John S. Klassen

Reliable determinations of protein-ligand interactions by direct ESI-MS measurements: Are we there yet?

The association-dissociation of noncovalent interactions between protein and ligands, such as other proteins, carbohydrates, lipids, DNA, or small molecules, are critical events in many biological processes. The discovery and characterization of these interactions is essential to a complete understanding of biochemical reactions and pathways and to the design of novel therapeutic agents that may be used to treat a variety of diseases and infections. Over the last 20 y, electrospray ionization mass spectrometry (ESI-MS) has emerged as a versatile tool for the identification and quantification of protein–ligand interactions in vitro. Here, we describe the implementation of the direct ESI-MS assay for the determination of protein–ligand binding stoichiometry and affinity. Additionally, we outline common sources of error encountered with these measurements and various strategies to overcome them. Finally, we comment on some of the outstanding challenges associated with the implementation of the assay and highlight new areas where direct ESI-MS measurements are expected to make significant contributions in the future.

Hydrophobic protein-ligand interactions preserved in the gas phase.

The results of time-resolved thermal dissociation measurements and molecular dynamic simulations are reported for gaseous deprotonated ions of the specific complexes of bovine beta-lactoglobulin (Lg) and a series of the fatty acids (FA): CH(3)(CH(2))(x)COOH, where x = 10, 12, 14, and 16. At the reaction temperatures investigated, 25-66 degrees C, the gaseous ions dissociate exclusively by the loss of neutral FA. According to the kinetic data, and confirmed by ion mobility measurements, the (Lg + FA)(7-) ions exist in two, noninterconverting structures designated the fast (Lg + FA)(f)(7-) and slow (Lg + FA)(s)(7-) components. The Arrhenius parameters for both components are sensitive to the length of the FA aliphatic chain. For the fast components, the activation energy (E(a)) increases in a nearly linear fashion, with each methylene group contributing approximately 0.8 kcal mol(-1) to E(a). This is similar to the contribution of -CH(2)- groups to the solvation of n-alkanes in nonpolar solvents. Furthermore, the magnitude of the E(a) values for the fast components is similar to the solvation enthalpies expected for the FA aliphatic chains in nonpolar and weakly polar solvents. The E(a) values determined for the slow components are larger than those of the fast components. Furthermore, the E(a) values do not vary in a simple fashion with the length of the aliphatic chain. Molecular dynamics simulations performed on the (Lg + PA) complex revealed that, depending on the charge configuration, the (Lg + PA)(7-) ion can exist in two distinct structures, which differ primarily by the position of the EF loop. In the open structure the EF loop is positioned away from the entrance to the hydrophobic cavity and the ligand is stabilized only through nonpolar intermolecular interactions. In the closed structure the EF loop covers the entrance of the cavity and the carboxylic group of PA participates in H-bonds with residues on the EF loop or residues located at the entrance of the cavity. The loss of ligand from the closed structure would require both the cleavage of the H-bonds and the nonpolar contacts. Taken together, these results suggest that the aliphatic chain of the FA remains bound within the hydrophobic cavity in the gas phase (Lg + FA)(7-) ions. Furthermore, the barrier to dissociation of the (Lg + FA)(f)(7-) ions reflects predominantly the cleavage of the nonpolar intermolecular interactions, while for the (Lg + FA)(s)(7-) ions the FA is stabilized by both nonpolar interactions and H-bonds.

Functional properties of the carboxy-terminal host cell-binding domains of the two toxins, TcdA and TcdB, expressed by Clostridium difficile

The biological and ligand-binding properties of recombinant C-terminal cell-binding domains (CBDs) and subdomains of the two large exotoxins, Toxin A (TcdA) and Toxin B (TcdB) expressed by Clostridium difficile were examined in the hemagglutination and Verocytotoxicity neutralization assays and by qualitative affinity chromatography using Sepharose-linked alpha Gal(1,3)betaGal(1,4)beta Glc as well as the direct electrospray ionization mass spectrometry (ES-MS) assay. These studies revealed that, whereas the full-length TcdA CBD agglutinated rabbit erythrocytes, neutralized TcdA-mediated Vero cell death and bound to alpha Gal(1,3)betaGal(1,4)beta Glc-derivatized Sepharose, the TcdB CBD was inactive in these functional assays. Moreover, retention by alpha Gal(1,3)betaGal(1,4)beta Glc-derivatized Sepharose corresponded to the number of available TcdA subdomain ligand-binding sites. By contrast, the ES-MS assays revealed that both the TcdA and TcdB CBD bind to 8-methoxycarbonyloctyl-alpha Gal(1,3)betaGal(1,4)beta Glc sequences with similar avidities. Additional ES-MS experiments using chemically altered alpha Gal(1,3)betaGal(1,4)beta Glc sequences also revealed that the TcdA and TcdB CBD will tolerate a fair amount of structural variation in their complementary glycan ligands. Although the studies are consistent with the known ligand-binding properties of the TcdA and TcdB holotoxins, they also revealed subtle heretofore unrecognized functional differences in their receptor recognition properties.

Trapping and characterization of covalent intermediates of mutant retaining glycosyltransferases

The enzymatic mechanism by which retaining glycosyltransferases (GTs) transfer monosaccharides with net retention of the anomeric configuration has, so far, resisted elucidation. Here, direct detection of covalent glycosyl-enzyme intermediates for mutants of two model retaining GTs, the human blood group synthesizing α-(1 → 3)-N-acetylgalactosaminyltransferase (GTA) and α-(1 → 3)-galactosyltransferase (GTB) mutants, by mass spectrometry (MS) is reported. Incubation of mutants of GTA or GTB, in which the putative catalytic nucleophile Glu(303) was replaced with Cys (i.e. GTA(E303C) and GTB(E303C)), with their respective donor substrate results in a covalent intermediate. Tandem MS analysis using collision-induced dissociation confirmed Cys(303) as the site of glycosylation. Exposure of the glycosyl-enzyme intermediates to a disaccharide acceptor results in the formation of the corresponding enzymatic trisaccharide products. These findings suggest that the GTA(E303C) and GTB(E303C) mutants may operate by a double-displacement mechanism.

Gas Phase Stabilization of Noncovalent Protein Complexes Formed by Electrospray Ionization

The use of gas phase additives to stabilize noncovalent protein complexes in electrospray ionization mass spectrometry (ES-MS) is demonstrated for two protein-ligand interactions, an enzyme-small molecule inhibitor complex, and a protein-disaccharide complex. It is shown that the introduction of gas phase imidazole into the ES ion source effectively protects gas phase protein-ligand complexes against in-source dissociation. The stabilizing effect of imidazole vapor is comparable to that observed upon addition of imidazole to the ES solution. The introduction of sulfur hexafluoride, at high partial pressure, into the source region also effectively suppresses in-source dissociation of protein complexes. It is proposed that evaporative cooling is the primary mechanism responsible for the stabilizing effects observed for the gas phase additives.

The bundlin pilin protein of enteropathogenic Escherichia coli is an N‐acetyllactosamine‐specific lectin

Synthetic N‐acetyllactosamine (LacNAc) glycoside sequences coupled to BSA competitively inhibit enteropathogenic Escherichia coli (EPEC) localized adherence (LA) to human intestinal biopsy specimens and tissue culture cell monolayers. The LacNAc‐specific adhesin appears to be associated with the bundle‐forming pili (BFP) expressed by EPEC during the early stages of colonization. Herein, we report that recombinant bundlin inhibits EPEC LA to HEp‐2 cells and binds to HEp‐2 cells. Recombinant bundlin also binds, with millimolar association constants (Kassoc), to synthetic LacNAc‐Benzene and LacNAc‐O(CH2)8CONH2 glycosides as assessed in the gas phase by nanoelectrospray ionization mass spectrometry. Furthermore, LacNAc‐BSA inhibits LA only of EPEC strains that express α bundlin alleles, suggesting putative locations for the LacNAc‐binding pocket in the α bundlin monomer. Collectively, these results suggest that α bundlin possesses lectin‐like properties that are responsible for LacNAc‐specific initial adherence of α bundlin‐expressing EPEC strains to host intestinal epithelial cells.

Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

A new electrospray ionization mass spectrometry (ES-MS) approach for quantifying protein—ligand complexes that are prone to in-source (gas-phase) dissociation is described. The method, referred to here as the reference ligand ES-MS method, is based on the direct ES-MS assay and competitive ligand binding. A reference ligand (Lref), which binds specifically to the protein (P), at the same binding site as the ligand (L) of interest, with known affinity and forms a stable protein—ligand complex in the gas phase, is added to the solution. The fraction of P bound to Lref, which is determined directly from the ES mass spectrum, is sensitive to the fraction of P bound to L in solution and enables the affinity of P for L to be determined. A mathematical framework for the implementation of the method in cases where P has one or two specific ligand binding sites is given. Affinities of two carbohydrate-binding proteins, a single chain fragment of a monoclonal antibody and the lectin concanavalin A, for monosaccharide ligands are reported and the results are shown to agree with values obtained using isothermal titration calorimetry.

Comparative study of substrate and product binding to the human ABO(H) blood group glycosyltransferases

The first comparative thermodynamic study of the human blood group glycosyltransferases, alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) and alpha-(1-->3)-galactosyltransferase (GTB), interacting with donor substrates, donor and acceptor analogs, and trisaccharide products in vitro is reported. The binding constants, measured at 24 degrees C with the direct electrospray ionization mass spectrometry (ES-MS) assay, provide new insights into these model GTs and their interactions with substrate and product. Notably, the recombinant forms of GTA and GTB used in this study are shown to exist as homodimers, stabilized by noncovalent interactions at neutral pH. In the absence of divalent metal ion, neither GTA nor GTB exhibits any appreciable affinity for its native donors (UDP-GalNAc, UDP-Gal). Upon introduction of Mn(2+), both donors undergo enzyme-catalyzed hydrolysis in the presence of either GTA or GTB. Hydrolysis of UDP-GalNAc in the presence of GTA proceeds very rapidly under the solution conditions investigated and a binding constant could not be directly measured. In contrast, the rate of hydrolysis of UDP-Gal in the presence of GTB is significantly slower and, utilizing a modified approach to analyze the ES-MS data, a binding constant of 2 x 10(4) M(-1) was established. GTA and GTB bind the donor analogs UDP-GlcNAc, UDP-Glc with affinities similar to those measured for UDP-Gal and UDP-GalNAc (GTB only), suggesting that the native donors and donor analogs bind to the GTA and GTB through similar interactions. The binding constant determined for GTA and UDP-GlcNAc (approximately 1 x 10(4) M(-1)), therefore, provides an estimate for the binding constant for GTA and UDP-GalNAc. Binding of GTA and GTB with the A and B trisaccharide products was also investigated for the first time. In the absence of UDP and Mn(2+), both GTA and GTB recognize their respective trisaccharide products but with a low affinity approximately 10(3) M(-1); the presence of UDP and Mn(2+) has no effect on A trisaccharide binding but precludes B-trisaccharide binding.

Discovery of Light-Responsive Ligands through Screening of a Light-Responsive Genetically Encoded Library

Light-responsive ligands are useful tools in biochemistry and cell biology because the function of these ligands can be spatially and temporally controlled. Conventional design of such ligands relies on previously available data about the structure of both the ligand and the receptor. In this paper, we describe de novo discovery of light-responsive ligands through screening of a genetically encoded light-responsive library. We ligated a photoresponsive azobenzene core to a random CX7C peptide library displayed on the coat protein of M13 phage. A one-pot alkylation/reduction of the cysteines yielded a photoresponsive library of random heptapeptide macrocycles with over 2 × 10(8) members. We characterized the reaction on-phage and optimized the yield of the modifications in phage libraries. Screening of the library against streptavidin yielded three macrocycles that bind to streptavidin in the dark and cease binding upon irradiation with 370 nm light. All ligands restored their binding properties upon thermal relaxation and could be turned ON and OFF for several cycles. We measured dissociation constants, Kd, by electrospray ionization mass spectrometry (ESI-MS) binding assay. For ligand ACGFERERTCG, the Kd of cis and trans isomers differed by 22-fold; an incomplete isomerization (85%), however, resulted in the apparent difference of 4.5-fold between the dark and the irradiated state. We anticipate that the selection strategy described in this report can be used to find light-responsive ligands for many targets that do not have known natural ligands.

Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

We describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based discovery (GE-FBD) uses selection of phage-displayed glycopeptides to dock a glycan fragment at the CRD and guide selection of synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10(8) glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40-50-fold enhancement in affinity over methyl α-d-mannopyranoside (MeMan). Lectin array suggested specificity: Man-WYD derivative bound only to 3 out of 17 proteins—ConA, LcH, and PSA—that bind to Man. An X-ray structure of ConA:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking, but their extra-CRD binding modes are significantly different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.