James R. Horn

A combinatorial histidine scanning library approach to engineer highly pH-dependent protein switches.

There is growing interest in the development of protein switches, which are proteins whose function, such as binding a target molecule, can be modulated through environmental triggers. Efforts to engineer highly pH sensitive protein–protein interactions typically rely on the rational introduction of ionizable groups in the protein interface. Such experiments are typically time intensive and often sacrifice the proteins affinity at the permissive pH. The underlying thermodynamics of proton‐linkage dictate that the presence of multiple ionizable groups, which undergo a pKa change on protein binding, are necessary to result in highly pH‐dependent binding. To test this hypothesis, a novel combinatorial histidine library was developed where every possible combination of histidine and wild‐type residue is sampled throughout the interface of a model anti‐RNase A single domain VHH antibody. Antibodies were coselected for high‐affinity binding and pH‐sensitivity using an in vitro, dual‐function selection strategy. The resulting antibodies retained near wild‐type affinity yet became highly sensitive to small decreases in pH, drastically decreasing their binding affinity, due to the incorporation of multiple histidine groups. Several trends were observed, such as histidine “hot‐spots,” which will help enhance the development of pH switch proteins as well as increase our understanding of the role of ionizable residues in protein interfaces. Overall, the combinatorial approach is rapid, general, and robust and should be capable of producing highly pH‐sensitive protein affinity reagents for a number of different applications.

An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop.

Conventional anti‐hapten antibodies typically bind low‐molecular weight compounds (haptens) in the crevice between the variable heavy and light chains. Conversely, heavy chain‐only camelid antibodies, which lack a light chain, must rely entirely on a single variable domain to recognize haptens. While several anti‐hapten VHHs have been generated, little is known regarding the underlying structural and thermodynamic basis for hapten recognition. Here, an anti‐methotrexate VHH (anti‐MTX VHH) was generated using grafting methods whereby the three complementarity determining regions (CDRs) were inserted onto an existing VHH framework. Thermodynamic analysis of the anti‐MTX VHH CDR1‐3 Graft revealed a micromolar binding affinity, while the crystal structure of the complex revealed a somewhat surprising noncanonical binding site which involved MTX tunneling under the CDR1 loop. Due to the close proximity of MTX to CDR4, a nonhypervariable loop, the CDR4 loop sequence was subsequently introduced into the CDR1‐3 graft, which resulted in a dramatic 1000‐fold increase in the binding affinity. Crystal structure analysis of both the free and complex anti‐MTX CDR1‐4 graft revealed CDR4 plays a significant role in both intermolecular contacts and binding site conformation that appear to contribute toward high affinity binding. Additionally, the anti‐MTX VHH possessed relatively high specificity for MTX over closely related compounds aminopterin and folate, demonstrating that VHH domains are capable of binding low‐molecular weight ligands with high affinity and specificity, despite their reduced interface.

Production and characterization of a genetically engineered anti-caffeine camelid antibody and its use in immunoaffinity chromatography☆

This work demonstrates the feasibility of using a camelid single domain antibody for immunoaffinity chromatographic separation of small molecules. An anti-caffeine VHH antibody was produced by grafting the complementarity determining sequences of a previously generated antibody onto an anti-RNase A antibody scaffold, followed by expression in E. coli. Analysis of the binding properties of the antibody by ELISA and fluorescence-based thermal shift assays showed that it recognizes not only caffeine, but also theophylline, theobromine, and paraxanthine, albeit with lower affinity. Further investigation of the effect of environmental conditions, i.e., temperature, pH, and ionic strength, on the antibody using these methods provided useful information about potential elution conditions to be used in chromatographic applications. Immobilization of the VHH onto a high flow-through synthetic support material resulted in a stationary phase capable of separating caffeine and its metabolites.

Principal determinants leading to transition state formation of a protein–protein complex, orientation trumps side-chain interactions

The binding transition state (TS) is the rate-limiting step for transient molecular interactions. This important step in the molecular recognition process, however, is largely understood only at a qualitative level. To establish a more quantitative picture of the TS structure, we exploit a set of biophysical techniques that have provided major insights in protein folding applications. As a model system representing the large class of “weakly charged” protein–protein interactions, we examine the binding of a variety of human growth hormone (hGH) variants to the human growth hormone receptor (hGHR) and the human prolactin receptor (hPRLR). hGH variants were chosen to probe different features of the TS structure, based on their highly reengineered interfaces. Both Eyring and urea (m value) analyses suggest that the majority of binding surface burial occurs after TS. A comprehensive φ analysis showed that individual hGH interface residues do not contribute energetically to the stability of the TS, but there is a TS “hot spot” in the receptor. Zinc dependence studies that take advantage of an endogenous tetracoordinated interfacial metal binding demonstrate that surfaces of the molecules have attained a high orientational complementarity by the time the TS is reached. The model that best fits these data are that a “knobs-into-holes” process precisely aligns the two molecular interfaces in forming the TS structure. Surprisingly, most of the thermodynamic character of the binding reaction is focused in the fine-tuning process occurring after TS.

Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series.

Discovery of Inhibitors of Burkholderia pseudomallei Methionine Aminopeptidase with Antibacterial Activity.

Evaluation of a series of MetAP inhibitors in an in vitro enzyme activity assay led to the first identification of potent molecules that show significant growth inhibition against Burkholderia pseudomallei. Nitroxoline analogs show excellent inhibition potency in the BpMetAP1 enzyme activity assay with the lowest IC50 of 30 nM, and inhibit the growth of B. pseudomallei and B. thailandensis at concentrations ≥ 31 μM.

A combinatorial approach to engineering a dual-specific metal switch antibody.

There is considerable interest in understanding how multiple binding events can be mediated through a single protein interface. Here, a synthetic library approach was developed to generate a novel dual-specific antibody. Using a combinatorial histidine-scanning phage display library, potential metal binding sites were introduced throughout an anti-RNase A antibody interface. Stepwise selection of RNase A and metal binding produced a dual-specific antibody that retained near wild-type affinity for its target antigen while acquiring a competitive metal binding site that is capable of controlling the antibody-antigen interaction. Structure analysis of the original antibody-RNase A complex suggested peripheral interface residues and loop flexibility are key contributors for obtaining the dual specificity.

Mutations in the coat protein-binding cis-acting RNA motifs debilitate RNA recombination of Brome mosaic virus.

Abstract We have previously described the efficient homologous recombination system between 5′ subgenomic RNA3a (sgRNA3a) and genomic RNA3 of Brome mosaic virus (BMV) in barley protoplasts (Sztuba-Solińska et al., 2011a). Here, we demonstrated that sequence alterations in the coat protein (CP)-binding cis-acting RNA motifs, the Bbox region (in the intercistronic RNA3 sequence) and the RNA3 packaging element (PE, in the movement protein ORF), reduced crossover frequencies in protoplasts. Additionally, the modification of Bbox-like element in the 5′ UTR region strongly debilitated crossovers. Along the lines of these observations, RNA3 mutants not expressing CP or expressing mutated CPs also reduced recombination. A series of reciprocal transfections demonstrated a functional crosstalk between the Bbox and PE elements. Altogether, our data imply the role of CP in sgRNA3a-directed recombination by either facilitating the interaction of the RNA substrates and/or by creating roadblocks for the viral replicase.

Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns.

In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the proteins surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first application of immunoaffinity chromatography for the analysis of MTX.

STRUCTURAL ENERGETICS OF SERINE PROTEASE INHIBITION

We have investigated the binding of the serine protease inhibitor, turkey ovomucoid third domain (OMTKY3), to the serine protease, porcine pancreatic elastase (PPE), using isothermal titration calorimetry and structural energetics calculations. The calculations predict that the binding at 25 8C is characterized by a negligible DH 8, a large and positive DS 8, and a large and negative DCp, resulting in a large and favorable DG 8. The experimental results indicate a significant contribution to the binding energetics from a change in the pK ao f an ionizable group, presumably His57 of PPE. The resulting proton linkage is manifest in the observed DH 8 and DCp of binding. However, a global analysis of binding data as a function of pH, buffer, and temperature yields the intrinsic binding energetics as well as the energetics of proton binding to the ionizable group in the free and bound PPE. The experimentally determined intrinsic energetics and the calculated values are in very good agreement, suggesting that the structural energetics calculations may be a useful tool for understanding protein-protein interactions in solution.