The 1.8 Å crystal structure of human tear lipocalin reveals an extended branched cavity with capacity for multiple ligands

In contrast with earlier assumptions, which classified human tear lipocalin (Tlc) as an outlier member of the lipocalin protein family, the 1.8-A resolution crystal structure of the recombinant apoprotein confirms the typical eight-stranded antiparallel beta-barrel architecture with an alpha-helix attached to it. The fold of Tlc most closely resembles the bovine dander allergen Bos d 2, a well characterized prototypic lipocalin, but also reveals similarity with beta-lactoglobulin. However, compared with other lipocalin structures Tlc exhibits an extremely wide ligand pocket, whose entrance is formed by four partially disordered loops. The cavity deeply extends into the beta-barrel structure, where it ends in two distinct lobes. This unusual structural feature explains the known promiscuity of Tlc for various ligands, with chemical structures ranging from lipids and retinoids to the macrocyclic antibiotic rifampin and even to microbial siderophores. Notably, earlier findings of biological activity as a thiol protease inhibitor have no correspondence in the three-dimensional structure of Tlc, rather it appears that its proteolytic fragments could be responsible for this phenomenon. Hence, the present structural analysis sheds new light on the ligand binding activity of this functionally obscure but abundant human lipocalin.


INTRODUCTION
Tear lipocalin (Tlc, also called Lcn1 or von Ebner's gland protein) was originally described as a unique major component of human tear fluid, comprising 15 to 33 % of its protein content and secreted by the lacrimal glands (1,2). Later on it was also found to be produced by a number of other secretory glands and tissues, including lingual glands, nasal mucosal glands, secretory glands of the tracheobronchial tract, sweat glands, mammary gland, adrenal gland, prostate, thymus, testis, and by corticotrophs of the pituitary gland (3)(4)(5)(6).
cDNA cloning and sequencing revealed that this protein component is a member of the lipocalin superfamily with significant amino acid similarity to β-lactoglobulin (7), one of the classical lipocalins. Since it lacks one of the three conserved amino acid motifs that are normally present in lipocalins, Tlc was initially classified as an outlier member of this protein family (8). However, a typical β-barrel fold was predicted from a hydropathy profile of the amino acid sequence (7), circular dichroism analysis (9), and also from an extended sitedirected tryptophan substitution and fluorescence study (10), revealing β-sheet and α-helical composition similar to other lipocalins.
Crystal structure of tear lipocalin Breustedt et al.,p. 7 equilibration against 0.5 ml of the precipitant solution. Several crystals were obtained at 12°C in the presence of 60 % (w/v) ethylene glycol, 200 mM Zn-acetate, 100 mM Tris-HCl pH 7.0 -7.9 after about six weeks. Protein crystals were harvested directly from the drop using Nylon loops (Hampton Research, Laguna Niguel, CA), cryo-protected by transfer to a drop of Paratone N (Hampton Research) -thereby removing excessive mother liquor -and frozen in a 100 K nitrogen stream (Oxford Cryosystems, Oxford, UK). Native diffraction data ( Table 1) were collected at the SLS beamline PX1 (PSI, Villigen, Switzerland). A three-wavelength anomalous dataset of the SeMet derivative ( Table 1) was collected at the synchroton beamline BM14 (ESRF, Grenoble, France). Data were processed with MOSFLM and SCALA (28). The space group was determined as C2 (a = 76.4 Å, b = 61.4 Å, c = 50.8 Å, β = 129.7°f or the native data set) with one protein molecule in the asymmetric unit.

Structure determination, model building, and refinement
The selenium substructure of a crystal grown at pH 7.9 was solved with SnB (29) using anomalous peak data, whereby three of the four SeMet sites that had to be expected from the primary structure were identified. Phasing was carried out with SHARP (30,31) using the three SeMet sites and the anomalous data from all three wavelengths (Table 1) to a resolution of 1.8 Å. Solvent flattening was subsequently performed with SOLOMON (32) by using a solvent content of 45 %. A partial structural model, comprising the β-barrel and the αhelix but lacking residues in the loop regions and at both polypeptide termini, was calculated with ARPwARP (33) as implemented in SHARP. Missing residues were then added in several sessions of manual rebuilding with QUANTA (34), alternating with restrained refinement cycles using REFMAC 5 (28). During this process three coordinated zinc ions and two chloride ions were identified and added to the model. Final refinement was carried out with the native data set of a crystal grown at pH 7.0. At this pH, two previously missing residues (Asn-32 and Leu-33) in loop #1 appeared in the electron density map and were built by guest on March 24, 2020 http://www.jbc.org/ Downloaded from Crystal structure of tear lipocalin Breustedt et al.,p. 8 accordingly. Water molecules were added where stereochemically plausible and the 2F O -F C and F O -F C difference Fourier maps revealed densities of more than 1.0 and 3.5 σ, respectively. The backbone dihedral angles fall mostly into the favoured regions of the Ramachandran plot. The only exception is residue Leu-33, which is situated C-terminal to the disordered part of loop #1 and has only weak electron density.

Analysis of the crystal structure
The few missing residues at the tips of loop #2 (Leu-56 and Ile-57) and loop #4 (Leu-105 and His-106) were inserted in QUANTA (34) with plausible stereochemistry (using the "regularize range" option). In order to model the stretch of seven residues in loop #1 without clear electron density (Asp-25 to Met-31), the Protein Data Bank was searched for a loop with identical length and matching conformation of the well resolved flanking segments (Cα positions 21 to 23 and 33 to 35, respectively) using INSIGHT II (Accelrys, San Diego, CA).
One nicely fitting peptide segment, corresponding to residues 117 to 125 of actophorin (PDB accession code 1CNU), was found and grafted onto the Tlc stump using least squares superposition of the flanking segments. Side chains were subsequently adjusted to match the Tlc sequence and oriented in standard conformation (35). The completed model was subjected to one round of refinement using REFMAC 5 with the occupancy of all three amended segments (residues 25 to 31, 56 and 57, 105 and 106) set to 0.01, resulting in an R-factor of 19.6 % and R free of 25.4 %. There were no structural clashes due to the new residues. Notably, the modelled loop #1 partly adopted a similar orientation as the corresponding loop of the lipocalin Bda, though shorter by two residues (see text).
Graphical representations were prepared using MOLSCRIPT (36), RASTER3D (37), and PyMOL (38). Secondary structure assignments were made with DSSP (39) and extracted with MAKEMOLS (40). For use with MOLSCRIPT, electron densities were converted from by guest on March 24, 2020 http://www.jbc.org/ Downloaded from Crystal structure of tear lipocalin Breustedt et al.,p. 9 CCP4 format (28) with MINIMAGE (41). Molecular surfaces were either prepared with PyMOL or calculated with GRASP (42) and exported for use in MOLSCRIPT with a modified version of UNGRASP (37). Superpositions were made with LSQMAN (43). Ligand complexes were modelled using INSIGHT II. The crystallographic coordinates of the apo-Tlc structure -without the modelled segments -have been deposited at the Protein Data Bank (44) under accession code 1XKI.

Crystallization, structure determination, and quality of the final model
Recombinant human Tlc, comprising residues 5 to 157 of the mature protein (7,19), with the free Cys-101 substituted by Ser, was fused to the OmpA signal peptide and secreted into the periplasm of E. coli, where processing, folding, and efficient disulphide bond formation can take place. One-step purification from the bacterial periplasmic extract was achieved by streptavidin affinity chromatography via the Strep-tag II (25), which had been fused to the Cterminus. Tlc was finally isolated as a homogeneous monomeric protein by gel filtration.
Crystallization of Tlc was achieved at pH 7.9 with ethylene glycol as precipitant in the presence of Zn-acetate. The crystal structure was determined by MAD phasing after in vivo labelling with selenomethionine (SeMet). Refinement against a data set for a crystal of the native protein, grown under similar conditions at pH 7.0, resulted in an R-factor of 18.9 % and an R free of 25.4 % with good stereochemistry ( Table 2). One molecule of the apo-protein was found in the asymmetric unit. The quality of the electron density map at a resolution of 1.8 Å allowed to unambiguously build 128 of the 152 residues of the recombinant Tlc Adjacent residues had elevated temperature factors but could be built unambiguously.
Hence, as in other lipocalins the β-barrel of Tlc appears rather rigid while extended loops and extraneous terminal segments of the polypeptide chain seem to be more flexible, probably also due to a lack of stabilizing crystal contacts. For purposes of illustration, the missing loop segments at the entrance to the ligand pocket were modelled with plausible stereochemistry (for details see Materials and Methods) and included in the Tlc structure described here.

Structure of tear lipocalin
Tlc exhibits the typical lipocalin fold, consisting of an eight-stranded, strictly antiparallel βbarrel that forms a central cavity. The cavity opens to one end of the barrel, where four loops (#1 -#4) connect neighbouring β-strands (dubbed A -H) in a pairwise manner (Fig. 1). A Cterminal α-helix (residues Leu-127 to Ala-136), which is followed by an almost extended peptide segment (strand I), packs against one side of the β-barrel. The N-terminal peptide segment of Tlc covers the bottom of the barrel between the loops connecting strands B/C and F/G and thereby buries Trp-17, one of the few residues conserved in all lipocalins (8).
Three of the loops at the open end of the β-barrel are rather long and partially disordered in the crystal structure, hence allowing access to the central cavity even for voluminous ligands.
Especially the Ω-type loop #1 with its altogether 13 residues is one of the largest loops by guest on March 24, 2020 http://www.jbc.org/ Downloaded from Crystal structure of tear lipocalin Breustedt et al.,p. 11 among the prototypic lipocalins that have been structurally characterized so far (16). On its N-terminal side this loop is likely to form a short α-helix comparable to those observed in Bda and Blg (Fig. 2). Loops #2 und #4 appear as β-hairpins, whereas the tip of the rather short loop #3 connecting β-strands E/F (cf. Fig. 1A) locally assumes the conformation of a 3 10 -helix (residues Asp-80 to Gly-82).
Even though Tlc represents one of the smallest members of the lipocalin family -with 158 residues for the native, mature polypeptide (cf. Fig. 2) -it exhibits an unusually large cavity inside its β-barrel. Nevertheless, the β-barrel itself, with the three loops that tie it up at its closed end and with the α-helix attached to it, appears conformationally rigid and is nicely superimposable to the supersecondary structure of other lipocalins.
Interestingly, apart from the wide mouth formed approximately at the middle between the loops #1 to #4 there is a second, smaller entry to the cavity of Tlc, which appears between βstrands A and H (Fig. 1D). The main entrance of the calyx-like pocket measures about 10 Å in diameter and is lined with negatively charged (Glu-34 and Asp-80) as well as hydrophobic residues. The cavity itself exhibits a bifurcated shape, is about 15 Å deep, and its inside is slightly positively charged (Fig. 3). This positive charge arises from the side chains of residues His-86 and Lys-114 together with the dipoles of main chain amide groups of up to ten residues in the vicinity.
The remaining side chains that line the pocket are hydrophobic in nature, particularly residues Phe-99 and Met-39, which separate the two lobes at the bottom of the calyx.
Although the cavity is accessible to solvent, the lower end and, most notably, the two lobes at its bottom are free of ordered water molecules in the crystal structure. This may be due to the absence of suitable H-bond donor/acceptor groups in order to fix the polar molecule (46).

Implications for ligand binding
There are three bound zinc ions in the crystal structure of Tlc (Fig. 1B). Zn-H2 is complexed by His-96 and Glu-125 together with two chloride ions. Zn-H3 is coordinated by His-92 and Asp-129, also located in the region around the N-terminus of the α-helix, as well as by two water molecules. In contrast, Zn-H1 is involved in a crystal packing contact between two Tlc molecules and complexed by the side chains of Glu-73 and Lys-76 of one polypeptide and Glu-102 of a symmetry-related molecule. The fourth coordination site is occupied by a water molecule.
All three bound Zn(II) ions exhibit the typical tetrahedral coordination geometry, and their distances to the closest side chain atoms of Tlc (Table 3) are in accordance with the published parameters for Zn-binding sites (47). Notably, when the Zn(II) that had been used in the precipitant solution (see Experimental Procedures) was substituted with Mg(II), no crystal growth was observed. However, preliminary binding studies between the recombinant Tlc and Zn(II) revealed a rather weak dissociation constant of approximately 1.33 mM (determined as half-maximal metal ion concentration to achieve saturation during CD titration at 212 nm; Skerra, Cirl and Breustedt, to be published).
The unusual shape of the Tlc binding pocket prompted us to investigate possible modes of interaction with two of its known ligands, retinoic acid and rifampin (7,15). Unfortunately, corresponding co-crystallisation trials remained unsuccessful. However, molecular docking with rifampin (CSD entry RIFAMP) clearly revealed that the bulky molecule occupies almost the entire pocket and that it fits the cavity with the two lobes astonishingly well (Fig. 3B). In contrast, two possible binding modes were identified for retinoic acid (CSD entry VITAAC01), which is significantly smaller than rifampin and has a more extended shape. In both by guest on March 24, 2020 http://www.jbc.org/ Downloaded from Crystal structure of tear lipocalin Breustedt et al.,p. 13 orientations, the β-ionone ring of retinoic acid can be placed into the larger of the two lobes at the bottom of the cavity while the terminal carboxylate group is alternatively arranged at the two different exits (Fig. 3C,D).

Structural comparison with other lipocalins
A search through the PDB using the DALI server (48)  showed lower similarity. Due to its comparably high sequence homology of 23 % the latter lipocalin was previously employed as a template structure for the homology modelling of Tlc (10). Even though Tlc and Bda display merely 17.8 % sequence identity (Fig. 2B), superposition of the 58 Cα atoms of the β-barrel (16) gave rise to a low R.m.s. deviation of 1.5 Å while a corresponding value of 2.3 Å was calculated for all 128 superimposable Cα atoms.
Despite the high structural similarity of the β-barrel and α-helix, major differences were found in the loop regions at the open end of the lipocalin calyx ( Fig. 2A). Loop #1, which is largely disordered in Tlc but well defined in the Bda crystal structure (45), runs almost across the opening of the calyx, thereby controlling accessibility of the ligand-binding site. The spatial orientation of the segments flanking this loop, which could be built with confidence in the Tlc crystal structure, together with the comparable loop lengths in both lipocalins (with two residues less in Tlc than in Bda; cf. The structure of Blg, which has served as a homologue of Tlc so far (7,10), is highly similar to that of Bda. Again, the β-barrel and α-helix superimpose well with those of Tlc ( Fig. 2A) whereas the loop regions of Blg resemble more closely those of Bda. Loops #1, #2, and #3 are found in almost the same conformation as in Bda while loop #4 is shifted away from the β-barrel axis in Blg compared with both Bda and Tlc. The N-terminal peptide segment, which is disordered in Tlc and in Bda, is well defined in Blg and runs across the bottom of the βbarrel as observed for most other lipocalins (16).

Comparison of tear lipocalin with protease inhibitors
Previous experimental evidence indicated that full length Tlc as well as a synthetic peptide comprising residues Leu-3 to Ala-21 exert inhibitory activity toward the cysteine proteinase papain (17,52). Three sequences were identified within the first 17 N-terminal residues of Tlc that are homologous to the motifs known to be essential for the inhibitory activity of cystatins and the closely related stefins (53). Although the sequence motifs are more than 40 residues apart in the primary structures of these well characterized inhibitors, they are spatially close  (56)) were superimposed onto each other and with the Nterminal segment of Tlc (Fig. 4). While mutual superposition of the different cystatin-type inhibitors revealed a clear pattern of conserved main chain and side chain orientation, the proposed inhibitory peptide of Tlc did not exhibit reasonable structural similarity. Especially Trp-17, which was considered functionally homologous to the central tryptophan residue of the cystatins in the third sequence motif and occurs there exposed at the tip of a hairpin loop, is buried in the hydrophobic core of Tlc. In fact, this amino acid is even part of the Gly-Xaa-Trp signature characteristic for all lipocalin structures (8) and therefore not likely to participate in an inhibitor-proteinase interaction, at least as long as the native fold is adopted.

DISCUSSION
As predicted based on its amino acid sequence (7) neighbouring residues are all solvent-exposed in the crystal structure, with concomitant irregularities in the β-strand backbone. Interestingly, the three residues following the β-bulge in strand A (Met-22 to Val-24) form a short antiparallel β-sheet with the short strand I (Ser-143 to Leu-145) that is part of the extended C-terminal polypeptide segment, which is found in most lipocalins and attaches to one side of the β-barrel.
The predicted short loop #3 connecting β-strands E/F becomes also apparent from the crystal structure. Yet, its tip points outward from the β-barrel axis, in contrast with the situation in Blg (cf. Fig. 2A). For residues Pro-29 to Thr-37 as part of loop #1 and residues Glu-9 to Ser-14 close to the N-terminus of Tlc α -helical and 3 10 -helical conformation, respectively, was predicted. These assumptions could not be confirmed in the crystal structure, even though it must be pointed out that some of the residues in these regions are not well resolved in the electron density.
Determination of the dissociation constants between various Tlc mutants and 16-doxylstearic acid was used to identify residues that play a role in ligand recognition and are therefore likely to be located inside the calyx (10). In these experiments, three alanine residues (Ala-51, Ala-66, and Ala-86) were found that could not be replaced by tryptophan without significantly diminishing ligand affinity. A fourth residue, Ala-79, was furthermore proposed to play a role in this context, but did not exhibit a similar experimental effect. In the crystal structure, all four Ala residues, as well as another two residues (Cys-101 and Gly-103) that were previously found to interact with a ligand of Tlc (57), are indeed located within the cavity.
Apart from that, energy minimization of the homology model for Tlc led to the assumption that Tyr-97 is exposed in the ligand pocket (10). Contrasting, in the crystal structure this Also, no tight packing contacts with a neighbouring molecule were observed in the Tlc crystal so that an explanation for the previously described Tlc dimerization remains obscure. The unpaired Cys-101 residue does not seem to play a role in this respect because in contrast with other lipocalins that display a free Cys residue for covalent complex formation, such as NGAL (58), its side chain is buried sterically inaccessible inside the binding pocket of Tlc.
In the presently described crystal structure of the apo-form of human Tlc a stretch of seven residues in loop #1, the tips of loop #2 and loop #4 as well as the disulphide bond linking the C-terminus of the polypeptide chain to loop #2 were not defined in the electron density map.
Adjacent residues showed high temperature factors, indicating that the whole loop region at the entrance to the cavity exhibits enhanced mobility. Previous investigation of the structural by guest on March 24, 2020 http://www.jbc.org/ Downloaded from Crystal structure of tear lipocalin Breustedt et al.,p. 19 changes in Tlc upon ligand binding via CD spectroscopy revealed that ligand complexation induces the formation of additional β-sheet structure and of a more rigid state of the protein (59). Thus, it is conceivable that the loops at the open end of the β-barrel can at least to some degree adapt to different ligands.
The structure of Tlc is most similar to Bda, a lipocalin with yet unknown biological function produced by the sweat glands of domestic animals. In this protein the loop regions are more rigid and some minor structural differences with Tlc are found. Loop #1 partially forms an αhelix in Bda and runs almost straight across the entrance to the ligand-binding site. Also, loops #2 and #3 are shifted towards the central axis of the β-barrel in Bda compared with Tlc.
As result, the ligand pocket is essentially obstructed in this allergen, and in fact no endogenous ligands, except of two water molecules, were found inside its cavity (45).
Unexpectedly, two zinc-binding sites were identified in the crystal structure of Tlc, together with a third one that occurs at a crystal packing contact. Spectrophotometric titration experiments revealed that there is a measurable affinity for Zn(II) in the low millimolar range.
Hence, the zinc-binding effect may not just be an artefact of crystallisation in the presence of 200 mM Zn(II), even though the apparent dissociation constant seems to be above physiological levels. While Zn(II)-binding activity was not yet described for Tlc, zincresponsive promoter elements were identified in the Lcn1 gene (60). Furthermore, it was shown that gene expression is induced in knockout mice for MTF-1 (metal-responsive transcription factor 1), a highly conserved protein involved in the transcriptional response to heavy metal exposure (61).
Further to its broad ligand-binding function, which is a more or less typical feature of lipocalins, Tlc was reported to inhibit the proteolytic activity of papain. Van Breustedt et al.,p. 20 could show that Tlc inhibits papain to the same extent as cystatin S, an inhibitor also present at high concentration in tear fluid. However, the target proteinase of cystatin S has not yet been identified and its inhibitory activity towards papain is much lower than that of cystatin C.
In following experiments with recombinant Tlc -and cystatin C serving as positive controlits inhibitory function was confirmed, but it also became evident that inhibition of papain occurs only in a narrow concentration range (52).
Still, a synthetic 18 amino acid peptide derived from the N-terminus of Tlc exhibited comparable proteinase inhibitory activity as a peptide that contained the three inhibitory motifs of cystatin S linked by single Ala residues, although both peptides inhibited papain only at much higher concentration than their respective parent proteins (17). In fact, at increased papain concentration Tlc is used as a substrate (52). N-terminal sequencing revealed that two cleavage sites lie within the N-terminal Leu-Leu-Ala-Ser motif (residues 3 to 6 of Tlc), but not in the conserved Gln-X-Val-X-Gly motif (residues 11 to 15 of Tlc) as it is typical for cystatin-type inhibitors.
Structural comparison revealed (Fig. 4B) that while in various cystatins the three inhibitory sequence motifs form a contigous interface that binds to the catalytic cleft of the target proteinase (also reviewed in (53)) the N-terminal segment of Tlc cannot be superimposed onto this structural element in a meaningful way. Whereas the N-terminal Leu-Leu-Ala-Ser sequence and the first residue of the Gln-X-Val-X-Gly motif are not resolved in the electron density and probably not conformationally ordered in Tlc, the third sequence motif containing Trp-17 is buried within its hydrophobic core. Since the Trp motif seems to be of minor importance for the inhibitory activity of cystatin-derived peptides (62) it is possible that the flexible N-terminus of Tlc acts in a peptide-like manner. This would also explain why Tlc is cleaved at higher papain concentration within one of the inhibitory motifs, similarly as the cystatin-derived peptides but in contrast to the full-length cystatins.  Breustedt et al.,p. 21 Notably, there are several other proteinase inhibitors which rely on an eight-stranded β-barrel fold similar to Tlc and for which the mode of interaction with their respective target enzymes is known in detail (Fig. 4C,D). Triabin (PDB accession code 1AVG (63)), a 142 residue protein from the saliva of the blood-sucking insect Triatoma pallidipennis, prevents blood clot formation by inhibiting the serine proteinase thrombin via binding to its exosite I. Unlike the prototypic lipocalin fold, β-strands A to D are arranged in an up-up-down-down topology while the following β-strands are antiparallel.
Recently, the structure of staphostatin B of Staphylococcus aureus, the inhibitor of the cysteine proteinase staphopain B, was elucidated (PDB accession code 1PXV (64)). In contrast to Tlc with its eight strictly antiparallel β-strands, neighboring strands A and B of staphostatin B have parallel topology. The inhibitory site of staphostatin B, which was cocrystallized with its target proteinase, is located in the loop connecting the two C-terminal βstrands. Residues 97 to 101 of staphostatin B interact with the catalytic cleft of staphopain.
Interestingly, this peptide stretch does not bind in a substrate-like manner but in an inverted (antiparallel) orientation.
Like Tlc, the metalloproteinase inhibitor Inh (PDB accession code 1SMP (65)) from Erwinia chrysanthemi folds into a strictly antiparallel β-barrel. This protein was shown to inhibit the 50 kDa proteinase from Serratia marcescens and its presumed physiological role is the protection of periplasmic proteins against proteolysis (66). In the crystallized complex with this proteinase, the five N-terminal residues of the inhibitor extend away from the β-barrel and bind to the catalytic cleft of the enzyme. Faint electron density for residue Ser-1 of the inhibitor, together with N-terminal sequencing, revealed that this residue is cleaved by ca. 50 % in the proteinase-inhibitor complex.  Breustedt et al.,p. 22 In contrast with Tlc, none of these proteinase inhibitors possesses a cavity that appears suitable for ligand binding, rather the interior of each β-barrel is densely packed with mostly hydrophobic side chains. Furthermore, they lack the C-terminal α-helix typical for lipocalins and, due to their varying β-barrel topologies, they exhibit structural differences in the loop regions. However, comparison with Tlc also reveals some similarities. The Cα positions of the β-barrel residues superimpose astonishingly well, resulting in R.m.s. deviations of 1.6 Å, 2.1 Å, and 2.2 Å for triabin, staphostatin B, and Inh, respectively.
Moreover, the Gly-Xaa-Trp motif, which is strictly conserved in the lipocalins, is also found in these three inhibitors at approximately the same position (Fig. 4C), close to the bottom of the β-barrel with the Trp side chain buried in the hydrophobic core. Indeed, triabin and Inh have recently been ascribed to the calycin protein superfamily (67). Nevertheless, a structural explanation for the inhibitory activity of Tlc towards a thiol proteinase is not evident from comparison with either of these three proteins (Fig. 4D).
Even though experimental evidence exists that the N-terminus of Tlc can act as a weak inhibitor of a thiol-proteinase in vitro, this probably does not correspond to a relevant physiological function. There are different, more potent cystatins present in the same secretory fluids and tissues, which, in contrast to Tlc, have also been isolated from tear samples in complex with various proteinases (68). Consequently, the major function of Tlc seems to lie in the binding of multiple physiological or pathological compounds. In order to gain further insight into the mode of interaction between this lipocalin and its presumed ligands, co-crystallization experiments are currently under way.    Blg. All three amino acid sequences were aligned based on structural similarities. Equivalent β-barrel residues (as defined for Bda and Blg in (16)) that were used for superposition are boxed. Secondary structure elements are indicated as interpreted by DSSP (39): β-strands A to I are depicted with green background, α-helices with orange background. In case of Tlc βstrands and α-helices are also marked as green arrows and orange cylinders, respectively.
Residues His-1 to Leu-4 of Tlc, which were not present in the recombinant protein, as well as the Cys-101→Ser replacement and the C-terminal Strep-tag II are shown as lower-case characters. Residue Asp-158 of native Tlc was substituted by Ala as linker to the Strep-tag II.  Tlc: Tlc: α  Fig. 2