I. Introduction: The Concept of Ligand-Selective Carrier Proteins

Top Previous Next References

Many active agents used in pharmaceutics, food, agriculture, and chemical processes require temporal stabilization and protection against degradation or oxidation (Hattori et al., 1995; Iametti et al., 1995). Alternatively and/or additionally, a sustained or triggered release may be a prerequisite. Finally, the efficacy of such agents may be improved by increasing their solubility or by masking unwanted properties, such as toxicity or bad taste, at least before the target environment is reached (Poznansky and Juliano, 1984; Tomlinson, 1987; Jain, 1989; Vingerhoeds et al., 1994; Pothakamury and Barbosa-Cánovas, 1995; Risch and Reineccius, 1995). In many cases, a combination of requirements may apply (Fig. 1A). For example, the oral administration of an unstable, insoluble, and bitter-tasting drug would call for 1) the prevention of early drug degradation, 2) an improvement of the solubility or dispersability of the drug in water, 3) a masking of its bitter taste, 4) a more efficient routing to its target environment (leading to a reduction of generic toxicity), and 5) a controlled release once the target environment has been reached. To meet (part of) such demands, carrier systems have been developed (Fig. 1B), including particulate systems like nano- and microcapsules, nano- and microspheres, liposomes (Felgner, 1990; Kreuter, 1992; Karsa and Stephenson, 1993, 1996; Risch and Reineccius, 1995), and even resealed erythrocytes (Dale, 1980). Whereas liposomes consist of (phospho)lipids, the other particulate systems normally consist of natural or synthetic biocompatible polymers. These can be proteins, such as gelatin, albumin, casein, or fibrin (Thies and Bissery, 1984; Chen et al., 1987; Wolkoff, 1987; Gupta and Hung, 1989; Senderoff et al., 1991; Banga, 1995; Narayani and Rao, 1996; Yu et al., 1996), but more often other polymers are used (Hsieh et al., 1981; Kost et al., 1989; Davis et al., 1991; Leung et al., 1991; Sanders, 1991; Hirabayashi et al., 1996; Mi et al., 1997) usually because they are more stable or less immunogenic. Several systems are used for the specific targeting (homing) of the entrapped agents (Poznansky and Juliano, 1984; Tomlinson, 1987; Felgner, 1990; Vingerhoeds et al., 1994; Hirabayashi et al., 1996), whereas most systems are designed to control the kinetics of release of the active agent in a site- and time-dependent manner. Where targeting rather than controlled release is the sole or prime goal, covalent attachment of the active agent to the carrier system is often used (Poznansky and Cleland, 1980; Trouet et al., 1982; Fiume et al., 1986; Franssen et al., 1994; Molema and Meijer, 1994; Narayani and Rao, 1996; Pirrung and Huang, 1996; Dosio et al., 1997; Lebbe et al., 1997). Alternatively, encapsulation of free or noncovalently bound drugs in liposomes and attachment of antibodies to the liposome is applied in drug targeting (Crommelin et al., 1992; Vingerhoeds et al., 1993, 1994; Storm et al., 1995). Many controlled release systems are designed to create a sustained release (Bagshawe et al., 1988; Merlin, 1991; Karsa and Stephenson, 1993; Vingerhoeds et al., 1993; Risch and Reineccius, 1995). However, release of the active compounds can also be designed to be burst-like, as triggered by the local conditions in the target environment (Brownlee and Cerami, 1979; Hsieh et al., 1981; Jeong et al., 1985; Albin et al., 1987; Fischel-Ghodsian et al., 1988; Kost et al., 1989; Merlin, 1991; Pinnaduwage and Huang, 1992; Kim et al., 1994) or by externally applied triggers (Jain, 1989; Kreuter, 1992; Karsa and Stephenson, 1993, 1996; Vingerhoeds et al., 1994; Pothakamury and Barbosa-Cánovas, 1995).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   A, schematic representation of the possible fates of active agents in view of the various requirements for their functional application. The active agent is represented by black triangles. Ad, site of administration; U, unwanted interaction (for example, bad taste resulting from interaction with taste receptors, toxicity resulting from interaction with hormone receptors at unintended sites, etc.); P, (undesirable) precipitation; D, (undesirable) degradation and/or oxidation; T, target site. B, attachment of the active agent to a carrier system (a single protein, a polymer capsule, or a more complex system, schematically represented by gray-speckled balls) may prevent unwanted side effects and loss of the agent. Furthermore, it may result in improved delivery and in controlled release of the active agent at the target site (T). (Again, the agent is represented by black triangles.).

The field of controlled release, which is of prime importance in drug delivery, especially in cancer therapy and the treatment of endocrinological disorders (e.g., Karsa and Stephenson, 1993), finds increasing application also in cosmetics and household materials (e.g., Schaeffer and Brooks, 1992; Joshi, 1996; Withenshaw et al., 1996), food (Yolles, 1973; Risch and Reineccius, 1995), agrochemicals/crop protectants (e.g., Allan and Neogi, 1971; Kuderna and Saliman, 1974; Cohen et al., 1977; Knight et al., 1995; Scher, 1999), fertilizers (e.g., Fersch and Stearns, 1976; Knight et al., 1995), industrial and other reactants (Mestetsky, 1973; Segalman and Wallace, 1995), and office supplies, e.g., "carbonless carbon paper" (see Karsa and Stephenson, 1993). In crop protection, triggers like rainfall, heat, photolysis, natural plant-associated molecules, or infestation-induced generation of by-products can be used. In cosmetics, as in pharmaceutical applications, carrier systems consisting of polymers and proteins with a low immunogenicity are advantageous, e.g., for the slow or stimulated release of fragrances, radical scavengers, etc. In food-oriented applications, food-grade protein-, polysaccharide-, or lipid-mediated carrier systems are particularly appropriate (e.g., lactoglobulin, albumin, starch, lecithin). Food-associated compounds that can profitably be protected and targeted by binding to carrier proteins include flavoring agents, colorants, vitamins, antioxidants, or preservatives.

In most traditional systems, the control of release and the stabilization of the agent is based on encapsulation (Brownlee and Cerami, 1979; Hsieh et al., 1981; Fischel-Ghodsian et al., 1988; Kost et al., 1989; Karsa and Stephenson, 1993; Kim et al., 1994; Vingerhoeds et al., 1994; Pothakamury and Barbosa-Cánovas, 1995; Risch and Reineccius, 1995) and/or on nonspecific reversible interactions between the carrier and the active agent (e.g., Chen et al., 1987; Wolkoff, 1987; Gupta and Hung, 1989; Senderoff et al., 1991; Chuo et al., 1996; Lubbe et al., 1996). However, the application of carrier components that selectively and reversibly bind the active agent with high affinity offers additional possibilities to stabilize the agent and improve the control of its release (Cohen et al., 1977, 1979; Mitchell, 1986; Schaeffer and Brooks, 1992; Hattori et al., 1995), reducing unwanted side effects of the active agent (Fig. 2). This is especially the case when the binding site and binding mechanism is well characterized and accessible to engineering. Thus, agent-specific ligand-binding (bio)polymers can be considered as a special class of carrier components either to be used as such or, alternatively, as agent-selective parts of more complicated (multicomponent) particulate carrier systems. Particularly, ligand-binding proteins could be potentially promising as such agent-specific binding components. The ligand-binding sites of a great number of proteins are well characterized, with known crystal structures, and well accessible to engineering via modern genetic techniques (site-directed mutagenesis, directed evolution, and/or screening of large numbers of more or less random mutants). In natural systems, the occurrence of high selectivity and binding affinity for a specific small ligand is almost invariably attributable to the presence of proteins. Examples are the binding of biotin by (strept)avidin, the binding of antigens by specific antibodies, and the binding of small molecules to receptors. In fact, the binding of a molecule to a protein is the basic step in most biological processes and functions. The first step in biological catalytic conversions is the binding of the target substrate by an enzyme. Cell signaling, DNA replication, gene expression, and the sorting and trafficking of any cellular component to the various compartments of the cell all depend on the specific binding of smaller and larger molecules to proteins and proteinaceous receptors. The cascade of events in the immune response is triggered by the recognition of an antigen molecule by an antibody. Carrier proteins bind small molecules to transport them through the intra- and extracellular compartments.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Use of carrier molecules (C) that selectively bind the active agent with high affinity. Ligand- (i.e., agent-) binding proteins are especially suitable for this purpose. They can be selected or genetically engineered to be sensitive to, for example, pH, temperature changes, and other molecules that displace the ligand by competitive or allosteric binding. Such selective high-affinity carrier molecules (proteins) can in turn be incorporated in more complex carrier systems that also include targeting devices like antibodies, stabilizing agents, and a selectively permeable capsule (schematically represented here by an interrupted black line). B, bound agent (ligand); F, free (unbound) agent; CD, competitive displacer; AD, allosteric displacer; Temp, temperature sensitivity; T, targeting device (e.g., antibody); St, stabilizing molecule.

Although agent-carrying controlled release systems may comprise several additional carrier components (see Fig. 2), the central function of the ligand-binding protein, with its high affinity and selectivity for the active agent and its ligand-stabilizing power (e.g., Hattori et al., 1995), would be to provide control over the binding, stabilization, and release of the active agent. Such application has already been proposed by others for some specific proteins and specific applications (e.g., Geisow, 1992; He and Carter, 1992; Kost and Langer, 1992; Batt et al., 1994; Stayton et al., 1995; Verhoeyen et al., 1995). We would like to consider this idea as a more general concept, potentially applicable in pharmacology and cosmetics but also in food technology, crop protection, industrial chemical processes, and various household purposes.

	II. Survey of Ligand-Binding Protein Classes

Top Previous Next References

One of the aims of this review is to give a general overview of different classes of ligand-binding proteins in view of their possible application in protein-mediated transport and controlled release of small molecules. Because many excellent reviews have already been devoted to the structural and ligand-binding properties of individual proteins and protein classes, we will give an overview of various classes and key proteins in terms of their ligand-binding properties and possibilities for modification. We concentrate on protein groups that can be used to bind small, chemically attractive, and commercially important molecules such as pharmaceuticals, flavors, pesticides, and/or insect pheromones that are used to control insect plagues. Excluded are proteins that bind metals, single-element ligands, or very large biopolymeric ligands such as nucleic acids and proteins. Thus, we will discuss 1) biotin-binding proteins, 2) lipid-binding proteins (LBPs)²/transporters of hydrophobic molecules, 3) bacterial periplasmic binding proteins, 4) lectins, 5) serum albumins, 6) immunoglobulins, 7) inactivated enzymes, 8) odorant-binding proteins (OBPs), 9) immunosuppressant-binding proteins, and 10) phosphate- and sulfate-binding proteins (PiBPs and SBPs). The treatment of each group could not possibly be exhaustive but merely serves to give an overview of the basic properties that may provide new ideas and may be relevant to the development of protein-mediated controlled release systems.

Of all ligand-binding proteins, the biotin-binding proteins avidin and streptavidin are perhaps most thoroughly studied. Their exceptionally high affinity for biotin (K_d = 6 × 10¹⁶ M for avidin and 4 × 10¹⁴ M for streptavidin) has lead to many biotechnological applications (Wilchek and Bayer, 1990). The novel approach of Stayton et al. (1995) in conjugating the protein to a stimuli-responsive polymer is likely to find applications in biotechnological and medical areas. The coupling of the protein to a temperature-sensitive polymer enables reversible, environmentally-triggered binding and release of the ligand. Normal binding of biotin is possible at temperatures below 32°C, but at temperatures above this, the coupled polymer collapses and blocks the binding site (Fig. 3). Another possibility is to engineer the loop involved in locking in the bound ligand or the monomer-monomer interfaces to render the protein susceptible to specific protease activity, temperature, pH, etc., thus making the release of ligand triggerable by these stimuli. In conjunction with the detailed molecular dynamics simulations performed using the biotin-binding system (Leckband et al., 1994; Moy et al., 1994; Vajda et al., 1994; Berendsen, 1996; Grubmüller et al., 1996), such studies bring these proteins closer to being ready for application in the area of ligand transport and release. It may, however, be difficult to engineer the binding site itself to obtain radical changes of ligand specificity (when molecules unrelated to biotin should be bound) because the molecular shapes and interacting groups of the binding site and its natural ligand are tightly complementary. The versatility of the (strept)avidin system is thus very limited. In this respect, the group of LBPs, which is discussed in the next section, offers much broader possibilities for adaptation.

View larger version (48K): [in this window] [in a new window]   Fig. 3.   Avidin modified with a temperature-sensitive polymer that blocks the binding site for biotin at high temperature, as described by Stayton et al. (1995). Left, high temperature. Right, low temperature. This figure was not taken from Stayton et al. (1995) but was newly designed to illustrate the principle. A, avidin; B, bound biotin; F, free avidin; P, polymer (indicated schematically with a black line).

The highly stable avidin-biotin complex (Green, 1990) has found actual application in drug targeting rather than in controlled release (Yoshikawa and Pardridge, 1992; Bickel et al., 1993; Kang and Pardridge, 1994; Shin et al., 1997; Schechter et al., 1999). It is an alternative to covalent attachment of drug to a carrier protein, which often requires harsh methods that can lead to denaturation, unwanted modification, and loss of functionality of the carrier and/or drug. Normally, a combination of biotinylated drug and an avidin-containing targeting system is used. The biotinylation of some peptide drugs can be obtained under relatively mild conditions, for example, by disulfide linkage (e.g., Yoshikawa and Pardridge, 1992; Bickel et al., 1993). Although the biotin-(strept)avidin complex is highly stable, the biotinylated drug can still be released at the target site. The release is easier in cases where coupling of avidin to a targeting device leads to a decreased affinity for biotin (Shin et al., 1997). The biotinyl group is small enough to allow in many cases a proper functioning of the drug at the target site. For targeting, the avidin has been chemically conjugated to antibodies or to cationized albumin (Bickel et al., 1993; Kang and Pardridge, 1994). Alternatively, antibodies and avidin have been fused to a single protein via genetic engineering (Shin et al., 1997), avoiding the need for chemical modification of the carrier (Fig. 4). The clearance of the fusion protein was more than 10 to 20 times slower than the clearance of free avidin (Shin et al., 1997). Probably due to its cationic nature and to glycosylation, free avidin has a very short plasma half-life (approximately 1 min) and is rapidly taken up by the kidney and liver. Removal of the glycosyl group of avidin and chemical modification may result in a prolonged half-life (Kang et al., 1995). Chemically modified avidin has also been used as a targeting device itself, negating the need for antibodies. Thus, trinitrophenylated streptavidin was used to effectively target 5-fluorouridine to the liver in mice (Schechter et al., 1999). The immunogenic nature of (strept)avidin is a point of concern. However, because most people have been exposed to egg avidin and because oral antigens are known to be tolerogenic, a certain degree of tolerance to avidin can be expected (Weiner, 1994). Antibody-avidin, cationized albumin-avidin fusion proteins (obtained by genetic engineering or chemical modification), and more complicated avidin-containing systems have been used for the successful delivery of biotin, biotinylated bioactive peptide, biotinylated nucleic acid, and other molecules to the brain as well as to cancer cells (e.g., Bickel et al., 1993; Kang and Pardridge, 1994; Shin et al., 1997; Penichet et al., 1999; Vinogradov et al., 1999). Because of the so-called blood-brain barrier, brain delivery is notoriously difficult (Halmos et al., 1997).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Avidin and antibody genes can be fused to encode a single fusion protein molecule with bispecific function (biotin binding and antigen recognition/targeting). Ab, antibody; Av, avidin. A similar principle can be applied to combinations of other ligand- (i.e., agent-) binding proteins.

The function of biotin-binding proteins in nature still remains a puzzle because, despite the apparent exquisite design, it is still unknown whether biotin is the sole or even the primary ligand for either protein (Linvah et al., 1993). Avidin is a basic tetrameric glycoprotein isolated from egg white (Fig. 5). Each monomer of 128 amino acids is capable of binding a single biotin molecule, giving a 62.4-kDa protein capable of binding four molecules of vitamin H (Pugliese et al., 1994). Streptavidin, secreted by Streptomyces avidinii, is slightly larger with 159 residues per subunit. However, such full-length molecules are rarely detected under the conditions used to culture the bacteria (Sano et al., 1995). The terminal regions of the protein are particularly susceptible to proteolysis, and truncated core streptavidins are more commonly formed, considerably improving solubility properties. Natural core streptavidin consists of 127 residues, forming a monomer of 13.3 kDa and a biotin-binding tetramer of ~53 kDa. Streptavidin lacks the carbohydrate chain present in avidin and has a lower isoelectric point. The resultant decrease of nonspecific binding has lead to increased application of the bacterial protein. Another difference between the two proteins is their sulfur content; avidin contains a disulfide bridge and two methionine residues per subunit, whereas streptavidin is free of sulfur-containing amino acids (Sano and Cantor, 1990). With a view to possible modification, it is essential for the protein to be expressed in a suitable vector. Both avidin and streptavidin have been successfully expressed in Escherichia coli (Sano and Cantor, 1990; Thompson and Weber, 1993; Airenne et al., 1994). Although the streptavidin gene is extremely lethal to the host cells, it can be expressed efficiently using T7 RNA polymerase/T7 promoter expression systems. As generally observed in E. coli overexpression systems, most of the streptavidin is insoluble in the cell, forming inclusion bodies.

View larger version (84K): [in this window] [in a new window]   Fig. 5.   Detail of a crystal structure of an avidin molecule complexed with biotin. The structure was obtained from the Brookhaven protein database (PDB), as deposited by O. Livnah and J. Sussman (see Livnah et al.,1993). The PDB data were processed using software from Quanta/Charmm Molecular Simulations Inc. (San Diego, CA).

The primary structures of the two proteins are similar (Linvah et al., 1993), and major structural motifs are conserved as well as the significant residues in the binding site. The proteins are constructed of eight antiparallel -strands forming the classic -barrel. The biotin-binding site is positioned toward one end of the barrel, where a number of aromatic and polar amino acids are involved in ligand binding being positioned to provide a precise fit for biotin. It has been reported (Linvah et al., 1993) that in the unoccupied binding site, the structure of the bound solvent molecules resembles that of the substrate. During the course of binding, solvent molecules are replaced by biotin and an exposed loop, residues 36 through 44 in avidin, orientates itself to lock the substrate in the binding site. Within the binding site, a number of aromatic amino acids form a "hydrophobic box" surrounding the biotin molecule. The two biotin-binding proteins differ somewhat in this area; in avidin, a phenylalanine, Phe⁷⁹, is involved in the binding, but this is replaced by a tryptophan, Trp⁹², in streptavidin. Also, an additional aromatic amino acid, Phe⁷², is involved that has no equivalent in streptavidin. The importance of the regions of monomer-monomer interaction is also emphasized in the binding site; for instance, the residues in the binding site of avidin comprise Trp⁷⁰, Phe⁷², Phe⁷⁹, and Trp⁹⁷ from one monomer and Trp¹¹⁰ from another. Similarly, the binding site of streptavidin contains only four aromatic residues, but these are again derived from adjacent monomers with Trp⁷⁹, Trp⁹², and Trp¹⁰⁸ from one monomer and Trp¹²⁰ from the adjoining one. Several crucial hydrogen bonds are formed between biotin and the side chains of polar amino acid residues. Interestingly, the biotin carboxyl group is involved in five hydrogen bond interactions in avidin but only two in streptavidin. Together with the additional aromatic residue in the avidin-binding site, this has been used (Linvah et al., 1993) to explain the 100-fold tighter binding of biotin to avidin compared with streptavidin. Another contributing factor may be the length of the loop that becomes ordered on binding and locks the ligand in the binding site. The longer loop in avidin contains nine residues, compared with six residues in streptavidin, and provides tighter closure.

The importance of the tryptophans in the binding site of streptavidin has been investigated (Chilkoti et al., 1995b) using site-directed mutants. This work confirmed the hypothesis that van der Waals and hydrophobic interactions, which are crucial to the binding process, are largely mediated by the aromatic side chains of the tryptophan residues. Similarly, the role played by the tryptophan residue Trp¹²⁰ of one subunit in binding a biotin molecule held by an adjoining subunit has been investigated (Sano and Cantor, 1995). Mutation of this residue to a phenylalanine had no effect on the biotin-induced formation of streptavidin tetramers or on the amount of binding sites per tetramer but resulted in a drastic drop in biotin-binding affinity, verifying the significance of the role played by Trp¹²⁰ in the tight binding of biotin. It has been proposed that the mutation of Trp¹²⁰ to Phe may have introduced additional structural transformations in the protein at or near the binding site, considerably lowering the affinity for biotin (Sano and Cantor, 1995).

The question of whether binding of biotin to the streptavidin tetramer is cooperative has been addressed (Jones and Kurzban, 1995), and it was found that despite the large binding energy involved, biotin slowly equilibrates between tetramers, and the equilibrium distribution indicates that binding is not cooperative. The degeneracy of the streptavidin tetramer can be abolished by the construction of chimeric tetramers composed of unmodified wild-type subunits and genetically engineered subunits (Chilkoti et al., 1995a). The chimeric streptavidin tetramers are produced by mixing wild-type streptavidin and site-directed mutants, followed by denaturation and slow renaturation. This approach allows tetramers to be produced in which one or more monomers retain the high affinity for biotin and one or more monomers possess a novel property such as reduced affinity due to a greater off-rate, enabling enhanced recovery in applications where streptavidin is used as a capture molecule. In applications where streptavidin is conjugated to functional moieties such as antibodies, a mutant subunit can be incorporated that spatially directs the assembly of the tetramer to encourage conjugation to a particular face of the protein, leaving another face free for biotin binding.

The widespread interest in avidin and streptavidin largely results from their high specificity for biotin. However, other molecules are known to bind to these proteins, albeit with much lower affinity. A number of peptide ligands have been reported that contain the consensus tripeptide sequence HPQ (Giebel et al., 1995; Katz, 1995). The amino acids flanking this sequence play important roles, but the interactions with the HPQ portion of the peptide are all very similar, involving common hydrogen bonds and van der Waals interactions. Some disulfide-bonded cyclic peptides bind with several hundred-fold greater affinity than their linear counterparts, and binding is seen to be greater at neutral pH than at pH 5 (Weber et al., 1992). The relationship between 1) the structural differences of biotin and the peptides studied so far and 2) their different binding characteristics is not clear at this moment. The stereochemical features of biotin that lead to the high binding affinity are not easily derived from the corresponding bound peptide structures. The binding of other ligands, such as synthetic azobenzenes, has also been reported (Weber et al., 1994). Biotin accounts for half the avidin-binding material in human serum with the remaining 50% comprising biotin metabolites (Mock et al., 1995).

The effective transport of hydrophobic molecules within a cell system is mediated in part by LBPs. These low-molecular weight proteins, which bind, among other compounds, fatty acids and retinol analogs (Newcomer, 1995), are present in a diverse range of cell types. Like many other binding proteins, their exact role is unclear, but there is presently little to suggest a function other than solubilization and transport of hydrophobic ligands.

The large and expanding group of LBPs includes commonly known members such as the milk whey protein -lactoglobulin as well as lesser known proteins such as ₁-microglobulin (Åkerström and Lögdberg, 1990), plasma or tear transthyretin (formerly called prealbumin) (Redl et al., 1992; Garibotti et al., 1995; Monaco et al., 1995), proteins secreted by the mammalian von Ebner's salivary glands of the tongue, soluble OBPs secreted by nasal glands, and mammalian aphrodisin (Kruhoffer et al., 1997; Magert et al., 1999). The OBPs appear to function as cofactors in olfaction and taste sensing (Pevsner et al., 1990; Korsching, 1991; Kock et al., 1994; Garibotti et al., 1995). Distantly related members of the group may have less than 20% amino acid sequence identity, but many have highly similar tertiary structures as will be discussed below. The LBPs (also called "calycins" because of the shape of the binding pocket) can be divided into two groups: the intracellular LBPs (iLBPs) and the extracellular LBPs (eLBPs; also referred to as "lipocalins"). A number of LBPs has been studied crystallographically (for example, see Table 1) and/or by NMR (e.g., Lassen et al., 1995). Many LBPs still await characterization.

A detailed description of the structural features of individual proteins is unnecessary here because this has been the subject of an excellent review by Banaszak et al. (1994). However, because the family members share many structural motifs, a brief overview will be useful to anyone interested in using or modifying these proteins. The eLBPs are composed of approximately 175 amino acid residues, whereas the iLBPs are slightly smaller with about 130 residues. The principal feature of both groups is an antiparallel -barrel with a repeated +1 topology, composed of 10 strands in the iLBPs and 8 in the lipocalins. The iLBP barrel is more elliptical than that of the eLBPs and is not continuously hydrogen bonded. The binding site has been found to exist almost exclusively within this -barrel, and moreover, the stoichiometry of binding is generally one hydrophobic molecule per molecule of protein. Comparison of the group structures shows that the initial three and final two strands of the iLBP barrel correspond well to the initial three and final two strands of the eLBPs (Flower et al., 1993). The strands of both families are mostly connected by tight turns, except the first two strands of the iLBPs, which are connected by a helix-turn-helix motif that is absent in the corresponding region of the eLBPs. Also, a "gap region" exists in the iLBP structure between strands 4 and 5 that is missing in the eLBP structure (Banaszak et al., 1994).

The conformational similarity between members of the LBP group is remarkably high; the -barrel, the link regions, and the binding cavity are virtually superimposable. Despite the high degree of structural homology, the sequence identity is generally low, particularly in the eLBPs where it is often less than 20%. However, there are regions where primary structure conservation is greater. Within the eLBPs, it has been reported (Katakura et al., 1994) that the only completely conserved residue is Trp¹⁹ and that this residue, although not vital for ligand binding, was critical for maintaining the environment surrounding the bound ligand for correct positioning of the ligand and for stabilizing the overall protein structure. Compared with the eLBPs, the iLBPs show a greater range of intragroup homology from 20 to over 80%, the average being 20 to 30%. Even though the number of homologous residues is low, a number of conserved amino acids do occur, many of these being involved in the formation of a structural backbone. The conserved residues appear to be either internal hydrophobic residues or neighboring hydrophilic amino acids that help to stabilize the backbone structure. Some of the hydrophobic residues within this backbone also form part of the wall of the binding site (Banaszak et al., 1994).

As a group, the LBPs are able to bind a diverse range of ligands, but also the individual members of the group bind a wide range of ligands (Richieri et al., 1994). Different LBPs can have ligand binding spectra that overlap to various degrees as summarized by Banaszak et al. (Banaszak et al., 1994). The affinities of various proteins for fatty acid ligands are similar (Wootan et al., 1990), ranging from approximately 2.5 × 10⁵ to 5 × 10⁶ M¹ and showing a general trend of decreasing affinity for shorter hydrophobic chain lengths (Banaszak et al., 1994).

Unlike the biotin-binding proteins, the unoccupied binding site in the hydrophobic transporter molecules does not reflect the shape of the fatty acid ligand. The volume of the binding cavity is considerably larger than any prospective ligand with perhaps only one-third of the cavity volume being occupied by the bound fatty acid or retinoid. This leaves space for a number of ordered water molecules, with 16 being identified in holo-adipocyte LBP (ALBP) (Xu et al., 1993). In the absence of ligand, there is space for 40 disordered solvent molecules (Banaszak et al., 1994). There are more than 35 amino acids lining the binding cavity, of which perhaps only half are hydrophobic; the remainder are polar and/or ionizable. Generally, the polar head of a fatty acid ligand lies at the bottom of the cavity, and binding of ligand results in little conformational change in the protein.

By far, the most studied of the hydrophobic transporter molecules is the 18-kDa globular milk whey protein, -lactoglobulin. The exact role of this protein is unknown. It is believed to function as a transporter of retinol (vitamin A) to the neonate in mammals, although its absence in the milk of many mammals, including humans, indicates that this role is not essential. Apart from retinol, it binds retinoic acid and other retinol analogs (Cho et al., 1994a), -carotene, -ionone compounds, saturated (Fig. 6) and unsaturated fatty acids (O'Neill and Kinsella, 1987), aliphatic hydrocarbons such as heptane and pentane, and carbonyl-based compounds such as 2-octanone (O'Neill and Kinsella, 1987). The affinities for the different compounds are widely different. For example, the K_a values of heptane and 2-heptanone are 4.8 × 10⁵ and 1.5 × 10² M¹, respectively, whereas that of retinol is 5 x 10⁷ M¹ (O'Neill and Kinsella, 1987). It has been previously proposed that -lactoglobulin could be used as a versatile carrier of small hydrophobic molecules in controlled delivery applications (Batt et al., 1994). This application is likely to be facilitated by expression of -lactoglobulin in the food-grade yeast Kluyveromyces lactis (Rocha et al., 1996) and by the recently obtained high-yield expression in the methylotrophic yeast Pichia pastoris (Kim et al., 1997; Denton et al., 1998, de Wolf et al., 1998), which is food-grade according to the FDA and, although not officially approved, also seems to be a safe species for humans (Scrimshaw and Murray, 1995). The crystal structure of bovine -lactoglobulin, exhibiting the characteristic -barrel structure of eLBPs, has been known for some time (Papiz et al., 1986), and differences in the structure between the naturally occurring genetic variants have been studied (Dong et al., 1996). The variants A and B differ in just two positions with no significant effect on the binding affinity or crystal structure. In contrast to the other eLBPs, -lactoglobulin contains two disulfide bonds and a free thiol group. For some time, there has been uncertainty over the location of the retinol-binding site. The initial observations based on its sequence and structural similarity to retinol-binding protein (RBP) indicated the retinol to be bound within the -barrel. This hypothesis was questioned when crystallographic evidence seemed to indicate the retinol to be bound in a surface groove (Monaco et al., 1987). However, further studies, using selective amino acid modifications, confirmed the retinol-binding site to be within the central calyx (Cho et al., 1994a). After binding to -lactoglobulin, retinol and other hydrophobic molecules are protected from (oxidative) degradation (Hattori et al., 1995; Iametti et al., 1995). Site-directed mutagenesis studies have been facilitated by the expression of -lactoglobulin in E. coli (Batt et al., 1990; Chatel et al., 1996) and in the yeast K. lactis (Rocha et al., 1996; Kim et al., 1997; Denton et al., 1998). These highlighted the importance of individual residues for retinol binding (Zanotti et al., 1993b) and protein stability (Katakura et al., 1994). Much work has been devoted to the denaturation (Cho et al., 1994b; Dumay et al., 1994; Hong et al., 1994; Jeyarajah and Allen, 1994; Macleod et al., 1995; Qi et al., 1995; Boye et al., 1996) and renaturation (Dufour et al., 1994) behavior of -lactoglobulin. Hattori et al. (1993) used -lactoglobulin as a model protein to show that, following denaturation, specific moieties within a protein cannot return to the native conformation from a denatured state. The results were based on the inability of monoclonal antibodies to recognize renatured -lactoglobulin. However, the incomplete refolding of the protein did not affect its biological properties. -Lactoglobulin also exhibits the ability to form heat-induced aggregates and gels (Hong et al., 1994; Matsuura and Manning, 1994) both separately and in combination with other macromolecules such as polysaccharides (Ndi et al., 1996a,b), a property which could be valuable in the development of protein-mediated transport systems.

View larger version (76K): [in this window] [in a new window]   Fig. 6.   Crystal structure of bovine -lactoglobulin with palmitic acid (C16:0) in the binding pocket. The structure was obtained from the Brookhaven protein database (PDB), as deposited by S.-Y. Wu and L. Sawyer (see Wu SY et al., 1999). The PDB data were processed using software from Quanta/Charmm Molecular Simulations Inc.

Another important eLBP is the plasma RBP (pRBP) found in the circulatory system bound to transthyretin (Monaco et al., 1995). This specific retinol carrier has been isolated from several vertebrates, including mammals, birds, and fish, although most work has been done on bovine and human pRBP (Zanotti et al., 1993a,b, 1994; Sivaprasadarao and Findlay, 1994). Recently, an E. coli expression system for pRBP has also been described (Müller and Skerra, 1993) that should make future mutagenesis possible. Bovine and human pRBP are found to be quite similar, exhibiting 92% sequence similarity and practically identical three-dimensional structure (Zanotti et al., 1993a). They exhibit the typical eLBP -barrel structure with the retinol molecule completely encapsulated within the -barrel with its -ionone ring in the center, its tail pointing outward, parallel to the barrel axis, and the end of its tail close to the protein surface. In accordance, it was found that alteration of the cyclohexene ring of retinol may result in a lowering of binding affinity, whereas substantial modifications of the hydroxyl end do not preclude high-affinity binding to pRBP (Zanotti et al., 1993b). The binding of retinol to pRBP is thus stabilized mainly by hydrophobic interactions with the ring moiety playing the dominant role.

Interestingly, the orientation of the ligand in eLBPs is opposed to that in iLBPs, where the polar end is buried inside the cavity. Accordingly, the binding appears to be particularly sensitive to modifications of the polar end, e.g., the hydroxyl of retinol in the case of intracellular RBP possibly in addition to changes of the isoprene chain (Malpeli et al., 1995). A member of the iLBP family that binds exclusively fatty acids (no retinoids) is the intestinal fatty acid-binding protein (I-FABP). The 15-kDa protein is synthesized in the enterocytes of the small intestine. Each molecule binds a single saturated, monounsaturated, or polyunsaturated fatty acid. The apo and holo forms of I-FABP from the rat have been characterized to high resolution (1.2 and 2.0 Å, respectively) (Scapin et al., 1992). The protein has the common structural features of the iLBP family, the carboxylate of the fatty acid being buried in the -barrel of the binding cavity. A comparison of apo and holo forms of the rat I-FABP shows that ligand binding does not induce clear conformational changes. The ligand-binding cavity of I-FABP is different from that of other proteins in the family in that the amino acid side chains that constitute the binding cavity are predominantly hydrophobic, and those that are in close contact with the ligand are quite different from those of related proteins of known structure. The protein has been successfully expressed in E. coli with no loss of function and an absence of co- or post-translational modifications. Studies based on such systems have yielded detailed information about the nature of the interaction between protein and fatty acid ligand (Sacchettini and Gordon, 1993) that deal comprehensively with the combination of X-ray crystallography and mutagenesis to analyze the binding forces. They propose that fatty acid binding results from a series of weak forces, including a complex range of electrostatic and dipolar forces between the fatty acid carboxylate, polar, and ionizable groups of the protein and solvent molecules, as well as a range of van der Waals contacts involving residues from both the -sheets and the helices. The importance of a number of individual residues has been implicated in playing distinct roles in various aspects of the binding interaction (Scapin et al., 1992; Zanotti et al., 1992; Sacchettini and Gordon, 1993; Banaszak et al., 1994).

The ALBP is a 14.6-kDa protein found in adipose cells. It is known to bind long-chain fatty acids and retinol (LaLonde et al., 1994a,b), and its function is believed to be the transport and solubilization of fatty acids. It has been well characterized and shown to possess the typical LBP properties such as the -barrel binding cavity and the retention of conformation between apo and holo forms. The specificity is generated as the result of interaction of the fatty acid carboxylate with two arginines and a tyrosine. In some adipocytes, the protein is phosphorylated in response to insulin (Xu et al., 1991). The effects of tyrosyl phosphorylation on the cellular function of ALBP is unknown, but its in vitro effect is the modulation of ligand binding, with the phosphorylated protein showing much reduced affinity for immobilized long-chain fatty acids (Xu et al., 1993). Murine ALBP has been successfully cloned and expressed in E. coli (Xu et al., 1991) and characterized crystallographically to 1.6 Å. The recombinant protein has been used for site-directed mutagenesis studies of the binding site (Sha et al., 1993). Increased insight into structure-function relationships of fatty acid-binding proteins is now coming from molecular modeling and dynamics studies (Woolf, 1998). Compared with the (strept)avidin system, the induction of triggered release or uptake of ligands is probably much more difficult to elicit in LBPs, even after modification of the protein structure by genetic means. However, the LBP system can be used with a broader range of (apolar or amphiphilic) ligands and allows easier switching between (such) ligands.

These proteins, found in the periplasmic space of Gram-negative bacteria, serve as initial high-affinity receptors in the active uptake of specific nutrients. After binding their specific substrate molecule, they interact with a membrane bound complex, triggering a series of events that results in translocation of the substrate. The mechanism of the transport system has been reviewed by Ames (1986), and although the number of binding proteins reported has increased since then, this work still provides a valuable basis for the understanding of bacterial periplasmic transport. More recently, Tam and Saier (1993) reviewed the mutual relationships between extracellular solute-binding receptors of bacteria, providing a clear overview of the various protein "clusters". The family of approximately 50 periplasmic binding proteins is valuable with respect to development of a system of protein-mediated delivery in that they collectively bind a wide range of ligands. This provides versatility (viz., when use can be made of several members of the group) without sacrificing ligand specificity, which is conferred by the individual proteins. The periplasmic binding proteins exhibit specificity for carbohydrates, amino acids, peptides, metals, or vitamins. Some binding proteins are highly ligand-specific, whereas others are known to bind several related ligands, each with comparable affinity. In general, the largest proteins bind the largest ligands, the protein molecular masses ranging from 20 to 58 kDa, with the average being approximately 33 kDa. The sequence similarity between members of the group is generally low. Nevertheless, the different proteins tend to exhibit a similar affinity for their respective ligands, the K_d being of the order of 5.0 × 10⁷ M (Spurlino et al., 1991). The crystal structures of a growing number of periplasmic binding proteins have been elucidated, and, despite the low sequence homology, the overall three-dimensional structures are similar. The structural features responsible for different functions (binding of ligands, binding to membrane receptors) are located in structurally distinct regions in each protein. All the proteins are composed of two lobes, joined by two or three peptide strands, which function as hinges. The ligand-binding site is located within a cleft between the two lobes (Fig. 7). The bound ligand is typically buried within this cleft, where it is almost inaccessible to bulk solvent. This form of the protein is designated the "closed" form. The "open" form is obtained by rotation of the lobes relative to one another by means of the peptide hinge region (see Fig. 7). This allows the access of the solvent to the binding cleft as well as the entry or exit of the ligand. The lobes themselves do not change their conformation significantly during the rotatory movement. On closure, residues from both lobes are involved in the formation of a network of interactions with the bound ligand, interactions that involve polar and nonpolar side chains as well as the protein backbone and charged side chains. Thus, the presence of the bound ligand shifts the relative energies of the two conformations (i.e., their probabilities of occurrence) in favor of the closed form, which is stabilized by the ligand. There is still some doubt whether the empty proteins continually alternate between the open and closed form or whether the conformational change occurs only once on binding of the ligand. For example, the equilibrium conformational free energies of the two forms could be very different, implying that the chance of occurrence of the closed form would be low (or virtually zero) in the empty protein. Alternatively, the activation energy could be relatively large in the absence of ligand, preventing the empty protein from closing. Although not unambiguously proven, it is generally thought that a (net) reversion of the conformation from the closed to the open state and a subsequent (directional) release of ligand are specifically induced by the binding of the protein-ligand complex to a corresponding membrane protein (Ames, 1986; Dean et al., 1992; Mowbray, 1992; Shilton and Mowbray, 1995). The membrane protein translocates the ligand to the cytoplasm (Ames, 1986; Shilton and Mowbray, 1995). It specifically recognizes the closed conformation (top of the two lobes) of the periplasmic binding protein with its complexed ligand (Spurlino et al., 1991; Sharff et al., 1992). Interestingly, modifications of the hinge region have been shown to affect the ability of the periplasmic proteins to bind their ligands. For example, in the case of maltose-binding proteins, the bound ligand was found to directly interact with at least one hinge-region residue, viz., Glu¹¹¹ (Spurlino et al., 1991; Sharff et al., 1995). In this light, the "hinge" region represents an interesting target area for protein engineering studies exploring the possibility of making periplasmic binding proteins stimulus- (e.g., protease-, pH-) responsive. Such developments could well find application in stimuli-responsive ligand release systems (pest control, hygiene, technical applications, and special therapeutic applications). However, it is not known whether the hinge region can still influence the binding after completion of the transition from the open to the closed conformation because the closed conformation is stabilized by interactions between the two lobes and the ligand. Such an influence could occur if the ligand-containing protein were subject to frequent alternation between the closed (major) and the open (minor) conformations, which would then be in equilibrium, in a manner similar to what has been proposed in relation to the empty protein. Obviously, this is an interesting subject that requires further research.

View larger version (79K): [in this window] [in a new window]   Fig. 7.   Crystal structure of E. coli periplasmic maltodextrin-binding protein. The molecule represented as a ribbon was complexed with maltodextrin; the molecule represented as a thread is the empty protein without ligand. The structures were obtained from the Brookhaven protein database (PDB), as deposited by J. C. Spurlino and F. A. Quiocho for the complex (see Quiocho et al., 1997) and by A. J. Scharff and F. A. Quiocho for the unliganded protein (see Sharff et al., 1992). The PDB data were processed using software from Quanta/Charmm Molecular Simulations Inc.

Examples of periplasmic binding proteins include those with specificities for maltodextrin [maltodextrin-binding protein (MBP), Fig. 7] (Spurlino et al., 1991; Dean et al., 1992; Sharff et al., 1992, 1995), histidine [histidine-binding protein (HBP)] (Oh et al., 1994b; Wolf et al., 1994, 1995), lysine/arginine/ornithine [lysine/arginine/ornithine-binding protein (LAOBP)] (Kang et al., 1991; Oh et al., 1993, 1994a), leucine/isoleucine/valine (leucine/isoleucine/valine-binding protein) (Ohla et al., 1993), glutamine (glutamine-binding protein) (Hing et al., 1994), dipeptides [dipeptide-binding protein (DPP)] (Dunten and Mowbray, 1995; Nickitenko et al., 1995), oligo-peptides [oligopeptide-binding protein (OPP)] (Hiles et al., 1987; Kashiwagi et al., 1990), arabinose (arabinose-binding protein) (Kehres and Hogg, 1992), galactose/glucose (galactose/glucose-binding protein) (Mowbray, 1992), sulfate (SBP) (Wang et al., 1994), phosphate (PiBP) (Wang et al., 1994), and ribose (ribose-binding protein) (Binnie et al., 1992; Björkman et al., 1994). The binding proteins with known tertiary structure have little in common with respect to size, amino acid composition, and specificity for the primary ligands, with the exception of the HBP and LAOBP pair, which show sequence identities of 70%. HBP and LAOBP are probably the most extensively studied of the periplasmic binding proteins (Oh et al., 1994a; Wolf et al., 1995). In addition to a high degree of sequence similarity, they contain an identical number of residues and have closely resembling domains that interact with common membrane-bound components of the transport system. Comparison of HBP and LAOBP is interesting because LAOBP also binds histidine, albeit with reduced affinity. The conformations of ligand-bound forms of LAOBP are essentially the same for lysine, arginine, and ornithine, with minor variations restricted to the area directly around the binding site. All of the residues surrounding the ligand in HBP are identical with those in LAOBP with the exception of residue 52, where a leucine in HBP is replaced with a phenylalanine in LAOBP. Furthermore, two other residues interact in a different way with the ligand in the two proteins (Oh et al., 1994b), reflecting the difference in shape and length of the ligands. Ser⁶⁹ forms a hydrogen bond from its carbonyl oxygen in HBP, but in LAOBP, a hydrogen bond originates from its hydroxyl group. Asp¹¹ in LAOBP makes an ionic and hydrogen bonding contribution, but in HBP, it is not close enough to the bound ligand to make a contribution.

Comparison of the residues lining the binding cleft in HBP and LAOBP show that only five amino acids differ between the two proteins, and it has been proposed that the differences in substrate affinities may be due to one or more of these residues. As mentioned above, only one of these five residues, residue 52, interacts with the ligand, although it is still possible that one or more of the other residues directly or indirectly contributes to the binding interaction. However, it is not apparent why a leucine at position 52 in HBP should result in this protein having a higher affinity for histidine than LAOBP, which has a phenylalanine in the corresponding position. Oh et al. (1994b) proposed that the presence of the bulkier phenylalanine prevented the formation of an essential ionic interaction. Hydrogen bonds play an important role in the recognition of the respective ligands, and their directional nature helps confer specificity on the binding site. In these particular binding proteins, the binding pocket seems to be large enough to accommodate the maximum common volume of the four ligands plus three water molecules. Residues within the binding pocket undergo small conformational changes to achieve geometric fit and most favorable interactions with the ligand. To achieve optimal fit, protein-bound water molecules can be displaced by the ligand.

To further understand the functions of individual amino acid residues in the periplasmic binding proteins, Wolf et al. (1995) carried out a series of studies on mutant proteins. They found that in many cases, the substitution of a single amino acid had quite pronounced effects, generally leading to the inability of the protein to assume the closed conformation of the liganded form. A total of 12 residues are involved in binding the histidine molecule, six of which have hydroxyl functions. In total, the histidine molecule is held in place by 10 hydrogen bonds, 2 salt links, and more than 60 van der Waals contacts (Yao et al., 1994). Tyr¹⁴ is the singly most involved protein residue, interacting with seven different histidine atoms.

Of the four sugar-binding proteins, the broadest specificity (or highest versatility) can be attributed to the MBP. It is able to bind linear maltodextrins of two to seven (1-4)-linked glucosyl units as well as cyclodextrins such as cyclomaltohexose and cyclomaltoheptose. The other three well characterized sugar-binding proteins are able to bind only monopyranosides, although their specificity is not limited to an individual sugar. Arabinose-binding protein binds L-arabinose, D-galactose, and D-fucose; the galactose/glucose-binding protein binds D-galactose and D-glucose; and the ribose-binding protein binds D-ribose. Of these, MBP is the most extensively studied. As with the amino acid-binding proteins, its binding site is located at the base of a groove between the two domains, almost completely shielded from solvent and bound by a combination of hydrogen bonds and van der Waals interactions. Every polar and nonpolar atom of the bound sugar molecule forms extensive hydrogen bond and van der Waals interactions, respectively, within the binding site. Thus, the number of van der Waals contacts is unusually high, e.g., 65 in MBP. The sugar hydroxyl groups act as simultaneous hydrogen bond donors and acceptors. MBP has a relatively high number of aromatic residues, many of which are located in or near the binding groove and take part in stacking interactions with the carbohydrate pyranose ring. Together with the deep binding, which shields more than 96% of the sugar molecule from the bulk solvent, these interactions make the complexes between the binding proteins and carbohydrates some of the tightest.

Sharff et al. (1992, 1995) have studied MBP in detail both crystallographically and by the use of mutations of the malE gene that encodes MBP. They observed the classic rigid body "hinge-bending" between the two domains to reveal the sugar-binding site, which is characteristic of the periplasmic binding proteins, and proposed that the hinge-mediated closing is triggered by the ligand-induced exclusion of water from the binding site, which is in agreement with the lower number of ordered water molecules found in the closed, maltose-containing site. Using mutant proteins, it was shown that, apart from the binding site, the hinge region of the protein is particularly sensitive to minor changes, whereas several other regions were tolerant to substantial modifications. In one example, a helix was deleted with little effect on sugar binding, binding to the membrane, and general structure of the protein.

Other periplasmic binding proteins are less well studied. DPP and OPP are two of the largest members of the periplasmic binding protein family. DPP from E. coli shows 24% sequence homology with the slightly larger OPP of Salmonella typhimurium. Certain structural features of the oligopeptides seem to be essential for their binding to OPP. These include a protonated primary amino group, L-stereochemistry, and a modified terminal carboxyl (Nickitenko et al., 1995). The nature of the side chains appears to make little contribution to ligand binding. These proteins are distinguished from the previous periplasmic binding proteins by their broad specificity. DPP binds dipeptides and some tripeptides as diverse as glycine-glycine, lysine-lysine, and phenylalanine-phenylalanine with similar affinity. OPP binds peptides that vary in length from two to five amino acids with little consideration to their side chains; it will not bind single amino acids. In both proteins, specific interactions seem to be restricted to the peptide backbone, and there are few specific interactions, if any, between the binding protein and the side chains of the peptide. Off the binding site, pockets are present that accept these side chains and that are large enough to accommodate any of the naturally occurring side chains. Unlike other periplasmic binders, these proteins are composed of three domains. Domains I and III are analogous to the domains in the other family members in that they form the two lobes of the "Venus flytrap", which close to entrap the ligand. Domain II has no counterpart in the bilobate binding proteins. Its function is not clear, but it appears to contribute residues to line the pocket that accepts the side chain of the initial peptide residue. This domain contains two -hairpins and consists of approximately 120 residues in OPP and approximately 150 residues in DPP; it makes few contacts with the ligand.

In contrast to the peptide transporters, the oxyanion transport molecules show extreme specificity (Wang et al., 1994). The structurally similar tetrahedral oxyanions, phosphate and sulfate, are bound, respectively, by the PiBP and the SBP. The high specificity for each oxyanion is vital in that one ligand cannot become an inhibitor for the transport of the other. The specificity is derived from the presence or complete absence of protons on the substrate molecule. The binding site of PiBP is designed to recognize the protons of weakly acidic mono- and dibasic phosphate, whereas the binding site of the SBP is constructed to receive only the fully ionized sulfate. Once bound, the phosphate is held in place by hydrogen bonds that involve 12 polar amino acid residues. The carboxyl function of one of these, Asp⁵⁶, serves as a charged hydrogen bond acceptor but disallows, by charge repulsion, the binding of a sulfate group. Site-directed mutagenesis studies by Wang et al. (1994) have shown that the specificity of PiBP can be even further enhanced. For instance, by replacement of Thr¹⁴¹ with an Asp residue, the binding can be limited to the monobasic form of phosphate (H₂PO₄), excluding the binding of the dibasic form (HPO₄²). Such exquisite modifications of the binding specificity provide information that can be applied in the tailoring of these proteins to carry other ligands.

Lectins are proteins that reversibly bind carbohydrates with high affinity and specificity. The binding involves hydrophobic interactions as well as hydrogen bonds. The lectins are able to induce cell agglutination and are thus believed to play a key role in plant defense mechanisms. These ubiquitous proteins are found in microorganisms, animals, and plants (Lis and Sharon, 1986; Brossmer et al., 1992; Sharon, 1993; Jordan and Goldstein, 1994; Iobst et al., 1994; Kennedy et al., 1995; Peumans and Van Damme, 1996). Plant lectins have been purified from leaves, fruits, roots, and tubers but primarily from seeds. There has been much discussion about the definition of a lectin; the current definition for plant lectins is related to function rather than structural criteria. All plant proteins possessing at least one noncatalytic domain that binds reversibly to a mono- or oligosaccharide are considered to be lectins. If this definition were extended to all carbohydrate-binding proteins, the aforementioned periplasmic binding protein MBP would also fulfill the criteria of a functional lectin. Lectins have been divided into three classes. The merolectins, found in certain microorganisms (Actinomyces, Myxococcus, and Mycoplasma among others) consist of a single carbohydrate-binding domain. These small single-chain proteins are incapable of agglutinating cells. The hololectins behave as true agglutinins. They are similarly composed exclusively of carbohydrate-binding domains but contain at least two such domains. The chimerolectins are fusion proteins composed of at least one sugar-binding domain and an unrelated domain with a separate biological function, e.g., the membrane-anchoring domain of vertebrate lectins. The majority of currently known plant (holo)lectins can be further classified into four subgroups of related proteins. The legume lectins occur exclusively within the legume family. The monocot mannose-binding lectins occur in at least five different families and all have similar molecular structure and binding specificity. The chitin-binding lectins, found in unrelated taxonomic families, are composed of similar structural domains and exhibit comparable specificity. The type 2 ribosome-inactivating proteins are chimeric proteins composed of a catalytically active A chain covalently linked to a carbohydrate-binding B chain.

The lectins of leguminous origin are perhaps best characterized; the three-dimensional structures of a number of them are known (Einspahr et al., 1986; Shaanan et al., 1991). The primary structures are highly homologous, resulting in similar three-dimensional conformations. -Sheets dominate, whereas -helices are virtually absent. Another common feature is the presence of two metal ions, Ca²⁺ and Mn²⁺, that appear to be essential for sugar binding. The amino acids involved in metal binding are highly conserved throughout the legume lectin subgroup. The