilysin metalloprotease
Keywords: metalloprotease; X-ray crystal structure; cancer; ulilysin; IGF; metzincin; astacin; serralysin; IGFBP
protease; pappalysin; batimastat; enzyme-inhibitor complex.
2
Abstract
Human pappalysin-1 is a multi-domain metalloprotease engaged in the homeostasis of insulin-like growth factors and
the founding member of the pappalysin family within the metzincin clan of metalloproteases. We have recently identified an
archaeal relative, ulilysin, encompassing only the protease domain. It is a 262-residue active protease with a novel 3D
structure with two subdomains separated by an active-site cleft. Despite negligible overall sequence similarity, noticeable
similarity is found with other metzincin prototypes, adamalysins/ADAMs and matrix metalloproteinases. Ulilysin has been
crystallised in a product complex with an arginine-valine dipeptide occupying the active-site S1’ and S2’ positions and in a
complex with the broad-spectrum hydroxamic acid-based metalloprotease inhibitor, batimastat. This molecule inhibits mature
ulilysin with an IC50-value of 61M under the conditions assayed. The binding of batimastat to ulilysin evokes binding to
vertebrate matrix metalloproteases but is much weaker. These data give insight into substrate specificity and mechanism of
action and inhibition of the novel pappalysin family.
Abbreviations: 3D, three-dimensional; ADAM, A disintegrin and a metalloprotease; CCP, complement control protein; IGF,
insulin-like growth factor; IGFBP, IGF-binding protein; IGFBPP, IGFBP protease; LNR, Lin12-Notch repeat; MP,
metalloprote(in)ase; MMP, matrix metalloproteinase; ZBCS, extended zinc-binding consensus sequence.
3
Introduction
The cell cycle and cell processes like survival/apoptosis, migration, differentiation and proliferation are regulated by
the circulating insulin-like growth factors (IGF)-I and –II [1, 2]. They act upon somatic growth and development but also on
several diseases, among them atherosclerosis and cardiovascular disease, diabetes and cancer [3, 4]. IGFs are mostly
sequestered in binary complexes with soluble IGF-binding proteins (IGFBP-1 to –6) [5, 6], which are widely spread factors with
distinct binding properties and distribution in physiological fluids. IGFBPs consist of conserved N- and C-terminal domains
linked by a non-conserved connecting mid-region and are both required for efficient growth-factor binding [7]. Due to their
higher affinity, IGFBPs antagonise binding of IGFs to their receptors and regulate IGF transport between intra- and
extravascular spaces [8]. In order to carry out their physiological mitogenic and metabolic tasks, IGFs must be released from
their IGFBP complexes. Proteolytic processing of IGFBPs liberates the cognate IGF and is exquisitely regulated by specific
IGFBP proteases (IGFBPPs), which are present both in circulation and in interstitial fluids and whose activity is kept in check
by endogenous protease activators and inhibitors [9-11]. Proteolytic susceptibility of IGFBPs may depend on ligand binding
[10] and cleavage can occur in an unspecific manner, i.e. under complete degradation of the IGFBP, or rather following
selective limited proteolysis, mostly in the mid-region. Among IGFBPPs studied in vitro, although not always at physiological
protease:substrate ratios, there are serine proteinases (complement protein 1s; kallikreins like prostate specific antigen, hK2
and 7S nerve growth factor; plasmin; a HtrA-related protease; seminal plasma trypsin; cathepsin G and neutrophil elastase;
and thrombin), cysteine proteinases (cathepsins and calpain), and metzincin metalloproteases (MPs) from the
adamalysin/ADAM (ADAM-9, –12 and -28) and the matrix metalloprotease (MMP) families (MMP-1, -2, -3, -7, -9, -11 and –19)
[8, 10, 12-15]. These two latter families have been implicated in the physiology of and pathologies associated with other
growth factor families as they mediate shedding of the ectodomains of membrane-anchored growth factors, cytokines and
receptors to increase their circulating forms [16-18].
In addition to MMPs and adamalysins/ADAMs, another MP has been shown to specifically inactivate IGFBPs, namely
human PAPP-A, alias pappalysin-1 orIGFBP-4 protease [19-21]. This MP was originally identified as an antigen present in
human plasma during pregnancy [22]. It is ubiquitously expressed and plays central roles in ovarian follicular development,
myogenesis, human embryo implantation and wound healing [23]. The importance of PAPP-A during foetal growth has been
demonstrated with knock out mice, which show severe dwarfism [24] and a strikingly similar phenotype to IGF-II null mice, and
in humans by the finding that neonatal weight correlates with levels of maternal PAPP-A [25]. Mature PAPP-A is a
glycosylated multidomain protein of 1,547 residues and 82 cysteine residues that specifically hydrolyses human IGFBP-4 in an
IGF-dependent manner. Only human IGFBP-5 and bovine and porcine IGFBP-2 have been identified as further substrates
[21]. Apart from its proteolytic domain, three Lin12-Notch repeats (LNR-1, -2 and -3), which are modules that regulate ligandinduced proteolytic cleavage of the Notch receptor, and five complement control protein modules (CCP1-5) have been
identified in PAPP-A [26]. This protease is the functionally best characterised IGFBPP and distinct from any of the MP families
known for their 3D structure [20]. Therefore, it has been appointed the founding member of the pappalysin family of MPs,
which further comprises the paralogue PAPP-A2, alias PAPP-E or pappalysin-2, which cleaves IGFBP-5 and IGFBP-3 [27-29].
4
These proteinases present an extended zinc-binding consensus sequence (ZBCS), HEXXHXXGXXH/D (amino-acid one-letter
code; X for any residue), that is characteristic for the metzincin clan of MPs and comprises three of the metal-liganding protein
residues (three histidines or two histidines and an aspartate) and the general-base glutamate [30]. The importance of this
latter residue in PAPP-A has been experimentally corroborated as substitution of alanine for the acidic residue abolishes
activity. Sequence analyses further suggested the presence of a conserved methionine residue as found in a further structural
hallmark element of metzincins, the Met-turn. These findings led to ascribe pappalysins to the later clan [20, 21, 30-32].
The 3D structural analysis of full-length PAPP-A or any fragment or vertebrate orthologue has not succeeded to date
hindered by (i) difficulties in obtaining sufficient amounts of homogenous protein, (ii) the large number of cysteine residues, (iii)
the high degree of glycosylation and (iv) a multimodular structure and therefore potential inherent flexibility due to linking
segments [33]. The probably most interesting function of the protein is its proteolytic potential because of its implications for
mechanistic studies and for the search of specific inhibitors with the aim of modulating the protease activity and thus the IGF
axis. Therefore, structural studies on the protease domain alone would already be of great interest. However, domain
boundary assignment within a high-molecular-weight polypeptide potentially harbouring several folding units and functions is,
in general, a formidable task [34]. An attempt to delimitate the protease domain within the 170-kDa PAPP-A has been
performed based on the 3D structures of the catalytic domains of astacin, matrilysin, adamalysin II and aeruginolysin and
secondary structure predictions for PAPP-A [20]. These studies suggested a ~300-residue protease domain to approximately
run from Lys352 to Tyr664 (see Fig. 1), but no experimental evidence is available nor could this segment be produced and
purified independently. It is in such cases that the study of a homologue potentially displaying the function of interest within a
single domain may prove helpful. To this aim, we have identified a whole series of novel putative pappalysins by bioinformatic
approaches and have analysed such a putative orthologue, Methanosarcina acetivorans ulilysin, the only archaeal pappalysin
found to date. We have recently shown that it undergoes a calcium-mediated autolytic activation entailing the loss of a 60residue potential pro-domain and of 20 residues at the C-terminus from the 38-kDa zymogen to render the 29-kDa mature
form. Ulilysin shows a preference for substrates with an arginine in P1’ [35]. After an initial report [35], we present hereby the
detailed full structural analysis of this protease in a product complex and in complex with the hydroxamic-acid-type
metalloprotease inhibitor, batimastat alias BB-94. This inhibitor was discovered by British Biotech Ltd. and it was originally
identified as a potent but unspecific inhibitor of certain MPs [36, 37]. It was shown to significantly inhibit ulilysin [35]. The
present studies enable us to assess the structural determinants of the specificity and inhibition of this model pappalysin. We
further discuss the implications for other members of the pappalysin family and the structural relation with other metzincins.
5
Materials and methods
2.1.Protein production and inhibitory analysis of batimastat
Full-length 38,370-Da protein MA3214 (referred to as proulilysin) with a cysteine-to-alanine mutation at position 269
(SwissProt database (SP) access code Q8TL28), as well as a variant in which methionine residues was replaced by
selemethione (SeMet), was overexpressed in Escherichia coli and purified as described [35]. Active 29-kDa ulilysin was
obtained through autolytic limited proteolysis of the zymogen upon incubation with 5mM CaCl 2 and subsequent purification
with size-exclusion chromatography. Inhibition studies in vitro with batimastat were performed with the DQTM EnzCheck assay
kit employing a fluorescein conjugate of pig-skin gelatin as a substrate (Molecular Probes). Therefore, purified ulilysin
(0.8mg/ml) was incubated to a final enzyme concentration of 1μM with 150μl of a reaction mixture containing 50μg/mL of
substrate in 20mM Tris·HCl pH 6.5. The cleavage reaction was allowed to proceed for 2h at 25ºC while the fluorescence was
monitored using a Victor microplate reader fluorimeter (Perkin Elmer Life Science) set for excitation at 490nm and for emission
detection at 520nm. Inhibitor concentrations ranged from 10μM to 2mM. The analysis was repeated thrice and the determined
average IC50-value was 60.63.3μM.
2.2.Crystallisation and X-ray diffraction
Morphologically indistinguishable prism-shaped crystals of two different types (form I and II) were obtained after
several weeks to months at 20°C by the vapour-diffusion crystallisation method from sitting drops consisting of 1μL of
proulilysin (5 mg/mL in 30mM Tris·HCl pH 7.5, 2mM dithiothreitol, 100mM NaCl), 1μL of reservoir solution (18% PEG 8000,
0.1M 2-morpholinoethanesulfonic acid, pH6.5, 0.2M CaCl2) and, optionally, 0.2μL of 0.1M spermidine or 30% 2,4methylpentanediol as additives. These crystals contained the mature 29-kDa ulilysin form as a result of autolytic activation
within the crystallisation drop favoured by the presence of calcium, as determined by N-terminal sequencing and mass
spectrometry [35]. Form I crystals could be easily indexed as C222. However, detailed inspection of the diffraction patterns
revealed that they actually belonged to the orthorhombic space group P21212 with same cell constants (a=49.6±0.6Å,
b=126.0±1.4Å and c=87.3±1.4Å for the different crystals measured; VM= 2.4; 47% solvent content [38]) and with two
molecules in the asymmetric unit, but with very week reflections corresponding to Miller-indices h+k≠2n. In several sections of
the reciprocal space sampled, these reflections were even completely missing. This pseudosymmetry correlated with a very
strong off-origin peak at u=v≈0.5, w=0 in a native Patterson map, accounting for a translation relating the two molecules in the
asymmetric unit in the primitive setting. This is equivalent to a non-crystallographic dyad parallel to cell axis c. Form II crystals
belonged to space group C2221 with cell constants a=118.8±0.5Å, b=60.2±0.5Å and c=169.3±2.9Å and also two molecules
per asymmetric unit (VM=2.6; 53% solvent content). A cryo-cooling protocol was established to protect crystals from radiation
damage during data collection consisting of a stabilisation in a harvesting solution (22% PEG 8000, 0.1M MES pH6.5, 0.2M
CaCl2) and then of a soak in a cryo-protector (22% PEG 8000, 0.1M MES pH6.5, 0.2M CaCl2, 26% glycerol) just before flash
cryo-cooling the crystals with liquid nitrogen. The complex between ulilysin and batimastat was obtained soaking form-II
crystals in a solution containing 1mM batimastat, 40% PEG 8000, 0.1M MES pH6.5, 0.2M CaCl2 for sixty hours. Cryo-
6
protection was achieved with the latter solution further implemented with 24% glycerol. Complete diffraction data sets were
collected each from a cryogenised single crystal at 100K on CCD-detectors at beamlines ID13, ID14-2, ID14-4, BM16 and
ID23-1 from the ESRF (Grenoble, France) and on X06SA beamline of the Swiss Light Source (Villigen, Switzerland). Data
were processed with MOSFLM [39] or XDS [40] and scaled, reduced and merged with SCALA [41].
2.3.Structure solution and refinement
To obtain heavy-atom/ion derivatives and solve the phase problem, ulilysin crystals were treated first for 3 hours with
the harvesting solution and then overnight with the same solution implemented with 10mM 1,10-phenathroline. Subsequently,
crystals were carefully washed with harvesting solution just preceding soaks with heavy-ion compounds. This strategy had
been previously employed to successfully remove the catalytic zinc ion from other MPs prior to derivatisation with mercury or
platinum compounds [42]. Complete multiple-wavelength anomalous diffraction (MAD) experiments were carried out on both
native (thus, zinc-containing) as well as putative mercury derivatives of both crystal forms. In all cases, XANES fluorescence
scans had previously confirmed the presence of the corresponding metal ions. However, although the positions of the
anomalous scatterers could be determined with programs XPREP/SHELXD [43] and SNB [44], the occupancy was too low to
render sufficient phasing power and interpretable electron density maps. The crystal structure of ulilysin was eventually solved
employing a SeMet derivative of crystal form I. These crystals were obtained under the same conditions as the native ones. A
three-wavelength MAD experiment to 2Å resolution was carried out at ESRF beamline BM16. Furthermore, a high resolution
dataset to 1.7Å was collected from the same crystal. Table 1 provides statistics on data collection and processing. The MAD
data enabled identification of 10 out of the 12 selenium sites present (six per monomer) with XPREP/SHELXD. Phases were
computed with SHARP [45] and subsequently subjected to density modification employing first SOLOMON [46] within the
SHARP package and then DM [47] within the CCP4 suite [48] (see Table 1). This procedure rendered phases of quality high
enough to calculate interpretable electron density maps. Thereupon, manual model building on a Silicon-Graphics graphic
workstation using TURBO-Frodo [49] alternated with crystallographic refinement with REFMAC5 [50] within CCP4 until the
final model was obtained. This model contains protein residues Arg61 to Ala322, as well as one zinc (numbered Zn999) and
two calcium cations (Ca998 and Ca997) for each of the two molecules present in the asymmetric unit (suffixes A and B,
respectively). All residues are placed in most favoured and additionally allowed regions of a Ramachandran plot. Three cispeptide bonds are found in each molecule, Pro218-Pro219, Tyr272-Pro273 and Gly280-Pro281. Each polypeptide chain
contains one disulphide bond (Cys250-Cys277). 587 solvent molecules (Hoh7W-Hoh604W), a fifth calcium cation engaged in
crystal contacts (Ca1W) and six (tentatively assigned) glycerol molecules (Gol2W-Gol7W) are also present in the structure. In
each active-site, a dipeptide (Arg401A-Val402A and Arg401B-Val402B) is found. Accordingly, this structure represents a
product complex of ulilysin.
The structure of ulilysin crystal form II was solved by Patterson-search techniques employing program AMoRe [51]
and data in the 15-to-4Å resolution range from a crystal for which diffraction data had been collected at the Zn 2+ K-edge
absorption maximum at beamline BM16. Table 1 provides statistics on data collection and processing. The protein dimer of
the refined co-ordinates of crystal form I was employed as a searching model. A unique solution was encountered at 103.8,
7
88.1, 276.0 (,, in Eulerian angles), 0.2716, 0.3027, 0.3803 (x,y,z, as fractional unit-cell co-ordinates) after rigid-body
refinement with the routine “fiting” within AMoRe [51]. This solution gave a correlation coefficient in structure factor amplitudes
(CCF) of 59.3% and a crystallographic Rfactor of 39.8% (for definitions, see [51]; second-highest unrelated peak, CCF 34.5%,
Rfactor 49.4%). Initial refinement with REFMAC5 of the appropriately rotated and translated dimer (Rfactor/free Rfactor values of
0.257/ 0.300; for definitions see Table 1) and electron density map calculations enabled to state that (i) the crystallographic
dimer was arranged in the same way as in crystal form I, (ii) the model spanned practically the same range of amino acids in
the two molecules within the asymmetric unit, and (iii) the S1’-pocket was likewise partially occupied by a dipeptide. Due to this
equivalence, this structure was not further refined but employed to solve the enzyme:inhibitor complex with batimastat within
crystal form II by Fourier synthesis. Model building and refinement proceeded as described. All residues of the final model
(Glu63-Leu321 of each chain A and B) are placed in the most favoured and additionally allowed regions of a Ramachandran
plot. The disulphide bond and the three cis-peptide bonds are equivalent to the enzyme:product complex. Two batimastat
moieties (Bat996A and Bat996B) at full occupancy, 460 solvent molecules (Hoh6W-Hoh465W), a fifth calcium cation engaged
in crystal contacts (Ca1W) and four (tentatively assigned) glycerol molecules (Gol2W-Gol5W) complete the structure.
2.4.Miscellaneous
Figures were prepared with TURBO-Frodo, GRASP [52] and MOLMOL [53]. Bioinformatic amino-acid sequence
similarity searches were performed within the MEROPS database (http://merops.sanger.ac.uk) and with servers ProDom
(http://protein.toulouse.inra.fr/prodom.html);
Pfam
(http://www.sanger.ac.uk/software/pfam)
and
PSI-BLAST
(http://www.ncbi.nlm.nih.gov/blast). For the latter, the sequence stretch of human PAPP-A shown in Fig. 1 encompassing the
putative MP region was employed as a bait after exclusion of the two LNR regions. Structural similarity searches were
undertaken
with
servers
DALI
(http://www.ebi.ac.uk/msd),
CE
(cl.sdsc.edu)
and
VAST
(www.ncbi.nlm.nih.gov/Structure/VAST/vastsearch.html). Quaternary structure considerations and interface analyses were
performed with the help of the PISA server (http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver) and with program CNS [54].
Surface complementarity was assessed by means of program SC within CCP4 [55]. Multiple sequence alignments and the
phylogenetic tree in Fig. 1 were calculated with MULTALIN ([56]; http://prodes.toulouse.inra.fr/multalin). The final structure coordinates have been deposited with the Protein Data Bank (PDB access codes 2cki and XXXX) at the European
Bioinformatics Institute (http://www.ebi.ac.uk/msd), Hinxton (UK). During the deposition procedure renaming/renumbering of
certain atoms/residues by the PDB proved unavoidable (see the REMARKS section of the PDB entries for equivalences).
8
Results and discussion
3.1.Polypeptide fold of mature ulilysin
Ulilysin with the point mutation C269A was identified in a systematic cysteine-to-alaine scanning as the construct that
rendered highest reproducibility in purification and crystallisation. The protein was crystallised as a product complex in two
crystal forms belonging to space groups P21212 (form I) and C2221 (form II), respectively, both with two protein molecules
(chains A and B) per asymmetric unit. In order to solve the phase problem, a selenomethionine derivative was obtained that
crystallised isomorphically to the native ones. The structure was solved by multiple-wavelength anomalous diffraction using
synchrotron radiation and form I crystals that led to an excellent experimental electron density map (Fig. 2a). The inhibitor
complex of ulilysin was solved by Patterson-search techniques employing form II crystals. The two molecules within the
asymmetric unit of the product complex crystals can be superimposed with an rmsd of 0.36Å, those of the inhibitor complex
with an rmsd of 0.24Å. Furthermore, the 518 C atoms defined by proper electron density in the product and in the inhibitor
complex dimers can be overlaid with an rmsd of 0.44Å. These numbers indicate close structural similarity of the polypeptide
chains within and between crystal structures. Accordingly, the presentation of results and discussion hereafter, in particular
concerning distance values, will refer to molecule A of the product complex if not otherwise indicated.
Ulilysin evinces an ellipsoidal shape with overall dimensions of about 61(height) x 40(width) x 48(depth) Å (Fig. 2bc)
and an / topology within a single polypeptide chain with a low overall content in regular secondary structure elements: 17%
of the residues are in -strand regions and 21% are helical. MPs can be displayed in a canonical standard orientation [42], i.e.
with the view onto the active-site cleft that traverses horizontally the molecular surface from where the substrate region
preceding the scissile peptide bond is bound (the non-primed side comprising the subsites S1, S2, S3, etc.; [57]) to the where
residues downstream of the target bond are anchored (primed side with subsites S1’, S2’, S3’, etc.; [57]). According to this view,
ulilysin features an electrostatic surface that is mainly negatively charged on the front side, i.e. with the view into the activesite groove (see Fig. 2c). The protein can be subdivided into two moieties, namely an N-terminal upper subdomain above the
active-site groove (NTSD; Arg61-Asn235), rich in regular secondary structure elements, and a rather irregular C-terminal
lower one below the cleft (CTSD; Leu236-Ala322). NTSD starts on the rear of the molecule and enters through strand 1 a
strongly-twisted mainly parallel five-stranded -sheet of strand connectivity –1x,+2x,+2,-1 according to [58] (Fig. 2b). The strands are approximately parallel to the active-site crevice. Following 1, helix 1 runs down diagonally on the back of the
molecule and finishes at the junction with the CTSD. Here, the polypeptide chain follows an extended loop (Lys100-Gly120),
featuring two short 310-helices (2 and 3; see Figs. 1 and 2b) and whose tip almost reaches the bottom of the molecule
resembling a cape covering the back. The peptide chain rejoins the NTSD at the second -strand that parallels 1 but is split
into two segments termed 2 and 3 separated by an insertion (Thr127-Thr136), hereby named LNR-like loop, on the convex
side of the -sheet (Fig. 2b). This insertion juts out from the molecular surface and is folded back such that residue Pro130, at
the tip of the loop, snugly fits into a hydrophobic pillow created by the side chains of Trp165 and Pro166 from the loopsegment connecting strands 3 and 4 (L34) and by Tyr170 immediately preceding 4. After 3, the protein chain runs
back on the convex surface of the sheet shaping the irregular L34 segment (residues Ser144-Tyr170). Of particular
9
importance here is a double main-chain to side-chain interaction performed by Trp165 with Asn172 (Asn172 O1-Trp165 N,
2.9Å; Asn172 N2-Trp165 O, 2.9Å) that contributes to stabilise and anchor L34 to the molecular moiety. At the end of 4,
the chain enters a small -ribbon, also on the convex side of the sheet, constituted by strands 5 and 6. After this ribbon
comes the outermost strand of the sheet and the only one that runs antiparallel, 7. After a bulky protrusion created by loop
L78, the chain enters 8 which leads to 4, the active-site helix that encompasses the first half of the ZBCS. The NTSD
finishes at Asn235 at the end of the latter helix. Structural cohesion of this subdomain is mainly achieved through an extended
hydrophobic cluster placed on the concave side of the -sheet that almost completely spans the width of the molecule. The
cluster is delimited by the internal faces of 1, the active-site helix 4 and the second half of the C-terminal helix 5 within
the CTSD. Hydrophobic residues constituting this cluster are provided by elements 1, L11, L32, 2, 3, 4, L78,
8, L84, 4 and 5. On the convex side of the sheet, a smaller hydrophobic cluster involves residues from the large
L34 segment, L78 and residues from the convex side of strands 1, 4, 5, 7 and 8.
After Leu236 the polypeptide chain enters the CTSD through a double-S loop structure (Trp240-Asp264) that serves
as a scaffold for two close calcium-binding sites (see below and Fig. 2e). This loop is anchored in the back to the main
molecular body by a strong hydrogen bond, Thr302 O1-Asp258 O2 (2.7Å). Subsequently, another loop (Asp264-Asn282)
runs widely on the molecular surface and is cross-linked with the double-S loop by means of a disulphide bond, Cys250Cys277, and with the downstream part of the molecule possibly via a second one, Cys269-Cys297. As the presently described
protein contains an alanine instead of a cysteine at position 269, this latter SS-bridge is not observed. However, atom Cys297
S is found with double occupancy in both molecules of the product complex asymmetric unit.In the inhibitor complex
structure, this atom is even covalently modified by an S-linked -mercaptoethanol moiety, possibly trapped during purification.
These observations account for accessibility and flexibility of the cysteinyl side chain in the present ulilysin mutant. Further
taking into account the distance between the C atoms of Cys269 and Ala297 (6.1Å), as well as the relative orientation of
their side chains, the former data strongly suggest that these two residues be covalently linked in the wild-type protein (Fig.
2b). After segment Asp264-Asn282, the polypeptide chain rejoins the molecular body at Gly283 and follows a trajectory
between the calcium-binding segment and the cape described before, eventually reaching a tight 1,4-turn made up by
residues Asn288-Tyr289-Met290-Asp291. A topologically equivalent turn is systematically found in metzincin MPs and is
termed the Met-turn [31]. Within this turn, Asn288 establishes a hydrogen bond with Gln305 of the C-terminal helix (see
below) and Tyr289 points into the hydrophobic cluster described for the NTSD. These two interactions contribute to stabilise
the Met-turn under the zinc-co-ordinating residues (see below), where it creates a hydrophobic pillow but does not contact the
cation nor its ligands. After the Met-turn, the polypeptide chain turns round to reach the back surface of the protein and enters
the C-terminal helix 5 (Fig. 2b). Approximately at half height, Arg308 points into the interior of the molecule contacting both
Asn235 O1 (3.2Å) and O (2.9Å) atoms, thus contributing with the previously mentioned Asn288-Gln305 interaction to
maintain the cohesion of the molecule. Helix 5 ends up at the molecular surface with the C-terminal Ala322 residue solvent
exposed and in proximity of the N-terminus (15.4Å between the respective C atoms).
10
Interestingly, the two molecules encountered in the asymmetric unit are related by a non-crystallographic rotation
(179.4º in the product complex; 180.1º in the inhibitor complex) around a rotation axis defined by the direction cosines 0.002, –
0.011, –1.000, i.e. almost parallel to cell axis c. This arrangement is observed in both crystal forms studied and positions the
active-site clefts (see below) in front of each other burying them within the dimer (Fig. 2d). The two molecules quite intimately
interact with each other through a total of 129 atoms/33 residues and 127 atoms/32 residues belonging to L78, L34, the
56-ribbon and the calcium-binding double S-loop of molecules B and A, respectively. The contact results in a calculated
iG value (according to [59]) of –13.2 kcal/mol and occludes a surface spanning 2,468Å2, i.e. more than 10% of the total
surface of both monomers (11,955Å2 for molecule A and 11,812Å2 for molecule B according to [54]). The calculated shape
complementarity is 72%, a value which indicates a good fit between interacting surfaces [55]. Both the surface area and the
shape complementarity are similar to those of the protease:protein inhibitor complex of human carboxypeptidase A4 with
latexin ([60]; 2,340Å2 and 71%). In both cases, the surface area values are rather large if compared with e.g. typical proteaseinhibitor complexes, which span between 1,250 and 1,750Å2 [61]. However, the number of direct intermolecular contacts is
significantly lower in the ulilysin dimer, comprising six hydrogen bonds, two bidentate aspartate-arginine salt bridges and
seven van-der-Waals residue pairs (Table 2). Furthermore, the complex traps a large number of solvent molecules and the
calculated complexation significance score of 0.128 accounts only for a weak interaction according to [59]. Finally, sizeexclusion chromatography studies with a calibrated column unambiguously revealed that ulilysin is a monomer in solution
even at high concentrations (data not shown). Accordingly, these findings most likely suggest a special case of extended
crystallographic packing interface, although a regulatory role to restrict access of larger substrates to the active-site cleft under
certain conditions favouring dimerisation cannot be ruled out completely.
3.2.A structural two-calcium site
Ulilysin evinces two calcium cations 9.1Å away from each other within the S-loop structure of the CTSD (Fig. 2e).
Binding of calcium ions to proteins usually encompasses from six to eight, mostly oxygen ligands, which on average are 2.4Å
away from the metal [62]. In ulilysin, the first site is centred on Ca998 and shows eight oxygen ligands, five approximately in a
plane with the cation and two and one, respectively, in the apical positions above and below the plane. Four ligands are
provided by the protein and four are solvent molecules and co-ordinating distances range between 2.3 and 2.6Å. The fact that
half of the ligands are superficial solvent molecules entails that there is a slight difference in the binding distances in the two
molecules present in the asymmetric unit. The number of co-ordinating oxygen atoms and the approximate ligand geometry is
reminiscent of the calcium site around Ca317 of thermolysin (PDB 8tln; [63]), the first metalloendopeptidase whose 3D
structure was solved, although in this case there is only one solvent ligand and a second calcium ion is just 3.8Å away. The
second calcium-binding site in ulilysin is centred on Ca997 and presents four ligands in a plane with the cation and, again, two
and one at the apical positions. Here, only one of the seven oxygen atoms is provided by a solvent molecule and thus the
distances diverge much less within the two molecules of the asymmetric unit. In this case, the site is reminiscent of EF hands
[64], proteins that reversibly bind calcium and thus modulate protein or enzymatic action. In particular, an identical type of
seven-fold co-ordination via identical oxygen-atom-types has been found in site I of the vitamin-D dependent intestinal
11
calcium-binding protein calbindin 9K [65]. However, the Ca997 site is not flanked by the characteristic helices of the helix-turnhelix motif found in EF hand calcium-sites. Taken together with structural similarity searches performed, these findings
suggest that this region encompassing two close calcium binding sites is novel. Finally, a further calcium cation (Ca1W) is
found at the interface between the two molecules of the asymmetric unit, octahedrally co-ordinated by Asp152 O2 of either
molecule and four solvent molecules, with liganding distances ranging from 2.2 to 2.4Å. This cation, however, is likely to be
merely required for crystal contacts.
3.3. The active site of ulilysin: a product complex with an oligopeptide
The active-site groove of ulilysin traverses the molecular surface paralleling helix 4 (Fig. 2b,c). The groove ceiling is
shaped by the upper-rim strand 7 of NTSD that runs in the opposite direction of a bound substrate. In this manner, the latter
is anchored to the active-site cleft through antiparallel -ribbon-like hydrogen bonds between the main chains. On the nonprimed side, the cleft is fronted by the 56-ribbon that contributes to a protrusion that narrows down the cleft cross-section to
~4-5Å, thus modulating the binding of substrates (Fig. 2b). The cleft basement is shaped by the CTSD segments Tyr237Trp240, Pro265-Gly268, the Met-turn and the subsequent four residues (Asn288-Asp295). Mainly on the non-primed side of
the active site, the polypeptide around the first part of the calcium-binding double S-loop (Asp242-Arg252) protrudes from the
molecular surface and probably participates in binding of substrate from the bottom. In the centre of the cleft base resides the
catalytic zinc cation (Zn999) that is tetrahedrally co-ordinated by a solvent molecule (Hoh8W, at 2.1Å) and the N2 atoms of
His228 (2.0Å), His232 (2.1Å) and His238 (2.0Å) from the active-site helix 4. These residues are imbedded within the ZBCS,
together with the general base, Glu229 (Figs. 2b and 3c,e). Interestingly, the strongly conserved ZBCS glycine residue is
replaced in ulilysin by Asn235, which is anchored through its side chain to the C-terminal helix 5 (see Fig. 3c and §3.4.). The
solvent molecule bound to the catalytic zinc is also bound to this acidic residue in unbound metzincins. Here, it is slightly
farther away (at 4.0Å from Glu229 O1 and at 3.8Å from O2). The side chain of Tyr292 is in close distance of the catalytic
cation (see Fig. 3b and §3.4.) but too far to participate in the first co-ordination sphere (4.0Å). The two latter findings are due
to the presence of a dipeptide occupying the primed side of the active-site cleft, probably left behind after a proteolytic event
during purification or crystallisation. Although two residues have been presently modelled based on the experimental data, it
cannot be excluded that a C-terminally extended peptide is actually bound but disordered beyond its P2’ position. The electron
density map unambiguously revealed the residue penetrating the deep S1’ or specificity pocket to be an arginine (Arg401),
followed by a possible valine nestling into S2’ (Val402).
This dipeptide is bound to the protein through an interaction involving 51 protein atoms from 18 residues and 18
peptide atoms. The interface area spans 336Å2 with a calculated iG value of –1.8 kcal/mol (according to [59]) and renders a
complexation significance score of 0.554, which accounts for a tight complex [59]. The interaction is characterised by three
hydrophobic contacts, four direct hydrogen bonds and three salt bridges, as well as four solvent mediated interactions. Main
contacts are two inter main-chain hydrogen bonds with upper-rim strand 7 (Arg401 N-Gly189 O, 2.7Å; Arg401 O-Leu188 N,
2.8Å; see Fig. 3e). The N-terminus of the dipeptide is attached to the general-base glutamate via a salt bridge (Arg401 NGlu229 O2, 2.8Å) and to the catalytic zinc ion and the Tyr292 Oatomthrough a solvent molecule (Hoh8W). Another
12
solvent molecule, Hoh343W, bridges the main-chain carbonyl oxygen atom of Tyr292 with product atoms Arg401 N and
Val402 O. The presence of Arg401 enabled to unambiguously identify the residues shaping the specificity pocket: Thr225 from
4, Leu188 from 7, Phe220 from L84, Met298 from the extended segment preceding 5 and the main chain from Ty292
to Asp295 (Fig. 3c). Of particular importance is the latter aspartate, as it is at the bottom of the pocket and strongly binds
through its O1 atom both Arg401 N1 and N2 atoms (2.7Å and 3.0Å away, respectively). This bidentate salt bridge mainly
explains the specificity of ulilysin for arginine residues in P1’. Arg401 N2 is further anchored to Val293 O (2.7Å) and Arg401
N1 to Thr225 O1 (2.7Å). Two hydrophobic interactions, with the ring of Phe220 (edge-to-face) and the Leu188 side chain,
complete the intricate interaction network of the P1’ residue. The nature of the surrounding residues, mostly hydrophobic
except for the pocket bottom, as well as their spatial arrangement and the depth of the pocket are ideally conceived to
accommodate an extended and basic residue like arginine, capable of participating in -stacking interactions. The bound
dipeptide further allows to assess that the S2’ pocket is much shallower and mainly created by Tyr292 from the Met-turn and
Gln185 plus Ile187 from ribbon 56. Two hydrophobic interactions between Val402 and the side chains of the former two
residues explain the preference for substrates with an at least partially hydrophobic character in P2’.
3.4.Structural relatives of ulilysin
A search for topological relatives revealed that ulilysin bears significant structural similarity with other metzincins, in
particular with members of the adamalysin/ADAM and the MMP families. As an example, the structures of ADAM-17/TACE
(PDB 1bkc; [66]) and ADAM-33 (PDB 1r54; [67]) can be superimposed with ulilysin according to program DALI for a total of
177 and 167 common residues, respectively, with an rmsd over all atoms of 3.0 and 2.8Å despite negligible sequence identity
(12% and 16%) (see Fig. 4a). Also the catalytic domains of MMPs give a good fit. 129 and 130 residues from MMP-12 (PDB
1jk3; [68]) and MMP-16/MT3-MMP (PDB 1rm8; [69]), respectively, render an rmsd of 3.5 and 3.4Å upon superimposition with
an equal number of ulilysin residues, again despite low sequence similarity (14 and 15% identity) (see Fig. 4b). These four
MPs are human proteases engaged in cancer, asthma, emphysema, inflammation and modulation of the immune response
and related family members are proven IGFBPPs. In addition, structural similarity can be found with other metzincins like
snapalysin, leishmanolysin, astacin and serralysins. Likewise, all these proteinases are subdivided into an NTSD and a CTSD
that are separated by an active-site cleft harbouring the catalytic zinc ion. Generally, the structural similarity is focussed on a
series of conserved regular secondary structure elements: a five-stranded -sheet, a long -helix in the back and an activesite helix of the NTSD, as well as a Met-turn and a C-terminal -helix of the CTSD (Fig. 4a,b). In detail, great similarity is
found within each polypeptide chain around the long ZBCS. The catalytic base and the catalytic ion-binding histidine residues
fit very well on top of each other. However, while adamalysins/ADAMs and MMPs strongly prefer bulky hydrophobic residues
in P1’ of substrates, ulilysin clearly favours an arginine. Further noteworthy is that three residues ahead of the second ZBCS
histidine there is a glycine in all metzincin structural prototypes known to date. The values of the main-chain angles of this
residue in the distinct prototypes (103º<<140º; -16º<<17º) indicate that a glycine is required to maintain the polypeptidechain conformation as any other amino acid would be in a high-energy conformation [30]. Contrary, this position is occupied
by an asparagine in ulilysin, whilst most pappalysins have a glycine (Fig. 1). Its main-chain angles are slightly different from
13
the aforementioned prototypes (=83º; =23º), now in the low-energy region corresponding to a left-handed -helix. Another
strongly conserved structural feature within metzincins is the Met-turn that can be perfectly well overlaid in all metzincins up to
the methionine side chain atoms. The function of this residue is still a matter of debate. Mutation studies in PAPP-A have
revealed that substitution of leucine for it causes a dramatic reduction in activity [20]. This finding correlates well with similar
studies performed on other metzincin prototypes like the matrixin MMP-8 [70] and the serralysin Erwinia crysanthemi protease
C [71]. Finally, astacins and serralysins have been reported to present besides the three histidines a slightly more distal
tyrosine zinc ligand, so that the metal co-ordination sphere is rather trigonal bipyramidal than tetrahedral [30]. This residue is
placed within the Met-turn two positions downstream of the methionine and it is swung out from the metal co-ordination sphere
upon substrate binding. It has been suggested to contribute to stabilisation of the tetrahedral negatively-charged reaction
intermediate [71, 72]. Ulilysin also evinces a tyrosine residue, Tyr292, in an equivalent position within the Met-turn. However,
as there is currently no unbound structure available, the aromatic side chain is only observed in its flipped conformation. Here
it is engaged in indirect stabilisation of the product and the inhibitor complexes (see §3.3. and §3.5.).
3.5.An inhibitor complex with batimastat
Hydroxamic acids are capable of inhibition of a variety of metalloenzymes due to their ability to chelate the active-site
zinc and to form hydrogen bonds with general-base residues. In this manner, they interfere with substrate binding and
turnover [73]. They constitute the most extensively studied class of small-molecule metalloprotease inhibitors and, in fact,
most of the lead compounds against MMPs and adamalysins/ADAMs currently under clinical development possess such a
functional group and are derived from molecules displaying a peptidomimetic architecture [74]. Among these inhibitors are the
succinyl hydroxamic-acid derivatives that contain a methylene spacer between the hydroxamic group and the position
equivalent to the P1’ position of a substrate. These derivatives were shown to be more potent and efficient inhibitors than their
counterparts lacking this spacer and those with longer ones [75]. Even further, substituents of one of the methylene hydrogen
atoms, referred to as P1 substituents, conferred a broad-spectrum activity against a variety of MMPs and variations of such
substituents gave rise to the discovery of batimastat, alias BB-94, with a tienylthiomethylene in this position ([37, 76]; see Fig.
3a). This is a very potent inhibitor, with IC50 values against MMPs and adamalysins/ADAMs mostly in the low nanomolar range
due to the presence of a leucine-like side chain that penetrates S1’, thus meeting the specificity-pocket preferences of the
latter MP families. In contrast, batimastat is a modest inhibitor of other MPs like ACE, meprin and thermolysin. Batimastat was
effectively employed to block human tumour growth in vivo in nude mice with no toxic effects, thus pointing to a potential role
as an adjunct in cancer therapies [77]. Furthermore, early clinical studies unveiled a response to treatment in about half the
evaluable patients with advanced malignant ascites [78]. Subsequently, it paved the way for the development of a more
soluble derivative, marimastat, that is readily absorbed upon oral administration. This compound underwent advanced clinical
trials for the treatment of several cancers [73]. However, further investigation with these compounds, as well as with several
other chemically-related first- and second-generation anticancer drugs, was eventually discontinued as they failed to show
superior efficacy over either standard chemotherapy or placebo in the clinic [79]. The breakdown of the expectations set on
these hydroxamates must be blamed both on the insufficient information on the precise role of each of the more than 50
14
identified human MMPs and ADAMs during the different stages of the distinct cancer types, as well as on the lack of specificity
of the inhibitors [80]. Current efforts have thus been redirected towards the development of subtype selective inhibitors starting
from the currently available armamentarium of lead compounds [81].
Our preliminary studies showed that in addition to the unspecific zinc-chelators o-phenanthroline and EDTA and an
excess of zinc, ulilysin was significantly inhibited by batimastat in vitro but not by other MP inhibitors like phosphoramidon,
captopril and galardine or those targeting other classes of proteases [35]. Accordingly, we determined the inhibitory potential
of batimastat against ulilysin with an assay employing a fluorescein conjugate of pig-skin gelatine and obtained an IC50-value
value of 61±3M. This accounts for a rather modest inhibition, attributable to the presence of a hydrophobic isopropyl side
chain occupying the ulilysin S1’ pocket instead of the preferred arginine. To obtain the complex structure with proper inhibitor
occupancy, a six-fold higher concentration of inhibitor over protein was employed to perform crystal soaks. These crystals
rendered an electron density omit-map that unambiguously showed the inhibitor bound to the active sites of both molecules of
the asymmetric unit (Fig. 3d). The two ulilysin:batimastat complexes in the asymmetric unit are equivalent (see §3.1.) so that
discussion, values and parameters hereafter will refer to complex A if not otherwise specified.
Batimastat occupies the active-site cleft from subsites S1 to S3’ thus providing subsite structural information that
complements the studies on the product complex (§3.3.). The protein:inhibitor interface spans 411Å2 under participation of 62
protein atoms coming from 20 residues under establishment of eight hydrogen bonds. In addition, seven protein residues are
contacted by hydrophobic interactions and all this results in a calculated iG of –3.6 kcal/mol following [59]. The complexation
significance score is 0.321, which accounts for a rather weak interaction [59]. The inhibitor binds in an extended conformation
mimicking a substrate (Fig. 3d,f) and is held in place via three inter main-chain-like interaction with upper-rim strand atoms
(Bat996 N1-Gly189 O, 3.0Å; Bat996 O3-Leu188 N, 2.8Å; Bat996 N3-Asp186 O, 3.2Å; for a numbering scheme of the inhibitor
atoms, see Fig. 3a). In addition, the complex is stabilised by hydrogen bonds involving the hydroxamic group and the general
base (Bat996 O2-Glu229 O1, 2.5Å and Bat996 N1-Glu229 O2, 2.8Å) and the zinc (Bat 996 O2-Zn999, 2.3Å and Bat996
O1-Zn999, 2.2Å). Interactions with residues shaping the active-site cleft basement, i.e. that are placed below the inhibitor, are
restricted to Tyr292 and Leu244. A further hydrogen bond is established between Bat996 S1 and Gly189 O (3.8Å; the
estimated upper-limit distance for hydrogen bonds under participation of sulphur atoms is about 0.6Å larger than hydrogen
bonds with N and O donors/acceptors; see [82, 83]). All these contacts are complemented by interactions with protein
residues shaping the distinct subsites (see also §3.3.): The thienylthiomethylene group of batimastat mimicking a P1 side chain
establishes a weak hydrogen bond with Tyr292 O (4.1Å in complex A, 3.9Å in complex B) and hydrophobic interactions with
the side chains of Ile187 and Ser181; the isopropyl group, made up by atoms C8 through C12 and occupying the S 1’ subsite,
with Tyr292, His228 and Leu188; the benzyl moiety, occupying S2’ and featuring atoms C14 and C17 through C23, with
Tyr292, Ile187, Leu244 and Gln185; and the terminal methyl group at S3’ (atom C16) with Leu188. Accordingly, batimastat
presents a leucine-like group in the position that penetrates the specificity pocket of ulilysin. Due to its much shorter length
compared with an arginine side chain, the deep pocket is not filled and the free space is occupied by three solvent molecules,
Hoh277W, Hoh374W and Hoh375W that establish a hydrogen bond network with Thr225, Tyr292, Val293 and Asp295 (Fig.
3f; see §3.3.). As foreseeable, this fact probably explains the high IC50 value of batimastat against ulilysin.
15
The binding mode of batimastat to ulilysin is reminiscent of the complexes of the inhibitor with MMP catalytic domains
like MMP-12 (PDB 1jk3; [68]) and MMP-16 (PDB 1rm8; [69]). In these complexes, exemplified by the MMP-16 complex
structure, the inhibitor is bound practically in the same conformation as in ulilysin except for the thienylthiomethylene group in
P1 that is rotated outwards in ulilysin due to interactions with Ser181 and Tyr292 (Fig. 4c). Likewise, the inhibitor is bound by
interactions with the zinc cation, the general base and the upper-rim strand. In MMPs, the upper-rim strand is preceded by the
S-loop that shapes a bulge on top of the primed side of the active-site cleft and thus penetrates deeper into the cleft. In
addition, in MMPs the inhibitor is further anchored to the active-site basement by two inter main-chain hydrogen bonds with
the so-called S1’-wall forming segment immediately after the Met-turn methionine in the lower subdomain (Fig. 4c). This
segment precedes the specificity loop and paves the floor of the active-site [84]. These additional interactions confer a higher
stability onto the MMP:batimastat complex and much higher potency, with IC50/Ki values in the nanomolar range. A direct
structural effect is that while the hydroxamate groups superimpose neatly, the region of the inhibitor occupying the primed side
of the cleft approaches more the upper-rim strand in ulilysin and the terminal methyl group carbon atoms (C16 in ulilysin) are
2.7Å apart (Fig. 4c).
3.6. Structural and functional implications for the pappalysin family of MPs
Bioinformatic sequence similarity searches suggest pappalysins be grouped into protease family M43 within
MEROPS database, family PD332581 within ProDom and Pfam family PF05572. These searches permitted the identification
of two groups. The first included close relatives of human PAPP-A, with E-values (for a definition, see [85]) below 3E-20, from
mammals (human, chimpanzee, mouse, rat, cattle, water buffalo, sheep, pig, dog and horse), birds (chicken), fish (zebrafish
and green-spoted pufferfish), amphibians (African clawed frog and pipid frog) and echinoderms (sea urchin). Besides, a
second group comprised sequences with higher values, 9E-12 > E-value > 7E-4, from fungi (Pleurotus ostreatus PoMTP,
Coccidioides posadasii MEP1, Ustilago maydis, Aspergillus nidulans and fumigatus, Magnaporthe grisea, Neurospora crassa,
Gibberella zeae and Metarhizium anisopliae), bacteria (Cytophaga sp. and hutchinsonii cytophagalysin and sequences from
Gloiobacter violaceus, Shewanella sp. and amazonensis) and archaea (M. acetivorans ulilysin). Accordingly, sequence
comparisons group ulilysin to the group of more distant relatives of human PAPP-A, which makes sense giving the
evolutionary distance between the organisms harbouring the proteins (see Fig. 1). Among these identified (putative)
pappalysins, Mep1 (SwissProt sequence database access code (SP) Q71H76) from the fungal pathogen Coccidioides
posadasii, which causes the respiratory San-Joaquín-Valley fever in immunocompetent humans and animals, is the only
biochemically and in vivo analysed member besides human PAPP-A and –A2. Mep1 is a 283-residue (pro)MP and is secreted
during endospore differentiation within the host and digests an immunodominant host cell surface antigen thus preventing
recognition of endospores by host phagocytes [86]. A further member studied in vitro is PoMTP metalloprotease from the
edible oyster mushroom, Pleurotus ostreatus (SP Q5Y972). The mRNA of this 290-residue (pro)protein has been shown to be
abundant at primordial and fruit body stages, thus suggesting to play an important role in mushroom fruiting. This protein was
proposed to be grouped together with a series of putative fungal orthologues into a separate metzincin family termed
eucolycins [87]. Finally, another studied member is cytophagalysin, a bacterial collagenase obtained from Cytophaga sp. L43-
16
1 [88]. It is a single-chain MP synthesised from gene cog as a polypeptide of 1,282 amino acid residues, putatively as a
zymogen. It is capable of digesting both insoluble and acid-soluble collagens and gelatin, as well as casein [89]. Whereas
most prokaryotic and fungal forms merely span the catalytic domain (plus a probable pro-domain), cytophagalysin
encompasses additional ~950 residues C-terminally of the catalytic domain, similarly to mammalian pappalysins thus likewise
potentially harbouring additional domains with distinct functions (Fig. 1). Detailed inspection of selected sequences reveals
that pappalysins evince a higher similarity within their CTSDs. This correlates well with most determinants of substrate
specificity being found within this subdomain in ulilysin (Fig. 2b). Overall, residues that are key for catalysis and substrate
binding and others establishing important interactions to maintain structural stability are absolutely conserved, such as
Asp295, determining the substrate specificity for arginine in P1’; Asn288, at the beginning of the Met-turn and establishing with
Gln305 a hydrogen bond required for structural integrity; Arg308, engaged in hydrogen binding of the main chain at Asn235;
and the aforementioned duos, Thr302-Asp258 and Trp165-Asn172. In the NTSD, the main structural features for domain
integrity are the two internal hydrophobic clusters on either side of the central -sheet. In this sense, strict residue
conservation is not indispensable as compensating substitutions might still suffice to keep these clusters together.
Nevertheless, several residues in this subdomain are invariant, Tyr289, Phe301 and Leu171, and others are highly conserved.
Ulilysin is the first pappalysin whose structure has been solved and its structural characteristics render an explanation
of some hallmarks of human PAPP-A but also new questions that arise requiring an answer. A first aspect deals with the
cysteine residues and the disulphide-bond patterns. Comparison of the start of the mature ulilysin sequence with the full gene
sequence suggests that a pro-domain of variable length be present in pappalysins (Fig. 1). Inspection of the first 60 residues
of ulilysin reveals a residue tandem, Cys23-Glu24, that is highly conseved among pappalysins. This strongly reminds of MMPs
and adamalysins/ADAMs, for which a conserved cysteine has been involved in maintenance of latency following a so-called
“cysteine-switch” or “velcro” mechanism [90, 91]. This points to a potentially similar solution for the zymogenic state in
pappalysins. Further to this cysteine, the disulfide-bond pattern of ulilysin and, most likely, the closer bacterial and fungal
relatives includes two SS-bonds in the CTSD, between the zinc-binding consensus sequence and immediately after the Metturn (Fig. 1). Although this double clamp affects the region where all pappalysins bear closest sequence similarity, the SSpattern of PAPP-A and, thus, probably of the closer vertebrate members, diverges [92]. This difference may be intrinsic and
would be compatible with the maintenance of the overall fold. Alternatively, it has been suggested that the activity of PAPP-A
in vivo may be controlled by the environmental redox potential. It may depend on reducing agents that could account for
changes in the disulfide-bond pattern. In this sense it is worth mentioning that PAPP-A activity is regulated in vivo by its
intrinsic protein inhibitor, eosinophil major basic protein, that establishes a covalent complex with the protease via two SSbond following a previously unreported mechanism for a protease:inhibitor complex [23]. A total of eight cysteine residues
change their status from unbound to bound (or vice versa) and/or binding partners in both the protease and the inhibitor on
going from the unbound to the SS-bonded state to permit complex formation. This reaction is dependent on reducing and
oxidising agents, likely to cause conformational changes. This may have implications for PAPP-A function under pathological
conditions, where the redox potential is altered. Further, when PAPP-A is not trapped in its inhibitory complex, it forms
disulfide-linked dimers. This, in turn, recalls the finding that ulilysin crystallises as a dimer (see §3.1.). Accordingly, reduction
17
or formation of specific SS-bonds may act as a reversible switch in the regulation of the extracellular protein function and a
SS-bond pattern more in accordance with that of ulilysin may be conceived under specific conditions [23].
Second, PAPP-A activity against IGFBP-4 has been reported to depend on calcium [26]. In the archaeal protease,
inspection of the residues and protein atoms participating in the co-ordination of the two calcium cations reveals that most
ligands are provided by solvent molecules or main-chain carbonyl oxygen atoms (Figs. 1 and 2e). Only three side chains
participate in metal binding, Asp254, Thr259 and Glu243. The first two are strictly conserved, while the third one could be
easily replaced by another glutamate, Glu580 following the PAPP-A numbering (see Fig. 1), two position ahead and
conserved within vertebrate pappalysins. Third, the surface of ulilysin is mainly negatively charged on its front (see Fig. 2c).
This correlates well with the finding that human PAPP-A is inhibited by positively charged peptides derived from heparinbinding domains through direct binding of the PAPP-A moiety [93]. Fourth, inspection of the sequences around the identified
cleavage points of PAPP-A suggests a lack of consensus in the sequences around the described cleavage sites of PAPP-A
(see Fig. 1). Further, the enzyme was reported to require unusually long synthetic peptides for proper cleavage to occur and
no other protein or low-molecular-weight substrates have been identified besides certain IGFBPs. These evidences have led
to propose steric regulation as a mechanism to account for substrate specificity, possibly mediated by the three LNR motifs,
two of which are inserted into the catalytic protease domain (see Fig. 1) [20, 26, 94]. These motifs are absent from bacterial
orthologues (Fig. 1), but the region of insertion fully coincides with the LNR-like loop observed between strands 2 and 3 in
ulilysin Fig. 2b). Accordingly, this protruding ten-residue loop may be shorter or even absent (as probably in the homologues
from Gloeobacter violaceus and Magnaporthe grisea, see Fig. 1) or much larger, featuring the ~66-residue insertion of PAPPA, than in ulilysin. In any case, it is clear that this insertion could occur under maintenance of the overall protease structure
and be surface located, thus in a disposition to carry out binding functions. These and other questions will only be definitively
clarified when a structure of human PAPP-A or at least of its catalytic domain becomes available.
18
Acknowledgements
This study was supported by the following grants and fellowships: SAB2002-0102 from the former Spanish Ministry
for Education, Culture and Sports; BIO2003-00132, GEN2003-20642 and BIO2004-20369-E from the former Spanish Ministry
for Science and Technology; BIO2006-02668 and BFU2006-09593 from the Spanish Ministry for Education and Science;
ON03-7-0 from the “Fundació La Caixa”; EU FP6 Integrated Project LSHC-CT-2003-503297 “CANCERDEGRADOME”;
“AVON-Project” 2005X0648 from the Scientific Foundation of the Spanish Association Against Cancer; Grant 2005SGR00280
from the Generalitat of Catalunya; and grants 31-67253.01 and 3100AO-108262/1 from the Swiss National Science
Foundation. M.S. is a beneficiary of the “Ramon y Cajal” Program from the Spanish Ministry for Science and Education.
Batimastat was a kind gift from K. Maskos and W. Bode, Munich. We are grateful for the help provided by EMBL and ESRF
synchrotron local contacts, in particular by Gavin Fox, during data collection (ESRF, Grenoble). For these data collections,
funding was provided by ESRF.
19
Legends to the Figures
Fig. 1. Pappalysin catalytic domain sequence alignment. Members of distinct kingdoms have been aligned based on
phylogenetic distance according to the inserted tree (red frame in the second alignment block; the bar indicates 10 pointaccepted mutations (PAM) and a black triangle pinpoints the centre of the tree), except for the first two sequences which have
been swapped. In each case, the harbouring organism (in blue; first alignment block) and the sequence database access
code (SwissProt, SP, and GenBank, XP; in magenta, preceding the second alignment block) are provided, as is the
numbering for ulilysin and human PAPP-A in blue above and below the alignment, respectively. Additional N- and C-terminal
residues present in each sequence are indicated in parenthesis. Regular secondary structure elements of ulilysin are
displayed as red arrows for -strands (labels 1-8) and as green cringles for helical segments (labels 1-5). Strictly
conserved positions (>89%) and highly conserved positions (all hydrophobic or >56%) are shown in white over magenta and
black over light blue, respectively. The LNR-motifs present in the mammalian family members (black over orange) are
enclosed. The disulphide bonds (probably) present in native ulilysin (SS1 and SS2) and those identified in human PAPP-A
within the shown sequence region (encircled numbers; there is an ambiguity regarding bonds and ; see [92]) are
further displayed. Scissors indicate autolytic cleavage points in ulilysin (above the alignment blocks; see [35]) and human
PAPP-A (below the alignment blocks; see [20]).
Fig. 2. Overall ulilysin structure. (A) Representative detail of the initial experimental A-weighted electron density
map obtained after MAD-phasing contoured at 1 above threshold and superimposed with the final refined model shown as
blue sticks. (B) (Left) Richardson-diagram of ulilysin in standard orientation, i.e. viewed perpendicular to the active-site cleft.
Helices (labelled 1–5) are shown as yellow ribbons, -strands (1-8) as pale green arrows and irregular segments as
white coils. Furthermore, the three cations, Zn999, Ca998 and Ca997 are depicted as magenta and blue spheres,
respectively. Residues engaged in zinc binding and disulphide bonding, the general base and the Met-turn methionine are
represented as orange sticks. (Right) Same as before but with the view corresponding to a 90º-rotation around the vertical. (C)
Surface electrostatic potential of ulilysin mapped on its Connolly surface in standard orientation (left) and in three additional
orientations corresponding to 90º rotations around the vertical (colour coding: red, below –10 kBT/e; white, between –10 kBT/e
and 10 kBT/e; blue, above +10 kBT/e. kB is the Boltzmann constant and T the temperature in Kelvin). The bound dipeptide is
shown as a stick model and the zinc cation as a green sphere. (D) Cartoon illustrating the non-crystallographic ulilysin dimer
present in the crystal asymmetric unit. Molecule A is shown superimposed with its solvent-accessible molecular surface in
white and molecule B as a green rope. The zinc (magenta) and the calcium cations (red) are further shown as spheres. (E)
Detail of the structure of ulilysin around the double S-loop constituting the double calcium-binding site. Intervening residues
and solvent molecules as well as the two cations (red spheres) are labelled. Atom colour coding: C, yellow; S, green; N, blue
and O, red.
Fig. 3. Ulilysin product and inhibitor complexes. (A) Chemical structure of the hydroxamic-acid based batimastat
inhibitor alias BB-94, [4-(N-hydroxyamino)-2(R)-isobutyl-3(S)-(2-thienylthiomethyl)succinyl]-L-phenylalanine-N-methylamide.
The atom numbering adopted is shown in italics in parenthesis. (B) Detail in stereo of the initial A-weighted (2mFobs-DFcalc)-
20
type electron density map contoured at 1 above threshold around the active-site of ulilysin molecule A of the ulilysin:inhibitor
complex showing the continuous and unambiguous electron density of the bound inhibitor. The model employed for phasing
lacked the inhibitor and the catalytic zinc cation (small magenta sphere). The final refined model of the inhibitor is shown as a
stick model with atom colour coding as in Fig. 2e. The zinc-liganding protein histidine residues are further shown as cyan
sticks. (C) Close-up view of the ulilysin Richardson-plot centred on the active site of the product complex in standard
orientation. The bound Arg401-Val402 dipeptide is shown as a cyan stick model and labelled. Selected side chains discussed
in the text are further depicted as orange sticks and labelled. (D) Same view as in (C) of the enzyme:inhibitor complex.
Batimastat is shown as red sticks. (E) Scheme depicting the hydrogen bonds and salt bridges (as dashed lines) observed in
the active site of the product complex. The dipeptide is depicted with bold lines, key solvent molecules as small spheres and
the zinc ion as a large sphere. (F) Same as in (E) but for the enzyme:inhibitor complex.
Fig. 4. Structural relatives of ulilysin. (A) Superimposed C-representations in stereo of ulilysin and ADAM-17/TACE.
The structurally equivalent -helices and -strands are shown in red and blue, respectively, in both structures. The remaining
segments are coloured in green (ulilysin) and yellow (TACE), as are the respective zinc cations (central spheres). The
topology schemes of ulilysin (left) and TACE (right) with helices as rods, -strands as arrows, cations as spheres (zinc in
purple and calcium in orange), disulphide bonds as orange lines, selected amino acids as ellipses and selected hydrogen
bonds as dashed lines are further depicted. The topologically equivalent helices are shown in red and those characteristic for
each structure in magenta. Common -strands are in light green and characteristic ones in dark green. (B) Same as in (A) but
for ulilysin and MMP-16. Characteristic segments and zinc of MMP-16 are depicted in pink. The topology scheme of MMP-16
is shown on the right. (C) Detail of the superimposed structures of the batimastat complexes of ulilysin (cyan stick model;
inhibitor in dark blue) and MMP-16 (lilac stick model; inhibitor in fuchsia). Characteristic structural features for MMPs (the Sloop and the S1’-wall forming segment) are pinpointed. The inhibitors fully coincide at the zinc-binding hydroxamic acid moiety
but they are 2.7Å away from each other at the distal N-bound methyl group (C16 according to Fig. 3a; see text).
21
Table 1 – Crystallographic statistics on data collection and refinement
________________________________________________________________________________________________________________________________
Ulilysin dataset
C269A
C269A SeMet-derivative
C269A
(batimastat complex)
(high resolution)
(Zn2+ f’’max)
Form II / C2221
Form I / P21212
Form II / C2221
Form I / P21212
118.9, 60.7, 168.9
49.6, 126.1, 87.4
119.3, 60.1, 169.3
49.6, 126.1, 87.4
Wavelength (Å)
0.9330
0.9840
1.2831
0.9798
0.9796
0.9779
No. of measurements
154,616
362,539
157,339
147,781
150,171
150,925
No. of unique reflections
40,166
61,061
41,264
37,825
37,836
37,833
45.6-2.00 (2.11-2.00) a
39.0-1.70 (1.79-1.70)
42.3-2.00 (2.11-2.00)
96.6 (90.39) / -
99.8 (99.9) / -
99.3 (97.2) / - 99.9 (99.9) / 97.3 (96.6) 99.9 (100.0) / 97.5 (96.7) 99.9 (100.0) / 97.5 (96.7)
0.037 (0.070)
0.082 (0.438)
0.099 (0.402)
0.044 (0.119)
0.048 (0.112)
0.048 (0.112)
-
-
-
0.038 (0.086)
0.061 (0.101)
0.049 (0.102)
24.2 (13.9)
11.6 (3.4)
9.0 (2.9)
32.5 (17.4)
28.8 (17.0)
27.8 (16.0)
16.0
17.9
20.5
20.3
19.8
19.7
3.8 (3.8)
5.9 (5.7)
3.8 (3.7)
3.9 (3.9)4.0 (4.0)
4.0 (4.0)
Estimated values of f’ / f’’ (in electrons)
-
-
-
-8.8 / 2.7-7.5 / 4.8
-4.9 / 3.7
Resolution range used for phasing (Å)
-
-
-
46.1 – 2.00
Mean figure of merit (fom) d
-
-
-
0.55/ 0.79/ 0.81
Crystal form / Space group
Cell constants (a, b, c; in Å;  in º)
Resolution range (Å) (outer shell)
Compl. / Anom. Compl. (%)
Rmerge b
Ranomalous c
Average intensity (<[<I> / (<I>)]>)
B-Factor (Wilson) (Å2)
Average multiplicity
Position of selenium sites found e
C269A SeMet-derivative
(f’min)
(f’’max)
(high-energy remote)
46.1-2.00 (2.11-2.00)
0.710, 0.058, 0.384/ 0.790, 0.438, 0.388/ 0.729, 0.119, 0.313/
0.771, 0.379, 0.315/ 0.637, 0.394, 0.344/ 0.863, 0.102, 0.341/
0.762, 0.061, 0.433/ 0.740, 0.434, 0.438/ 0.661, 0.072, 0.413/
0.841, 0.424, 0.417
Resolution range used for refinement (Å)
45.5 – 2.00
32.1 – 1.70
No. of reflections used (test set)
39,606 (520)
60,401 (610)
Crystallographic Rfactor (free Rfactor) f
0.161 (0.211)
0.173 (0.241)
No. of protein atoms (asymmetric unit)
No. of solvent molecules / ions /
4,049
g
4,130
g
460 / 2 (Zn2+), 5 (Ca2+) / 597 / 2 (Zn2+), 5 (Ca2+) /
22
/ other molecules (asymmetric unit)
4 (glycerol), 2 (batimastat)
7 (glycerol)
0.009
0.012
1.18
1.29
0.38 / 1.20
0.91 / 2.34
16.6
18.4
18.6 / 19.6
-
R.m.s. deviation from target values
bonds (Å)
angles (°)
bonded B-factors
(Å2)
main-/side-chain
Average B-factors for protein atoms (Å2)
Average B-factors for batimastat A / B (Å2)
_________________________________________________________________________________________________________________________________
Friedel-pairs were treated separatedly in the processing of the MAD datasets.
a
Figures in parenthesis refer to the outermost resolution shell, unless otherwise indicated.
b
Rmerge= hkli |Ii(hkl) - <I(hkl)>| / hkli Ii(hkl), where Ii(hkl) is the i-th intensity measurement of reflection hkl, including symmetry-related reflections, and <I(hkl)> its average.
c
Ranomalous= hkl|<I(hkl)> - <I(-h-k-l)>| / hkl(<I(hkl)> + <I(-h-k-l)>).
d
fom = | F (hkl)best | / | F (hkl) | , with F (hkl)best = P () F hkl() / P (). After experimental phase calculation with SHARP (first value), density modification with SOLOMON (second value)
and density modification/averaging with program DM (third value).
e
In fractional cell co-ordinates.
f
Rfactor = hkl ||Fobs| - k |Fcalc|| / hkl |Fobs|, with Fobs and Fcalc as the observed and calculated structure factor amplitudes; free Rfactor, the same for a test set of reflections (> 500) not used during
refinement.
g
Including atoms present in double occupancy.
23
Table 2 – Direct inter-protomer interactions observed in the ulilysin product complex structure
Hydrogen bonds and salt bridges
Hydrophobic interactions
Molecule B
Dist.(Å)
Molecule A
Molecule B
Molecule A
Ser158 O
3.1
Glu199 O1
Pro265
Phe246
Ser158 O
3.0
Glu199 O2
Phe267
Phe246
Tyr237 O
3.1
Glu182 O
Phe246
Pro265
Arg245 N
2.9
Asp264 O1
Phe246
Phe267
Arg245 N1
2.7
Asp264 O2
Phe156
Pro197
Glu199 O1
3.1
Ser158 O
Phe156
Phe156
Glu199 O2
3.1
Ser158 O
Pro197
Phe156
Glu182 O
3.0
Tyr237 O
Asp264 O1
2.7
Arg245 N
Asp264 O2
2.8
Arg245 N2
24
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