Camelid immunoglobulins and nanobody technology.

It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from other species, these special antibodies are devoid of light chains and are composed of a heavy-chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma-genes. An immune response is raised in these so-called heavy-chain antibodies following classical immunization protocols. These HCAbs are easily purified from serum, and the antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. Since the antigen-binding site of the dromedary HCAb is comprised in one single domain, referred to as variable domain of heavy chain of HCAb (VHH) or nanobody (Nb), we designed a strategy to clone the Nb repertoire of an immunized dromedary and to select the Nbs with specificity for our target antigens. The monoclonal Nbs are well produced in bacteria, are very stable and highly soluble, and bind their cognate antigen with high affinity and specificity. We have successfully developed recombinant Nbs for research purposes, as probe in biosensors, to diagnose infections, and to treat diseases like cancer or trypanosomosis.

Degenerate interfaces in antigen-antibody complexes.

In most of the work dealing with the analysis of protein-protein interfaces, a single X-ray structure is available or selected, and implicitly it is assumed that this structure corresponds to the optimal complex for this pair of proteins. However, we have found a degenerate interface in a high-affinity antibody-antigen complex: the two independent complexes of the camel variable domain antibody fragment cAb-Lys3 and its antigen hen egg white lysozyme present in the asymmetric unit of our crystals show a difference in relative orientation between antibody and antigen, leading to important differences at the protein-protein interface. A third cAb-Lys3-hen lysozyme complex in a different crystal form adopts yet another relative orientation. Our results show that protein-protein interface characteristics can vary significantly between different specimens of the same high-affinity antibody-protein antigen complex. Consideration should be given to this type of observation when trying to establish general protein-protein interface characteristics.

Arsenate reductase from S. aureus plasmid pI258 is a phosphatase drafted for redox duty.

Arsenate reductase (ArsC) from Staphylococcus aureus plasmid pI258 plays a role in bacterial heavy metal resistance and catalyzes the reduction of arsenate to arsenite. The structures of the oxidized and reduced forms of ArsC were solved. ArsC has the PTPase I fold typical for low molecular weight tyrosine phosphatases (LMW PTPases). Remarkably, kinetic experiments show that pI258 ArsC also catalyzes the tyrosine phosphatase reaction in addition to arsenate reduction. These results provide evidence that ArsC from pI258 evolved from LMW PTPase by the grafting of a redox function onto a pre-existing catalytic site and that its evolutionary origin is different from those of arsenate reductases from Escherichia coli plasmid R773 and from Saccharomyces cerevisiae. The mechanism proposed here for the catalysis of arsenate reduction by pI258 ArsC involves a nucleophilic attack by Cys 10 on arsenate, the formation of a covalent intermediate and the transport of oxidative equivalents by a disulfide cascade. The reaction is associated with major structural changes in the ArsC.

Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae.

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII beta-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those beta-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 beta-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of beta-lactamases.

Weak protein-protein interactions in lectins: the crystal structure of a vegetative lectin from the legume Dolichos biflorus.

The legume lectins are widely used as a model system for studying protein-carbohydrate and protein-protein interactions. They exhibit a fascinating quaternary structure variation, which becomes important when they interact with multivalent glycoconjugates, for instance those on cell surfaces. Recently, it has become clear that certain lectins form weakly associated oligomers. This phenomenon may play a role in the regulation of receptor crosslinking and subsequent signal transduction. The crystal structure of DB58, a dimeric lectin from the legume Dolichos biflorus reveals a separate dimer of a previously unobserved type, in addition to a tetramer consisting of two such dimers. This tetramer resembles that formed by DBL, the seed lectin from the same plant. A single amino acid substitution in DB58 affects the conformation and flexibility of a loop in the canonical dimer interface. This disrupts the formation of a stable DBL-like tetramer in solution, but does not prohibit its formation in suitable conditions, which greatly increases the possibilities for the cross-linking of multivalent ligands. The non-canonical DB58 dimer has a buried symmetrical alpha helix, which can be present in the crystal in either of two antiparallel orientations. Two existing structures and datasets for lectins with similar quaternary structures were reconsidered. A central alpha helix could be observed in the soybean lectin, but not in the leucoagglutinating lectin from Phaseolus vulgaris. The relative position and orientation of the carbohydrate-binding sites in the DB58 dimer may affect its ability to crosslink mulitivalent ligands, compared to the other legume lectin dimers.

Antigen specificity and high affinity binding provided by one single loop of a camel single-domain antibody.

Detailed knowledge on antibody-antigen recognition is scarce given the unlimited antibody specificities of which only few have been investigated at an atomic level. We report the crystal structures of an antibody fragment derived from a camel heavy chain antibody against carbonic anhydrase, free and in complex with antigen. Surprisingly, this single-domain antibody interacts with nanomolar affinity with the antigen through its third hypervariable loop (19 amino acids long), providing a flat interacting surface of 620 A(2). For the first time, a single-domain antibody is observed with its first hypervariable loop adopting a type-1 canonical structure. The second hypervariable loop, of unique size due to a somatic mutation, reveals a regular beta-turn. The third hypervariable loop covers the remaining hypervariable loops and the side of the domain that normally interacts with the variable domain of the light chain. Specific amino acid substitutions and reoriented side chains reshape this side of the domain and increase its hydrophilicity. Of interest is the substitution of the conserved Trp-103 by Arg because it opens new perspectives to 'humanize' a camel variable domain of heavy chain of heavy chain antibody (VHH) or to 'camelize' a human or a mouse variable domain of heavy chain of conventional antibody (VH).

A microbial biosensor to predict bioavailable nickel in soil and its transfer to plants.

Ralstonia eutropha strain AE2515 was constructed and optimised to serve as a whole-cell biosensor for the detection of bioavailable concentrations of Ni2+ and Co2+ in soil samples. Strain AE2515 is a Ralstonia eutropha CH34 derivative containing pMOL1550, in which the cnrYXH regulatory genes are transcriptionally fused to the bioluminescent luxCDABE reporter system. Strain AE2515 was standardised for its specific responses to Co2+ and Ni2+. The detection limits for AE2515 were 0.1 microM Ni2+ and 9 microM Co2+, respectively. The signal to noise (S/N) bioluminescence response and the metal cation concentration could be linearly correlated: for Ni2+ this was applicable within the range 0.1-60 microM, and between 9 and 400 microM for Co2+. The AE2515 biosensor strain was found to be highly selective for nickel and cobalt: no induction was observed with Zn(II), Cd(II), Mn(II), Cu(III) and Cr(VI). In mixed metal solutions, the bioluminescent response always corresponded to the nickel concentrations. Only in the presence of high concentrations of Co2+ (2 mM), the sensitivity to nickel was reduced due to metal toxicity. AE2515 was used to quantify the metal bioavailability in various nickel-enriched soils, which had been treated with additives for in situ metal immobilisation. The data obtained with strain AE2515 confirmed that the bioavailability of nickel was greatly reduced following the treatment of the soils with the additives beringite and steel shots. Furthermore, the data were found to correlate linearly with those on the biological accumulation of Ni2+ in specific parts of important agricultural crops, such as maize and potato. Therefore, the test can be used to assess the potential transfer of nickel to organisms of higher trophic levels, in this case maize and potato plants grown on nickel-enriched soils, and the potential risk of transfer of these elements to the food chain.

Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains.

The antigen-binding site of antibodies from vertebrates is formed by combining the variable domains of a heavy chain (VH) and a light chain (VL). However, antibodies from camels and llamas are an important exception to this in that their sera contain, in addition, a unique kind of antibody that is formed by heavy chains only. The antigen-binding site of these antibodies consists of one single domain, referred to as VHH. This article reviews the mutations and structural adaptations that have taken place to reshape a VH of a VH-VL pair into a single-domain VHH with retention of a sufficient variability. The VHH has a potent antigen-binding capacity and provides the advantage of interacting with novel epitopes that are inaccessible to conventional VH-VL pairs.

Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs.

Single-domain antibodies against various antigens are isolated from the unique heavy-chain antibodies of immunized camels and llamas. These minimal sized binders are very robust and bind the antigen with high affinity in a monomeric state. We evaluated the feasibility to produce soluble, functional bispecific and bivalent antibodies in Escherichia coli with camel single-domain antibody fragments as building blocks. Two single-domain antibody fragments were tethered by the structural upper hinge of a natural antibody to generate bispecific molecules. This linker was chosen for its protease resistance in serum and its natural flexibility to reorient the upstream and downstream located domains. The expression levels, ease of purification, and the solubility of the recombinant proteins were comparable with those of the constituent monomers. The individual moieties fully retain the binding capacity and the binding characteristics within the recombinant bispecific constructs. The easy generation steps and the biophysical properties of these bispecific and bivalent constructs based on camel single-domain antibody fragments makes them particularly attractive for use in therapeutic or diagnostic programs.

Zinc/calcium- and cadmium/cadmium-substituted concanavalin A: interplay of metal binding, pH and molecular packing.

The crystal structures of cadmium/cadmium and zinc/calcium concanavalin A (con A) at pH 5.0 and pH 6.15, respectively, were determined. The structure of cadmium/cadmium con A confirms that the secondary Cd(2+)-binding site S3 is empty at pH 5. The metal-binding sites S1 and S2 are only very slightly affected by the substitution with cadmium. On the other hand, S1 and S2 and most of the protein surface of zinc/calcium con A at pH 6.15 differ from other fully metal-bound and carbohydrate-free structures. Most of these structural differences at the protein surface are a result of the interplay between metal binding, protonation and crystal packing. This interplay is expressed by relative rotations and translations of the con A units in alternative crystal packings and participation in space-group conversions inside crystals in situ. The particular crystal packing of zinc/calcium con A creates a novel zinc-binding site S4. The Zn(2+) ion in S4 ligates two aspartates from one tetramer and a histidine from a symmetry-related tetramer.

Structural basis of carbohydrate recognition by lectin II from Ulex europaeus, a protein with a promiscuous carbohydrate-binding site.

Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins. The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework.

Canonical antigen-binding loop structures in immunoglobulins: more structures, more canonical classes?

Grafting the antigen-binding loops onto a human antibody scaffold is a widely used technique to humanise murine antibodies. The success of this approach depends largely on the observation that the antigen-binding loops adopt only a limited number of canonical structures. Identification of the correct canonical structure is therefore essential. Algorithms that predict the main-chain conformation of the hypervariable loops using only the amino acid sequence often provide this information. Here, we describe new canonical loop conformations for the hypervariable regions H1 and H2 as found in single-domain antibody fragments of dromedaries or llama. Although the occurrence of these new loop conformations was not predicted by the algorithms used, it seems that they could occur in human or mouse antigen-binding loops. Their discovery indicates that the currently used set of canonical structures is incomplete and that the prediction algorithms should be extended to include these new structures.

The role of weak protein-protein interactions in multivalent lectin-carbohydrate binding: crystal structure of cross-linked FRIL.

Binding of multivalent glycoconjugates by lectins often leads to the formation of cross-linked complexes. Type I cross-links, which are one-dimensional, are formed by a divalent lectin and a divalent glycoconjugate. Type II cross-links, which are two or three-dimensional, occur when a lectin or glycoconjugate has a valence greater than two. Type II complexes are a source of additional specificity, since homogeneous type II complexes are formed in the presence of mixtures of lectins and glycoconjugates. This additional specificity is thought to become important when a lectin interacts with clusters of glycoconjugates, e.g. as is present on the cell surface. The cryst1al structure of the Glc/Man binding legume lectin FRIL in complex with a trisaccharide provides a molecular snapshot of how weak protein-protein interactions, which are not observed in solution, can become important when a cross-linked complex is formed. In solution, FRIL is a divalent dimer, but in the crystal FRIL forms a tetramer, which allows for the formation of an intricate type II cross-linked complex with the divalent trisaccharide. The dependence on weak protein-protein interactions can ensure that a specific type II cross-linked complex and its associated specificity can occur only under stringent conditions, which explains why lectins are often found forming higher-order oligomers.

The thermodynamic stability of the proteins of the ccd plasmid addiction system.

The two opponents, toxin (CcdB, LetB or LetD, protein G, LynB) and antidote (CcdA, LetA, protein H, LynA), in the plasmid addiction system ccd of the F plasmid were studied by different biophysical methods. The thermodynamic stability was measured at different temperatures combining denaturant and thermally induced unfolding. It was found that both proteins denature in a two-state equilibrium (native dimer versus unfolded monomer) and that CcdA has a significantly lower thermodynamic stability. Using a numerical model, which was developed earlier by us, and on the basis of the determined thermodynamic parameters the concentration dependence of the denaturation transition temperature was obtained for both proteins. This concentration dependence may be of physiological significance, as the concentration of both ccd addiction proteins cannot exceed a certain limit because their expression is controlled by autoregulation. The influence of DNA on the thermal stability of the two proteins was probed. It was found that cognate DNA increases the melting temperature of CcdA. In the presence of non-specific DNA the thermal stability was not changed. The melting temperature of CcdB was not influenced by the applied double-stranded oligonucleotides, neither cognate nor unspecific.

An unusual carbohydrate binding site revealed by the structures of two Maackia amurensis lectins complexed with sialic acid-containing oligosaccharides.

Seeds from the legume tree Maackia amurensis contain two lectins that can agglutinate different blood cell types. Their specificity toward sialylated oligosaccharides is unique among legume lectins; the leukoagglutinin preferentially binds to sialyllactosamine (alphaNeuAc(2-3)betaGal(1-4)betaGlcNAc), whereas the hemagglutinin displays higher affinity for a disialylated tetrasaccharide (alphaNeuAc(2-3)betaGal(1-3)[alphaNeuAc(2-6)]alphaG alNAc). The three-dimensional structure of the complex between M. amurensis leukoagglutinin and sialyllactose has been determined at 2.75-A resolution using x-ray crystallography. The carbohydrate binding site consists of a deep cleft that accommodates the three carbohydrate residues of the sialyllactose. The central galactose sits in the primary binding site in an orientation that has not been observed previously in other legume lectins. The carboxyl group of sialic acid establishes a salt bridge with a lysine side chain. The glucose residue is very efficiently docked between two tyrosine aromatic rings. The complex between M. amurensis hemagglutinin and a disialylated tetrasaccharide could be modeled from the leukoagglutinin/sialyllactose crystal structure. The substitution of one tyrosine by an alanine residue is responsible for the difference in fine specificity between the two isolectins. Comparison with other legume lectins indicates that oligosaccharide specificity within this family is achieved by the recycling of structural loops in different combinations.

The structural features of concanavalin A governing non-proline peptide isomerization.

The reversible binding of manganese and calcium to concanavalin A determines the carbohydrate binding of the lectin by inducing large conformational changes. These changes are governed by the isomerization of a non-proline peptide bond, Ala-207-Asp-208, positioned in a beta-strand in between the calcium binding site S2 and the carbohydrate specificity-determining loop. The replacement of calcium by manganese allowed us to investigate the structures of the carbohydrate binding, locked state and the inactive, unlocked state of concanavalin A, both with and without metal ions bound. Crystals of unlocked metal-free concanavalin A convert to the locked form with the binding of two Mn(2+) ions. Removal of these ions from the crystals traps metal-free concanavalin A in its locked state, a minority species in solution. The ligation of a metal ion in S2 to unlocked concanavalin A causes bending of the beta-strand foregoing the S2 ligand residues Asp-10 and Tyr-12. This bending disrupts conventional beta-sheet hydrogen bonding and forces the Thr-11 side chain against the Ala-207-Asp-208 peptide bond. The steric strain exerted by Thr-11 is presumed to drive the trans-to-cis isomerization. Upon isomerization, Asp-208 flips into its carbohydrate binding position, and the conformation of the carbohydrate specificity determining loop changes dramatically.

Camel heavy-chain antibodies: diverse germline V(H)H and specific mechanisms enlarge the antigen-binding repertoire.

The antigen-binding site of the camel heavy-chain antibodies devoid of light chain consists of a single variable domain (V(H)H) that obviously lacks the V(H)-V(L) combinatorial diversity. To evaluate the extent of the V(H)H antigen-binding repertoire, a germline database was constructed from PCR-amplified V(H)H/V(H) segments of a single specimen of Camelus dromedarius. A total of 33 V(H)H and 39 V()H unique sequences were identified, encoded by 42 and 50 different genes, respectively. Sequence comparison indicates that the V(H)Hs evolved within the V(H) subgroup III. Nevertheless, the V(H)H germline segments are highly diverse, leading to a broad structural repertoire of the antigen-binding loops. Seven V(H)H subfamilies were recognized, of which five were confirmed to be expressed in vivo. Comparison of germline and cDNA sequences demonstrates that the rearranged V(H)Hs are extensively diversified by somatic mutation processes, leading to an additional hypervariable region and a high incidence of nucleotide insertions or deletions. These diversification processes are driven by hypermutation and recombination hotspots embedded in the V(H)H germline genes at the regions affecting the structure of the antigen-binding loops.

Regulation of the cnr cobalt and nickel resistance determinant of Ralstonia eutropha (Alcaligenes eutrophus) CH34.

The linked resistance to nickel and cobalt of Ralstonia eutropha-like strain CH34 (Alcaligenes eutrophus CH34) is encoded by the cnr operon, which is localized on the megaplasmid pMOL28. The regulatory genes cnrYXH have been cloned, overexpressed, and purified in Escherichia coli. CnrY fractionated as a 10.7-kDa protein in in vitro translation assays. CnrX, a periplasmic protein of 16.5 kDa, was overproduced and purified as a histidine-tagged fusion protein in E. coli. His-CnrX was found to possess a secondary structure content rich in alpha-helical and beta-sheet structures. CnrH, a sigma factor of the extracytoplasmic function family, was purified as an N-terminally histidine-tagged fusion. In gel shift mobility assays, His-CnrH, in the presence of E. coli core RNA polymerase enzyme, could retard at least two different promoter DNA targets, cnrYp and cnrHp, localized within the cnrYXH locus. These promoters and their transcription start sites were confirmed by primer extension. Purified His-CnrX did not inhibit the DNA-binding activity of His-CnrH and is therefore unlikely to be an anti-sigma factor, as previously hypothesized (EMBL M91650 description entry). To study the transcriptional response of the regulatory locus to metals and to probe promoter regions, transcriptional fusions were constructed between fragments of cnrYXH and the luxCDABE, luciferase reporter genes. Nickel and cobalt specifically induced the cnrYXH-luxCDABE fusion at optimal concentrations of 0.3 mM Ni(2+) and 2.0 mM Co(2+) in a noncomplexing medium for metals. The two promoter regions P(Y) (upstream cnrY) and P(H) (upstream cnrH) were probed and characterized using this vector and were found to control the nickel-inducible regulatory response of the cnr operon. The cnrHp promoter was responsible for full transcription of the cnrCBA structural resistance genes, while the cnrYp promoter was necessary to obtain metal-inducible transcription from the cnrHp promoter. The zinc resistance phenotype (ZinB) of a spontaneous cnr mutant strain, AE963, was investigated and could be attributed to an insertion of IS1087, a member of the IS2 family of insertion elements, within the cnrY gene.

Development of a downstream process for the isolation of Staphylococcus aureus arsenate reductase overproduced in Escherichia coli.

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular As(V) (arsenate) to the more toxic As(III) (arsenite). In order to study the structure of ArsC and to unravel biochemical and physical properties of this redox enzyme, wild type enzyme and a number of cysteine mutants were overproduced soluble in Escherichia coli. In this paper we describe a novel purification method to obtain high production levels of highly pure enzyme. A reversed-phase method was developed to separate and analyze the many different forms of ArsC. The oxidation state and the methionine oxidized forms were determined by mass spectroscopy.