Erwin De Genst

Potent enzyme inhibitors derived from dromedary heavy‐chain antibodies

Evidence is provided that dromedary heavy‐chain antibodies, in vivo‐matured in the absence of light chains, are a unique source of inhibitory antibodies. After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic α‐amylase, it was demonstrated that a considerable amount of heavy‐chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream. In contrast, the conventional antibodies apparently do not interact with the enzymes active site. Next we illustrated that peripheral blood lymphocytes are suitable for one‐step cloning of the variable domain fragments in a phage‐display vector. By bio‐panning, several antigen‐specific single‐domain fragments are readily isolated for both enzymes. In addition we show that among those isolated fragments active site binders are well represented. When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays. The low complexity of the antigen‐binding site of these single‐domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors.

Receptor binding studies disclose a novel class of high‐affinity inhibitors of the Escherichia coli FimH adhesin

Mannose‐binding type 1 pili are important virulence factors for the establishment of Escherichia coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K‐12 strain, and affinity measurements with mannose, common mono‐ and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl α‐ d‐mannoside, bound in  the  crystal  structures,  exhibits  a  significantly better affinity for FimH (Kd = 0.15 µM) than mannose (Kd = 2.3 µM). Exploration of the binding affinities of α‐ d‐mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100‐fold improvement and 15‐fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti‐adhesive drugs to prevent anticipated and recurrent UTIs.

Camel single‐domain antibody inhibits enzyme by mimicking carbohydrate substrate

Whereas antibodies have demonstrated the ability to mimic various compounds, classic heavy/light‐chain antibodies may be limited in their applications. First, they tend not to bind enzyme active site clefts. Second, their size and complexity present problems in identifying key elements for binding and in using these elements to produce clinically valuable compounds. We have previously shown how cAb‐Lys3, a single variable domain fragment derived from a lysozyme‐specific camel antibody naturally lacking light chains, overcomes the first limitation to become the first antibody structure observed penetrating an enzyme active site. We now demonstrate how cAb‐Lys3 mimics the oligosaccharide substrate functionally (inhibition constant for lysozyme, 50 nM) and structurally (lysozyme buried surface areas, hydrogen bond partners, and hydrophobic contacts are similar to those seen in sugar‐complexed structures). Most striking is the mimicry by the antibody complementary determining region 3 (CDR3) loop, especially Ala104, which mimics the subsite C sugar 2‐acetamido group; this group has previously been identified as a key feature in binding lysozyme. Comparative simplicity, high affinity and specificity, potential to reach and interact with active sites, and ability to mimic substrate suggest that camel heavy‐chain antibodies present advantages over classic antibodies in the design, production, and application of clinically valuable compounds. Proteins 32:515–522, 1998.

Structure and Properties of a Complex of Alpha-Synuclein and a Single-Domain Camelid Antibody.

The aggregation of the intrinsically disordered protein α-synuclein to form fibrillar amyloid structures is intimately associated with a variety of neurological disorders, most notably Parkinsons disease. The molecular mechanism of α-synuclein aggregation and toxicity is not yet understood in any detail, not least because of the paucity of structural probes through which to study the behavior of such a disordered system. Here, we describe an investigation involving a single-domain camelid antibody, NbSyn2, selected by phage display techniques to bind to α-synuclein, including the exploration of its effects on the in vitro aggregation of the protein under a variety of conditions. We show using isothermal calorimetric methods that NbSyn2 binds specifically to monomeric α-synuclein with nanomolar affinity and by means of NMR spectroscopy that it interacts with the four C-terminal residues of the protein. This latter finding is confirmed by the determination of a crystal structure of NbSyn2 bound to a peptide encompassing the nine C-terminal residues of α-synuclein. The NbSyn2:α-synuclein interaction is mediated mainly by side-chain interactions while water molecules cross-link the main-chain atoms of α-synuclein to atoms of NbSyn2, a feature we believe could be important in intrinsically disordered protein interactions more generally. The aggregation behavior of α-synuclein at physiological pH, including the morphology of the resulting fibrillar structures, is remarkably unaffected by the presence of NbSyn2 and indeed we show that NbSyn2 binds strongly to the aggregated as well as to the soluble forms of α-synuclein. These results give strong support to the conjecture that the C-terminal region of the protein is not directly involved in the mechanism of aggregation and suggest that binding of NbSyn2 could be a useful probe for the identification of α-synuclein aggregation in vitro and possibly in vivo.

The fimbrial adhesin F17‐G of enterotoxigenic Escherichia coli has an immunoglobulin‐like lectin domain that binds N‐acetylglucosamine

The F17‐G adhesin at the tip of flexible F17 fimbriae of enterotoxigenic Escherichia coli mediates binding to N‐acetyl‐β‐d‐glucosamine‐presenting receptors on the microvilli of the intestinal epithelium of ruminants. We report the 1.7 Å resolution crystal structure of the lectin domain of F17‐G, both free and in complex with N‐acetylglucosamine. The monosaccharide is bound on the side of the ellipsoid‐shaped protein in a conserved site around which all natural variations of F17‐G are clustered. A model is proposed for the interaction between F17‐fimbriated E. coli and microvilli with enhanced affinity compared with the binding constant we determined for F17‐G binding to N‐acetylglucosamine (0.85 mM−1). Unexpectedly, the F17‐G structure reveals that the lectin domains of the F17‐G, PapGII and FimH fimbrial adhesins all share the immunoglobulin‐like fold of the structural components (pilins) of their fimbriae, despite lack of any sequence identity. Fold comparisons with pilin and chaperone structures of the chaperone/usher pathway highlight the central role of the C‐terminal β‐strand G of the immunoglobulin‐like fold and provides new insights into pilus assembly, function and adhesion.

Observation of spatial propagation of amyloid assembly from single nuclei

The crucial early stages of amyloid growth, in which normally soluble proteins are converted into fibrillar nanostructures, are challenging to study using conventional techniques yet are critical to the protein aggregation phenomena implicated in many common pathologies. As with all nucleation and growth phenomena, it is difficult to track individual nuclei in traditional macroscopic experiments, which probe the overall temporal evolution of the sample, but do not yield detailed information on the primary nucleation step as they mix independent stochastic events into an ensemble measurement. To overcome this limitation, we have developed microdroplet assays enabling us to detect single primary nucleation events and to monitor their subsequent spatial as well as temporal evolution, both of which we find to be determined by secondary nucleation phenomena. By deforming the droplets to high aspect ratio, we visualize in real-time propagating waves of protein assembly emanating from discrete primary nucleation sites. We show that, in contrast to classical gelation phenomena, the primary nucleation step is characterized by a striking dependence on system size, and the filamentous protein self-assembly process involves a highly nonuniform spatial distribution of aggregates. These findings deviate markedly from the current picture of amyloid growth and uncover a general driving force, originating from confinement, which, together with biological quality control mechanisms, helps proteins remain soluble and therefore functional in nature.

Engineering a Camelid Antibody Fragment That Binds to the Active Site of Human Lysozyme and Inhibits Its Conversion into Amyloid Fibrils

A single-domain fragment, cAb-HuL22, of a camelid heavy-chain antibody specific for the active site of human lysozyme has been generated, and its effects on the properties of the I56T and D67H amyloidogenic variants of human lysozyme, which are associated with a form of systemic amyloidosis, have been investigated by a wide range of biophysical techniques. Pulse-labeling hydrogen-deuterium exchange experiments monitored by mass spectrometry reveal that binding of the antibody fragment strongly inhibits the locally cooperative unfolding of the I56T and D67H variants and restores their global cooperativity to that characteristic of the wild-type protein. The antibody fragment was, however, not stable enough under the conditions used to explore its ability to perturb the aggregation behavior of the lysozyme amyloidogenic variants. We therefore engineered a more stable version of cAb-HuL22 by adding a disulfide bridge between the two beta-sheets in the hydrophobic core of the protein. The binding of this engineered antibody fragment to the amyloidogenic variants of lysozyme inhibited their aggregation into fibrils. These findings support the premise that the reduction in global cooperativity caused by the pathogenic mutations in the lysozyme gene is the determining feature underlying their amyloidogenicity. These observations indicate further that molecular targeting of enzyme active sites, and of protein binding sites in general, is an effective strategy for inhibiting or preventing the aberrant self-assembly process that is often a consequence of protein mutation and the origin of pathogenicity. Moreover, this work further demonstrates the unique properties of camelid single-domain antibody fragments as structural probes for studying the mechanism of aggregation and as potential inhibitors of fibril formation.

Chemical Basis for the Affinity Maturation of a Camel Single Domain Antibody

Affinity maturation of classic antibodies supposedly proceeds through the pre-organization of the reactive germ line conformational isomer. It is less evident to foresee how this can be accomplished by camelid heavy-chain antibodies lacking light chains. Although these antibodies are subjected to somatic hypermutation, their antigen-binding fragment consists of a single domain with restricted flexibility in favor of binding energy. An antigen-binding domain derived from a dromedary heavy-chain antibody, cAb-Lys3, accumulated five amino acid substitutions in CDR1 and CDR2 upon maturation against lysozyme. Three of these residues have hydrophobic side chains, replacing serines, and participate in the hydrophobic core of the CDR1 in the mature antibody, suggesting that conformational rearrangements might occur in this loop during maturation. However, transition state analysis of the binding kinetics of mature cAb-Lys3 and germ line variants show that the maturation of this antibody relies on events late in the reaction pathway. This is reflected by a limited perturbation of ka and a significantly decreased kd upon maturation. In addition, binding reactions and the maturation event are predominantly enthalpically driven. Therefore, maturation proceeds through the increase of favorable binding interactions, or by the reduction of the enthalpic penalty for desolvation, as opposed to large entropic penalties associated with conformational changes and structural plasticity. Furthermore, the crystal structure of the mutant with a restored germ line CDR2 sequence illustrates that the matured hydrophobic core of CDR1 in cAb-Lys3 might be compensated in the germ line precursor by burying solvent molecules engaged in a stable hydrogen-bonding network with CDR1 and CDR2.

Kinetic and Affinity Predictions of a Protein-Protein Interaction Using Multivariate Experimental Design

We measured the influence of 14 mutations and 5 environmental variables (buffer perturbation) on the association and dissociation rate of a camel single domain antibody (cAb-Lys3) interacting with hen egg white lysozyme using a surface plasmon resonance-based biosensor. Based on this data set, we constructed quantitative predictive models for both kinetic (ka and kd ) constants as for the affinity constant (K d ). Mutations, after parameterization by quantitative descriptors, and buffers were selected using multivariate experimental design. These models were able to predict the corresponding parameters of four new variants of cAb-Lys3. Moreover, the models provide insights to the important chemical aspects of the interacting residues, which are difficult to deduce from the crystal structure. Our approach provides useful physicochemical information of protein-protein interactions in general. The information obtained from this kind of analysis complements and goes beyond that of conventional methods like alanine scanning and substitution by closely related amino acids. The mathematical modeling may contribute to a rational approach in the optimization of bio-molecules of biotechnological interest.

Nanobodies Raised against Monomeric α-Synuclein Distinguish between Fibrils at Different Maturation Stages

Nanobodies are single-domain fragments of camelid antibodies that are emerging as versatile tools in biotechnology. We describe here the interactions of a specific nanobody, NbSyn87, with the monomeric and fibrillar forms of α-synuclein (αSyn), a 140-residue protein whose aggregation is associated with Parkinsons disease. We have characterized these interactions using a range of biophysical techniques, including nuclear magnetic resonance and circular dichroism spectroscopy, isothermal titration calorimetry and quartz crystal microbalance measurements. In addition, we have compared the results with those that we have reported previously for a different nanobody, NbSyn2, also raised against monomeric αSyn. This comparison indicates that NbSyn87 and NbSyn2 bind with nanomolar affinity to distinctive epitopes within the C-terminal domain of soluble αSyn, comprising approximately amino acids 118-131 and 137-140, respectively. The calorimetric and quartz crystal microbalance data indicate that the epitopes of both nanobodies are still accessible when αSyn converts into its fibrillar structure. The apparent affinities and other thermodynamic parameters defining the binding between the nanobody and the fibrils, however, vary significantly with the length of time that the process of fibril formation has been allowed to progress and with the conditions under which formation occurs, indicating that the environment of the C-terminal domain of αSyn changes as fibril assembly takes place. These results demonstrate that nanobodies are able to target forms of potentially pathogenic aggregates that differ from each other in relatively minor details of their structure, such as those associated with fibril maturation.