Crystallographic analysis of an "anticalin" with tailored specificity for fluorescein reveals high structural plasticity of the lipocalin loop region

The artificial lipocalin FluA with novel specificity toward fluorescein was derived via combinatorial engineering from the bilin-binding protein, BBP by exchange of 16 amino acids in the ligand pocket. Here, we describe the crystal structure of FluA at 2.0 A resolution in the space group P2(1) with two protein-ligand complexes in the asymmetric unit. In both molecules, the characteristic beta-barrel architecture with the attached alpha-helix is well preserved. In contrast, the four loops at one end of the beta-barrel that form the entrance to the binding site exhibit large conformational deviations from the wild-type protein, which can be attributed to the sidechain replacements. Specificity for the new ligand is furnished by hydrophobic packing, charged sidechain environment, and hydrogen bonds with its hydroxyl groups. Unexpectedly, fluorescein is bound in a much deeper cavity than biliverdin IX(gamma) in the natural lipocalin. Triggered by the substituted residues, unmutated sidechains at the bottom of the binding site adopt conformations that are quite different from those observed in the BBP, illustrating that not only the loop region but also the hydrophobic interior of the beta-barrel can be reshaped for molecular recognition. Particularly, Trp 129 participates in a tight stacking interaction with the xanthenolone moiety, which may explain the ultrafast electron transfer that occurs on light excitation of the bound fluorescein. These structural findings support our concept of using lipocalins as a scaffold for the engineering of so-called "anticalins" directed against prescribed targets as an alternative to recombinant antibody fragments.     

The 1.8-A 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.     

Exon-intron structure of outlier tick lipocalins indicate a monophyletic origin within the larger lipocalin family

All tick proteins assigned to the lipocalin family lack the structural conserved regions (SCRs) that are characteristic of the kernel lipocalins and can thus be classified as outliers. These tick proteins have been assigned to the tick lipocalin family based on database searches that indicated homology between tick sequences and the fact that the histamine binding protein (HBP2) from the hard tick Rhipicephalus appendiculatus (Ixodidae) shows structural similarity to the lipocalin fold. Sequence identity between kernel and outlier lipocalins falls below 20% and the question raised is whether the outlier and kernel lipocalins are truly homologous. More specifically in the case of the tick lipocalins, whether their structural fold is derived from the lipocalin fold or whether convergent evolution resulted in the generation of the basic lipocalin-like fold which consists of an eight stranded continuous anti-parallel beta-barrel terminated by a C-terminal alpha-helix that lies parallel to the barrel. The current study determined the gene structure for HBP2 and TSGP1, TSGP2 and TSGP4, lipocalins identified from the soft tick Ornithodoros savignyi (Argasidae). All tick lipocalins have four introns (A-D) with conserved positions and phases within the tick lipocalin sequence alignment. The positions and phase information are also conserved with regard to the rest of the lipocalin family. Phylogenetic analysis using this information shows conclusively that tick lipocalins are evolutionary related to the rest of the lipocalin family. Tick lipocalins are grouped within a monophyletic clade that indicates a monophyletic origin within the tick lineage and also group with the other arthropod lipocalins in a larger clade. Phylogenetic analysis of sequence alignments based on conserved secondary structure of the lipocalin fold support the conclusions from the gene structure trees. These results indicate that exon-intron arrangement can be useful for the inclusion of outlier lipocalins within the larger lipocalin family.     

Identification of functionally diverse lipocalin proteins from sequence information using support vector machine

Lipocalins are functionally diverse proteins that are composed of 120–180 amino acid residues. Members of this family have several important biological functions including ligand transport, cryptic coloration, sensory transduction, endonuclease activity, stress response activity in plants, odorant binding, prostaglandin biosynthesis, cellular homeostasis regulation, immunity, immunotherapy and so on. Identification of lipocalins from protein sequence is more challenging due to the poor sequence identity which often falls below the twilight zone. So far, no specific method has been reported to identify lipocalins from primary sequence. In this paper, we report a support vector machine (SVM) approach to predict lipocalins from protein sequence using sequence-derived properties. LipoPred was trained using a dataset consisting of 325 lipocalin proteins and 325 non-lipocalin proteins, and evaluated by an independent set of 140 lipocalin proteins and 21,447 non-lipocalin proteins. LipoPred achieved 88.61% accuracy with 89.26% sensitivity, 85.27% specificity and 0.74 Matthew’s correlation coefficient (MCC). When applied on the test dataset, LipoPred achieved 84.25% accuracy with 88.57% sensitivity, 84.22% specificity and MCC of 0.16. LipoPred achieved better performance rate when compared with PSI-BLAST, HMM and SVM-Prot methods. Out of 218 lipocalins, LipoPred correctly predicted 194 proteins including 39 lipocalins that are non-homologous to any protein in the SWISSPROT database. This result shows that LipoPred is potentially useful for predicting the lipocalin proteins that have no sequence homologs in the sequence databases. Further, successful prediction of nine hypothetical lipocalin proteins and five new members of lipocalin family prove that LipoPred can be efficiently used to identify and annotate the new lipocalin proteins from sequence databases. The LipoPred software and dataset are available at .     

NMR solution structure of lipocalin-type prostaglandin D synthase: evidence for partial overlapping of catalytic pocket and retinoic acid-binding pocket within the central cavity

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH(2), a common precursor of various prostanoids, to produce PGD(2), an endogenous somnogen and nociceptive modulator, in the brain. L-PGDS is a member of the lipocalin superfamily and binds lipophilic substances, such as retinoids and bile pigments, suggesting that L-PGDS is a dual functional protein acting as a PGD(2)-synthesizing enzyme and a transporter for lipophilic ligands. In this study we determined by NMR the three-dimensional structure of recombinant mouse L-PGDS with the catalytic residue Cys-65. The structure of L-PGDS exhibited the typical lipocalin fold, consisting of an eight-stranded, antiparallel beta-barrel and a long alpha-helix associated with the outer surface of the barrel. The interior of the barrel formed a hydrophobic cavity opening to the upper end of the barrel, the size of which was larger than those of other lipocalins, and the cavity contained two pockets. Molecular docking studies, based on the result of NMR titration experiments with retinoic acid and PGH(2) analog, revealed that PGH(2) almost fully occupied the hydrophilic pocket 1, in which Cys-65 was located and all-trans-retinoic acid occupied the hydrophobic pocket 2, in which amino acid residues important for retinoid binding in other lipocalins were well conserved. Mutational and kinetic studies provide the direct evidence for the PGH(2) binding mode. These results indicated that the two binding sites for PGH(2) and retinoic acid in the large cavity of L-PGDS were responsible for the broad ligand specificity of L-PGDS and the non-competitive inhibition of L-PGDS activity by retinoic acid.     

Evolution of the lipocalin family as inferred from a protein sequence phylogeny

The lipocalins constitute a family of proteins that have been found in eubacteria and a variety of eukaryotic cells, where they play diverse physiological roles. It is the primary goal of this review to examine the patterns of change followed by lipocalins through their complex history, in order to stimulate scientists in the field to experimentally contrast our phylogeny-derived hypotheses. We reexamine our previous work on lipocalin phylogeny and update the phylogenetic analysis of the family. Lipocalins separate into 14 monophyletic clades, some of which are grouped in well supported superclades. The lipocalin tree was rooted with the bacterial lipocalin genes under the assumption that they have evolved from a single common ancestor with the metazoan lipocalins, and not by horizontal transfer. The topology of the rooted tree and the species distribution of lipocalins suggest that the newly arising lipocalins show a higher rate of amino acid sequence divergence, a higher rate of gene duplication, and their internal pocket has evolved towards binding smaller hydrophobic ligands with more efficiency.     

Structural insight into the dual ligand specificity and mode of high density lipoprotein association of apolipoprotein D

Human apolipoprotein D (ApoD) occurs in plasma associated with high density lipoprotein. Apart from the involvement in lipid metabolism, its binding activity for progesterone and arachidonic acid plays a role in cancer development and neurological diseases. The crystal structures of free ApoD and its complex with progesterone were determined at 1.8A resolution and reveal a lipocalin fold. The narrow, mainly uncharged pocket within the typical beta-barrel accommodates progesterone with its acetyl side chain oriented toward the bottom. The cavity adopts essentially the same shape in the absence of progesterone and allows complexation of arachidonic acid as another cognate ligand. Three of the four extended loops at the open end of the beta-barrel expose hydrophobic side chains, which is an unusual feature for lipocalins and probably effects association with the high density lipoprotein particle by mediating insertion into the lipid phase. This mechanism is in line with an unpaired Cys residue in the same surface region that can form a disulfide cross-link with apolipoprotein A-II.     

'Anticalins': a new class of engineered ligand-binding proteins with antibody-like properties

The development of soluble receptor proteins that recognise given target molecules--ranging from small chemical compounds to macromolecular structures at a cell surface, for example--is of ever increasing importance in the life sciences and biotechnology. For the past century this area of application was dominated by antibodies, which were traditionally generated via immunisation of animals but have recently also become available by means of protein engineering methods. The so-called 'anticalins' offer an alternative type of ligand-binding proteins, which has been constructed on the basis of lipocalins as a scaffold. The central element of this protein architecture is a beta-barrel structure of eight antiparallel strands, which supports four loops at its open end. These loops form the natural binding site of the lipocalins and can be reshaped in vitro by extensive amino acid replacement, thus creating novel binding specificities. The bilin-binding protein (BBP) was employed as a model system for the preparation of a random library with 16 selectively mutagenized residues. Using bacterial phagemid display and colony screening techniques, several lipocalin variants--termed anticalins--have been selected from this library, exhibiting binding activity for compounds like fluorescein or digoxigenin. Anticalins possess high affinity and specificity for their prescribed ligands as well as fast binding kinetics, so that their functional properties are similar to those of antibodies. Compared with them, they exhibit however several advantages, including a smaller size, composition of a single polypeptide chain, and a simple set of four hypervariable loops that can be easily manipulated at the genetic level. Apart from haptenic compounds as targets, anticalins should also be able to recognise macromolecular antigens, provided that the random library is accordingly designed. Hence, they should not only serve as valuable reagents for bioanalytical purposes, but may also have a potential in replacing antibodies for medical therapy.     

Lipocalins - a family portrait

Lipocalins are a widely distributed group of proteins whose common feature is the presence of six-or eight-stranded beta-barrel in their tertiary structure and highly conservative motifs short conserved region, (SCR) in their amino acid sequences. The presence of three SCRs is typical for kernel lipocalins, while outlier lipocalins have only one or two such regions. Owing to their ability to bind and transport small, hydrophobic molecules, lipocalins participate in the distribution of such substances. However, the physiological significance of lipocalins is not limited to transfer processes. They play an important role in the regulation of immunological and developmental processes, and are also involved in the reactions of organisms to various stress factors and in the pathways of signal transduction. Of special interest is the enzymatic activity found in a few members of the lipocalin family, as well as the interaction with natural membranes, both directly with lipids and through membrane-localized protein receptors.     

Complexes of porcine odorant binding protein with odorant molecules belonging to different chemical classes

Porcine odorant binding protein (pOBP) is a monomer of 157 amino acid residues, purified in abundance from pig nasal mucosa. In contrast to the observation on lipocalins as retinol binding protein (RBP), major urinary protein (MUP) or bovine odorant binding protein (bOBP), no naturally occurring ligand was found in the beta-barrel cavity of pOBP. Porcine OBP was therefore chosen as a simple model for structure/function studies with odorant molecules. In competition experiments with tritiated pyrazine, the affinity of pOBP towards several odorant molecules belonging to different chemical classes has been found to be of the micromolar order, with a 1:1 stoichiometry. The X-ray structures of pOBP complexed to these molecules were determined at resolution between 2.15 and 1.4 A. As expected, the electron density of the odorant molecules was observed into the hydrophobic beta-barrel of the lipocalin. Inside this cavity, very few specific interactions were established between the odorant molecule and the amino acid side-chains, which did not undergo significant conformational change. The high B-factors observed for the odorant molecules as well as the existence of alternative conformations reveal a non-specific mode of binding of the odorant molecules in the cavity.     

Structure-guided engineering of Anticalins with improved binding behavior and biochemical characteristics for application in radio-immuno imaging and/or therapy

Modern strategies in radio-immuno therapy and in vivo imaging require robust, small, and specific ligand-binding proteins. In this context we have previously developed artificial lipocalins, so-called Anticalins, with high binding activity toward rare-earth metal–chelate complexes using combinatorial protein design. Here we describe further improvement of the Anticalin C26 via in vitro affinity maturation to yield CL31, which has a fourfold slower dissociation half-life above 2 h. Also, we present the crystallographic analyses of both the initial and the improved Anticalin, providing insight into the molecular mechanism of chelated metal binding and the role of amino acid substitutions during the step-wise affinity maturation. Notably, one of the four structurally variable loops that form the ligand pocket in the lipocalin scaffold undergoes a significant conformational change from C26 to CL31, acting as a lid that closes over the accommodated metal–chelate ligand. A systematic mutational study indicated that further improvement of ligand affinity is difficult to achieve while providing clues on the contribution of relevant side chains in the engineered binding pocket. Unexpectedly, some of the amino acid replacements led to strong increases – more then 10-fold – in the yield of soluble protein from periplasmic secretion in Escherichia coli.     

Tuning ligand affinity,specificity, and folding stability of an engineered lipocalin variant -- a so-called 'anticalin' -- using a molecular random approach

Anticalins are prepared by reshaping the ligand pocket of a natural lipocalin via protein engineering in order to recognize a prescribed ligand. In this manner, the anticalin DigA with specificity for digoxigenin was previously derived from the bilin-binding protein (BBP), a natural lipocalin from Pieris brassicae. The four peptide loops that form its ligand-binding site were randomized and a cognate variant was selected from the resulting library. Here, we propose a concept for improving the ligand-binding properties of this anticalin in an in vitro affinity maturation process by step-wise randomization of restricted areas of the loop region. Following selection on digoxigenin-binding activity via phage display and colony screening, several DigA variants were thus obtained. The recombinant proteins were thoroughly characterized in terms of ligand affinity and specificity, secondary structure and thermal stability against unfolding. The variant DigA16/19, which carries several new mutations, exhibits clearly improved affinity for digoxigenin, with K(D)=12.4 nM. Hence, it is suitable as a sensitive reagent in biochemical detection experiments, especially when produced as a functional fusion protein with alkaline phosphatase as reporter enzyme. In addition, DigA16/19 possesses enhanced ligand specificity and recognizes part of the linker that was used for fixing the steroid group to a carrier protein. Finally, the digoxigenin-binding anticalins appear to have high physico-chemical stability, with T(m) values in the 70 degrees C range. Our present findings support the notion that anticalins provide a useful class of compact and robust ligand-receptor proteins that can be tailored for practical demands.     

Exon-intron structure and evolution of the Lipocalin gene family

The Lipocalins are an ancient protein family whose expression is currently confirmed in bacteria, protoctists, plants, arthropods, and chordates. The evolution of this protein family has been assessed previously using amino acid sequence phylogenies. In this report we use an independent set of characters derived from the gene structure (exon-intron arrangement) to infer a new lipocalin phylogeny. We also present the novel gene structure of three insect lipocalins. The position and phase of introns are well preserved among lipocalin clades when mapped onto a protein sequence alignment, suggesting the homologous nature of these introns. Because of this homology, we use the intron position and phase of 23 lipocalin genes to reconstruct a phylogeny by maximum parsimony and distance methods. These phylogenies are very similar to the phylogenies derived from protein sequence. This result is confirmed by congruence analysis, and a consensus tree shows the commonalities between the two source trees. Interestingly, the intron arrangement phylogeny shows that metazoan lipocalins have more introns than other eukaryotic lipocalins, and that intron gains have occurred in the C-termini of chordate lipocalins. We also analyze the relationship of intron arrangement and protein tertiary structure, as well as the relationship of lipocalins with members of the proposed structural superfamily of calycins. Our congruence analysis validates the gene structure data as a source of phylogenetic information and helps to further refine our hypothesis on the evolutionary history of lipocalins.     

Structural mechanism of specific ligand recognition by a lipocalin tailored for the complexation of digoxigenin

DigA16 is an artificial digoxigenin-binding protein, which was derived from the bilin-binding protein, a lipocalin of Pieris brassicae, via reshaping of its natural ligand pocket. Here we report the crystal structures of DigA16 in the presence of either digoxigenin or digitoxigenin and for the apo-protein at resolutions below 1.9A. As a consequence of the altogether 17 amino acid substitutions within the binding site significant structural changes have occurred in the four loops that form the entrance to the ligand pocket on top of the structurally conserved beta-barrel framework. For example, one loop adopts a new alpha-helical backbone structure, which seems to be induced by few critical side-chain contacts. Digoxigenin becomes almost fully buried (by 95%) upon complexation, whereby specificity for the hydrophilic steroid is maintained through hydrogen-bonding networks and shape complementarity. The differential binding of the related steroid digitoxigenin is mainly governed by an internal histidine residue, whose side-chain undergoes significant induced fit. Among those amino acids that line the ligand pocket two tyrosine and one tryptophan residue provide the largest contacts. Interestingly, corresponding three side-chains are found with the same mutual orientation in the anti-digoxigenin antibody 26-10, even though the hapten orientation is quite different there and only 66% of the steroid surface is buried in the combining site. Hence, in the case of the engineered lipocalin DigA16 an example of convergent in vitro evolution is observed. Generally, the remarkable structural plasticity of the loop region and the role of polar residues in the binding site illustrate the potential of the lipocalin scaffold for the generation of specific receptor proteins towards a variety of ligands.     

Mouse oncogene protein 24p3 is a member of the lipocalin protein family.

Rigorous new methods of protein sequence analysis have been applied to the lipocalins, a diverse family of ligand binding proteins. Using three conserved sequence motifs to search for similar patterns in a large sequence database, the size and composition of this protein family have been defined in an automatic and objective way. It has allowed the identification of an existing sequence, mouse 24p3 protein, as a lipocalin and the possible rejection of other putative members from this protein family. On the basis of this newly discovered homology, a possible function for mouse 24p3 protein is proposed.     

A phylogenetic analysis of the lipocalin protein family

The lipocalins are a family of extracellular proteins that bind and transport small hydrophobic molecules. They are found in eubacteria and a great variety of eukaryotic cells, in which they play diverse physiological roles. We report here the detection of two new eukaryotic lipocalins and a phylogenetic analysis of 113 lipocalin family members performed with maximum-likelihood and parsimony methods on their amino acid sequences. Lipocalins segregate into 13 monophyletic clades, some of which are grouped in well-supported superclades. An examination of the G + C content of the bacterial lipocalin genes and the detection of four new conceptual lipocalins in other eubacterial species argue against a recent horizontal transfer as the origin of prokaryotic lipocalins. Therefore, we rooted our lipocalin tree using the clade containing the prokaryotic lipocalins. The topology of the rooted lipocalin tree is in general agreement with the currently accepted view of the organismal phylogeny of arthropods and chordates. The rooted tree allows us to assign polarity to character changes and suggests a plausible scenario for the evolution of important lipocalin properties. More recently evolved lipocalins tend to (1) show greater rates of amino acid substitutions, (2) have more flexible protein structures, (3) bind smaller hydrophobic ligands, and (4) increase the efficiency of their ligand-binding contacts. Finally, we found that the family of fatty-acid-binding proteins originated from the more derived lipocalins and therefore cannot be considered a sister group of the lipocalin family.     

Mutant bovine odorant-binding protein: Temperature affects the protein stability and dynamics as revealed by infrared spectroscopy and molecular dynamics simulations

Bovine odorant-binding protein (bOBP), a member of the lipocalin family, presents the so-called 3D "domain-swapped" protein structure. In fact, in solution, it appears as a dimer in which each monomer is composed by the classical lipocalin fold, with a central beta-barrel followed by a stretch of residues and the alpha-helix domain protruding out of the barrel and crossing the dimer interface. Recently, a deswapped mutant form of bOBP was obtained, in which a Gly residue was inserted after position 121 and the two residues in position 64 and 156 were replaced by Cys residues for restoring the disulfide bridge common to the lipocalin family. In this work, we used Fourier transform infrared spectroscopy and molecular dynamics simulations to investigate the effect of temperature on the structural stability and conformational dynamics of the mutant bOBP. The spectroscopic and molecular simulation data pointed out that the hydrophobic regions of the protein matrix appear to be an important factor for the protein stability and integrity. In addition, it was also found that the mutant bOBP is significantly stabilized by the binding of the ligand, which may have an impact on the biological function of bOBP. The obtained results will allow for a better use of this protein as probe for the design of advanced protein-based biosensors for the detection of compounds used in the fabrication of explosive powders.     

The bacterial lipocalins

The lipocalins were once regarded as a eukaryotic protein family, but new members have been recently discovered in bacteria. The first bacterial lipocalin (Blc) was identified in Escherichia coli as an outer membrane lipoprotein expressed under conditions of environmental stress. Blc is distinguished from most lipocalins by the absence of intramolecular disulfide bonds, but the presence of a membrane anchor is shared with two of its closest homologues, apolipoprotein D and lazarillo. Several common features of the membrane-anchored lipocalins suggest that each may play an important role in membrane biogenesis and repair. Additionally, Blc proteins are implicated in the dissemination of antibiotic resistance genes and in the activation of immunity. Recent genome sequencing efforts reveal the existence of at least 20 bacterial lipocalins. The lipocalins appear to have originated in Gram-negative bacteria and were probably transferred horizontally to eukaryotes from the endosymbiotic alpha-proteobacterial ancestor of the mitochondrion. The genome sequences also reveal that some bacterial lipocalins exhibit disulfide bonds and alternative modes of subcellular localization, which include targeting to the periplasmic space, the cytoplasmic membrane, and the cytosol. The relationships between bacterial lipocalin structure and function further illuminate the common biochemistry of bacterial and eukaryotic cells.     

Genome-wide identification and comparative analysis of lipocalin families in Lepidoptera with an emphasis on Bombyx mori

Lipocalins exhibit functional diversity, including roles in retinol transport, invertebrate cryptic coloration, and stress response. However, genome-wide identification and characterization of lipocalin in the insect lineage have not been thoroughly explored. Here, we found that a lineage-specific expansion of the lipocalin genes in Lepidoptera occurred in large part due to tandem duplication events and several lipocalin genes involving insect coloration were expanded more via tandem duplication in butterflies. A comparative analysis of conserved motifs showed both conservation and divergence of lepidopteran lipocalin family protein structures during evolution. We observe dynamic changes in tissue expression preference of paralogs in Bombyx mori, suggesting differential contribution of paralogs to specific organ functions during evolution. Subcellular localization experiments revealed that lipocalins localize to the cytoplasm, nuclear membrane, or nucleus in BmN cells. Moreover, several lipocalin genes exhibited divergent responses to abiotic and biotic stresses, and 1 lipocalin gene was upregulated by 300 fold in B. mori. These results suggest that lipocalins act as signaling components in defense responses by mediating crosstalk between abiotic and biotic stress responses. This study deepens our understanding of the comprehensive characteristics of lipocalins in insects.     

Generation of anticalins with specificity for a nonsymmetric phthalic acid ester

A set of engineered lipocalins, so-called anticalins, that bind benzyl butyl phthalate, a potential pollutant of environmental and food samples or medical plastic ware, has been generated. To this end, the synthesis of a derivative of the target analyte carrying an activatable carboxylate group at the end of an aliphatic spacer arm was established. This compound was covalently coupled to amino-functionalized paramagnetic beads. Using phage display technology three variants were selected from a random library of the bilin-binding protein (BBP), a prototypic lipocalin, which exhibit binding activity toward the nonsymmetric phthalic acid ester. These anticalins (denominated PhtA, PhtB, and PhtC) possess dissociation constants of 9.1, 6.2, and 11.6 microM, respectively. Specificity for the binding of other phthalic acid esters was studied. No cross-reactivity was found for diethyl phthalate, while binding to dibutyl phthalate was observed with higher dissociation constants. Interestingly, two differing types of binding behavior were observed among the three selected anticalins. Sequence comparison of these engineered lipocalins with the wild-type BBP revealed that all of the 16 randomized positions carried an amino acid exchange and that a certain sequence pattern had been selected, thus pointing toward a peculiar mode of structural interaction. Our data suggest that the generation of anticalins may provide an alternative to antibodies for the creation of stable receptor proteins against haptens with bioanalytical relevance.