Large Differences In Affinity Research Articles

Binding site plasticity regulation of the FimH catch-bond mechanism

The bacterial fimbrial adhesin FimH is a remarkable and well-studied catch-bond protein found at the tip of E. coli type 1 pili, which allows pathogenic strains involved in urinary tract infections to bind high-mannose glycans exposed on human epithelia. The catch-bond behavior of FimH, where the strength of the interaction increases when a force is applied to separate the two partners, enables the bacteria to resist clearance when they are subjected to shear forces induced by urine flow. Two decades of experimental studies performed at the single-molecule level, as well as x-ray crystallography and modeling studies, have led to a consensus picture whereby force separates the binding domain from an inhibitor domain, effectively triggering an allosteric conformational change in the former. This force-induced allostery is thought to be responsible for an increased binding affinity at the core of the catch-bond mechanism. However, some important questions remain, the most challenging one being that the crystal structures corresponding to these two allosteric states show almost superimposable binding site geometries, which questions the molecular origin for the large difference in affinity. Using molecular dynamics with a combination of enhanced-sampling techniques, we demonstrate that the static picture provided by the crystal structures conceals a variety of binding site conformations that have a key impact on the apparent affinity. Crucially, the respective populations in each of these conformations are very different between the two allosteric states of the binding domain, which can then be related to experimental affinity measurements. We also evidence a previously unappreciated but important effect: in addition to the well-established role of the force as an allosteric regulator via domain separation, application of force tends to directly favor the high-affinity binding site conformations. We hypothesize that this additional “local” catch-bond effect could delay unbinding between the bacteria and the host cell before the “global” allosteric transition occurs, as well as stabilizing the complex even more once in the high-affinity allosteric state.

Impact of the Syn/Anti Relative Configuration of Cryptophane-222 on the Binding Affinity of Cesium and Thallium.

We report in this article the synthesis, the X-ray crystal structure of compound syn-2, and its binding properties with cesium and thallium in aqueous solution under basic conditions. Compound syn-2 is the diastereomeric compound of anti-1 that shows very high affinity for cesium and thallium in aqueous solution under the same conditions. Despite the close structural similarities that exist between the syn-2 and anti-1 compounds, they show large discrepancy in their ability to bind cesium and thallium cations in the same conditions. Indeed, the syn-2 derivative has a lower affinity for these two cationic species and the binding constants are several orders of magnitude lower than those found for its congener. The large differences in affinity observed with these two compounds can be explained by the relative position of the six hydroxyl groups to each other.

The origins of binding specificity of a lanthanide ion binding peptide

Lanthanide ions (Ln3+) show similar physicochemical properties in aqueous solutions, wherein they exist as + 3 cations and exhibit ionic radii differences of less than 0.26 Å. A flexible linear peptide lanthanide binding tag (LBT), which recognizes a series of 15 Ln3+, shows an interesting characteristic in binding specificity, i.e., binding affinity biphasically changes with an increase in the atomic number, and shows a greater than 60-fold affinity difference between the highest and lowest values. Herein, by combining experimental and computational investigations, we gain deep insight into the reaction mechanism underlying the specificity of LBT3, an LBT mutant, toward Ln3+. Our results clearly show that LBT3-Ln3+ binding can be divided into three, and the large affinity difference is based on the ability of Ln3+ in a complex to be directly coordinated with a water molecule. When the LBT3 recognizes a Ln3+ with a larger ionic radius (La3+ to Sm3+), a water molecule can interact with Ln3+ directly. This extra water molecule infiltrates the complex and induces dissociation of the Asn5 sidechain (one of the coordinates) from Ln3+, resulting in a destabilizing complex and low affinity. Conversely, with recognition of smaller Ln3+ (Sm3+ to Yb3+), the LBT3 completely surrounds the ions and constructs a stable high affinity complex. Moreover, when the LBT3 recognizes the smallest Ln3+, namely Lu3+, although it completely surrounds Lu3+, an entropically unfavorable phenomenon specifically occurs, resulting in lower affinity than that of Yb3+. Our findings will be useful for the design of molecules that enable the distinction of sub-angstrom size differences.

A Bispecific Inhibitor of the EGFR/ADAM17 Axis Decreases Cell Proliferation and Migration of EGFR-Dependent Cancer Cells

Dysregulated epidermal growth factor receptor (EGFR) is an oncogenic driver of many human cancers, promoting aberrant cell proliferation, migration, and survival. Pharmacological targeting of EGFR is often challenged by acquired mechanisms of resistance. Ligand-dependent mechanisms in EGFR wild-type cells rely on ligand or receptor overexpression, allowing cells to outcompete inhibitors and perpetuate signaling in an autocrine manner. Importantly, EGFR ligands are synthesized as membrane-bound precursors that must be solubilized to enable receptor-ligand interactions. The A disintegrin and metalloproteinase 17 (ADAM17) is considered the main sheddase of several EGFR ligands, and a potential pharmacological target. However, its broad substrate range and ubiquitous expression complicate its therapeutic targeting. Here, we present a novel bispecific fusion protein construct consisting of the inhibitory prodomain of ADAM17 (TPD), fused to an EGFR-targeting designed ankyrin repeat protein (DARPin). TPD is a natural inhibitor of ADAM17, maintaining the protease in a zymogen-like form. Meanwhile, the high affinity anti-EGFR DARPin E01 binds to EGFR and inhibits ligand binding. The resulting fusion protein E01-GS-TPD retained binding ability to both molecular targets EGFR and ADAM17. The large difference in affinity for each target resulted in enrichment of the fusion protein in EGFR-positive cells compared to EGFR-negative cells, suggesting a possible application in autocrine signaling inhibition. Accordingly, E01-GS-TPD decreased migration and proliferation of EGFR-dependent cell lines with no significant increase in apoptotic cell death. Finally, inhibition of proliferation was observed through EGFR ligand-dependent mechanisms as growth inhibition was not observed in EGFR mutant or KRAS mutant cell lines. The use of bispecific proteins targeting the EGFR/ADAM17 axis could be an innovative strategy for the treatment of EGFR-dependent cancers.

Intrinsic DNA Shape Accounts for Affinity Differences between Hox-Cofactor Binding Sites

Transcription factors bind to their binding sites over a wide range of affinities, yet how differences in affinity are encoded in DNA sequences is not well understood. Here, we report X-ray crystal structures of four heterodimers of the Hox protein AbdominalB bound with its cofactor Extradenticle to four target DNA molecules that differ in affinity by up to ∼20-fold. Remarkably, despite large differences in affinity, the overall structures are very similar in all four complexes. In contrast, the predicted shapes of the DNA binding sites (i.e., the intrinsic DNA shape) in the absence of bound protein are strikingly different from each other and correlate with affinity: binding sites that must change conformations upon protein binding have lower affinities than binding sites that have more optimal conformations prior to binding. Together, these observations suggest that intrinsic differences in DNA shape provide a robust mechanism for modulating affinity without affecting other protein-DNA interactions.

Silicone rubber selection for passive sampling of pesticides in water

Silicone rubber can extract organic compounds with a broad range of polarities (logKow>2–3) from aqueous samples. Such compounds include substances of major concern in the protection of aquatic ecosystems and human health, e.g. pesticides. Silicone rubbers (SRs) with various characteristics have been successfully used in sorptive methods for water sample extraction in the laboratory (SPME, SBSE), and for passive sampling in aquatic environments. However, only few studies have evaluated variability in organic compound sorption due to the origin of SRs, particularly for pesticides. The aim of this study was to select an SR for the extraction of pesticides from water samples by passive sampling. To this end we measured the impact of seven SR formulations on sorption capacity, defined by the partition coefficient (Ksw). Kinetic experiments and sorption isotherms were performed to determine extraction recovery as a selection criterion for SRs, and pesticide partition coefficients. Very large differences in affinity for pesticides were found between two kinds of SRs: “Polymerized SR kits” and “Manufactured SRs”. One SR was chosen among the “Manufactured SRs”, and the Ksw values of 21 pesticides were determined, filling a gap in the literature (1.50<logKow<5.51). In light of sorption properties, literature data and additional economic and technical factors, we suggest using SR from Goodfellow in future work to reduce the variability of Ksw literature values.

Determination of the α-Conotoxin Vc1.1 Binding Site on the α9α10 Nicotinic Acetylcholine Receptor

α-Conotoxin Vc1.1 specifically and potently inhibits the nicotinic acetylcholine receptor subtype α9α10 (α9α10 nAChR) and is a potential novel treatment for neuropathic pain. Here, we used a combination of computational modeling and electrophysiology experiments to determine the Vc1.1 binding site on the α9α10 nAChR. Interactions of Vc1.1 with two probable binding sites, α9α10 and α10α9, were modeled. Mutational energies calculated by assuming specific interactions in the α10α9 binding site correlated better with electrophysiological recordings than those assuming interactions with the α9α10 binding site. Two novel Vc1.1 analogues, [N9F]Vc1.1 and [N9W]Vc1.1, were predicted to have large differences in affinity between the two binding sites. Data from functional studies were consistent with computational predictions that assumed preferred binding of Vc1.1 to the α10α9 pocket. Moreover, our modeling study suggested that a single hydrogen bond formed between Vc1.1 and position 59 of the α10α9 pocket confers specificity to rat versus human α9α10 nAChRs.

Binding trimethyllysine and other cationic guests in water with a series of indole-derived hosts: large differences in affinity from subtle changes in structure

The binding of a series of indole-derived hosts to various ammonium cations in pure, buffered water is investigated using both solution phase (1)H NMR studies and computational modeling. These hosts can engage their targets via the cation-pi interaction, electrostatic attraction, and the hydrophobic effect. The hydrophobic effect is shown to be a dominant influence in the strength of the binding interactions, both in terms of the hydrophobicity of the host and of the guest. Our findings show that small changes that reduce the host hydrophobic surface area without reducing either the number of negative charges or amount of aromatic surface area are found to significantly decrease binding. Additionally, the position of solubilizing charges is also shown to influence the preferred host geometry and resulting binding constants.

Role of Bacterial Biomass in the Sorption of Ni by Biomass-Birnessite Assemblages

Birnessites precipitated by bacteria are typically poorly crystalline Mn(IV) oxides enmeshed within biofilms to form complex biomass-birnessite assemblages. The strong sorption affinity of bacteriogenic birnessites for environmentally important trace metals is relatively well understood mechanistically, but the role of bacterial cells and extracellular polymeric substances appears to vary among trace metals. To assess the role of biomass definitively, comparison between metal sorption by biomass at high metal loadings in the presence and absence of birnessite is required. We investigated the biomass effect on Ni sorption through laboratory experiments utilizing the birnessite produced by the model bacterium, Pseudomonas putida. Surface excess measurements at pH 6-8 showed that birnessite significantly enhanced Ni sorption at high loadings (up to nearly 4-fold) relative to biomass alone. This apparent large difference in affinity for Ni between the organic and mineral components was confirmed by extended X-ray absorption fine structure spectroscopy, which revealed preferential Ni binding to birnessite cation vacancy sites. At pH ≥ 7, Ni sorption involved both adsorption and precipitation reactions. Our results thus support the view that the biofilm does not block reactive mineral surface sites; instead, the organic material contributes to metal sorption once high-affinity sites on the mineral are saturated.

The major determinant of exendin‐4/glucagon‐like peptide 1 differential affinity at the rat glucagon‐like peptide 1 receptor N‐terminal domain is a hydrogen bond from SER‐32 of exendin‐4

Exendin-4 (exenatide, Ex4) is a high-affinity peptide agonist at the glucagon-like peptide-1 receptor (GLP-1R), which has been approved as a treatment for type 2 diabetes. Part of the drug/hormone binding site was described in the crystal structures of both GLP-1 and Ex4 bound to the isolated N-terminal domain (NTD) of GLP-1R. However, these structures do not account for the large difference in affinity between GLP-1 and Ex4 at this isolated domain, or for the published role of the C-terminal extension of Ex4. Our aim was to clarify the pharmacology of GLP-1R in the context of these new structural data. The affinities of GLP-1, Ex4 and various analogues were measured at human and rat GLP-1R (hGLP-1R and rGLP-1R, respectively) and various receptor variants. Molecular dynamics coupled with in silico mutagenesis were used to model and interpret the data. The membrane-tethered NTD of hGLP-1R displayed similar affinity for GLP-1 and Ex4 in sharp contrast to previous studies using the soluble isolated domain. The selectivity at rGLP-1R for Ex4(9-39) over Ex4(9-30) was due to Ser-32 in the ligand. While this selectivity was not observed at hGLP-1R, it was regained when Glu-68 of hGLP-1R was mutated to Asp. GLP-1 and Ex4 bind to the NTD of hGLP-1R with similar affinity. A hydrogen bond between Ser32 of Ex4 and Asp-68 of rGLP-1R, which is not formed with Glu-68 of hGLP-1R, is responsible for the improved affinity of Ex4 at the rat receptor.

A role for tungsten in the biology of Campylobacter jejuni: tungstate stimulates formate dehydrogenase activity and is transported via an ultra‐high affinity ABC system distinct from the molybdate transporter

The food-borne pathogen Campylobacter jejuni possesses no known tungstoenzymes, yet encodes two ABC transporters (Cj0300-0303 and Cj1538-1540) homologous to bacterial molybdate (ModABC) uptake systems and the tungstate transporter (TupABC) of Eubacterium acidaminophilum respectively. The actual substrates and physiological role of these transporters were investigated. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry of the purified periplasmic binding proteins of each system revealed that while Cj0303 is unable to discriminate between molybdate and tungstate (K(D) values for both ligands of 4-8 nM), Cj1540 binds tungstate with a K(D) of 1.0 +/- 0.2 pM; 50 000-fold more tightly than molybdate. Induction-coupled plasma mass spectroscopy of single and double mutants showed that this large difference in affinity is reflected in a lower cellular tungsten content in a cj1540 (tupA) mutant compared with a cj0303c (modA) mutant. Surprisingly, formate dehydrogenase (FDH) activity was decreased approximately 50% in the tupA strain, and supplementation of the growth medium with tungstate significantly increased FDH activity in the wild type, while inhibiting known molybdoenzymes. Our data suggest that C. jejuni possesses a specific, ultra-high affinity tungstate transporter that supplies tungsten for incorporation into FDH. Furthermore, possession of two MoeA paralogues may explain the formation of both molybdopterin and tungstopterin in this bacterium.

Computational Redesign of the SHV-1 β-Lactamase/β-Lactamase Inhibitor Protein Interface

β-lactamases are enzymes that catalyze the hydrolysis of β-lactam antibiotics. β-lactamase/β-lactamase inhibitor protein (BLIP) complexes are emerging as a well characterized experimental model system for studying protein–protein interactions. BLIP is a 165 amino acid protein that inhibits several class A β-lactamases with a wide range of affinities: picomolar affinity for K1; nanomolar affinity for TEM-1, SME-1, and BlaI; but only micromolar affinity for SHV-1 β-lactamase. The large differences in affinity coupled with the availability of extensive mutagenesis data and high-resolution crystal structures for the TEM-1/BLIP and SHV-1/BLIP complexes make them attractive systems for the further development of computational design methodology. We used EGAD, a physics-based computational design program, to redesign BLIP in an attempt to increase affinity for SHV-1. Characterization of several of designs and point mutants revealed that in all cases, the mutations stabilize the interface by 10- to 1000-fold relative to wild type BLIP. The calculated changes in binding affinity for the mutants were within a mean absolute error of 0.87 kcal/mol from the experimental values, and comparison of the calculated and experimental values for a set of 30 SHV-1/BLIP complexes yielded a correlation coefficient of 0.77. Structures of the two complexes with the highest affinity, SHV-1/BLIP (E73M) and SHV-1/BLIP (E73M, S130K, S146M), are presented at 1.7 Å resolution. While the predicted structures have much in common with the experimentally determined structures, they do not coincide perfectly; in particular a salt bridge between SHV-1 D104 and BLIP K74 is observed in the experimental structures, but not in the predicted design conformations. This discrepancy highlights the difficulty of modeling salt bridge interactions with a protein design algorithm that approximates side chains as discrete rotamers. Nevertheless, while local structural features of the interface were sometimes miscalculated, EGAD is globally successful in designing complexes with increased affinity.

Assessing the catalytic activity of three different sources of tyrosinase: A study of the oxidation of mono- and difluorinated monophenols

Tyrosinase (tyr) purified from Pseudomonas putida F6, Streptomyces antibioticus, and Agaricus bisporus (mushroom) oxidises 3 regioisomers of fluorophenol as well as 3,4-difluorophenol (3,4FP). The catalytic efficiency ( k cat/ K m) of tyrosinase towards any one substrate is different for each enzyme source. Oftentimes a large difference in affinity ( K m), and turnover ( k cat) is observed for different enzyme sources with the same substrate. The best catalytic efficiency towards a fluorinated substrate was observed for P. putida F6 tyr with 4-fluorophenol (4FP). The presence of a second fluorine on the aromatic ring (3,4FP) resulted in a decrease in the catalytic efficiency of all three enzymes compared to values for 4FP. However, the K m value for 3,4FP decreased for P. putida F6 tyr indicating a higher affinity for P. putida F6 tyr for 3,4FP compared to 4FP. Furthermore the k cat value for 3,4FP increased for mushroom tyr in comparison to the value for 4FP indicating a higher maximum turnover of 3,4FP compared to 4FP for mushroom tyr. All three sources of tyr exhibited lower catalytic efficiencies for 3-fluorophenol (3FP) and 2-fluorophenol (2FP) compared to 4FP. However, the K m value for 2FP was lower than that for 3FP for both S. antibioticus and mushroom tyr indicating a higher affinity for 2FP over 3FP.

MDMX Overexpression Prevents p53 Activation by the MDM2 Inhibitor Nutlin

The p53 tumor suppressor plays a key role in maintaining genomic stability and protection against malignant transformation. MDM2 and MDMX are both p53-binding proteins that regulate p53 stability and activity. Recent development of the MDM2 inhibitor Nutlin 3 has greatly facilitated functional analysis of MDM2-p53 binding. We found that although MDMX is homologous to MDM2 and binds to the same region on p53 N terminus, Nutlin does not disrupt p53-MDMX interaction. The ability of Nutlin to activate p53 is compromised in tumor cells overexpressing MDMX. Combination of Nutlin with MDMX siRNA resulted in synergistic activation of p53 and growth arrest. These results suggest that MDMX is also a valid target for p53 activation in tumor cells. Development of novel compounds that are MDMX-specific or optimized for dual-inhibition of MDM2 and MDMX are necessary to achieve full activation of p53 in tumor cells.

Functional differences between NPFF 1 and NPFF 2 receptor coupling: High intrinsic activities of RFamide-related peptides on stimulation of [ 35S]GTPγS binding

By using an optimized [ 35S]GTPγS binding assay, the functional activities (potency and efficacy) of peptides belonging to three members of the RFamide family; Neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and 26RFamide, were investigated on NPFF 1 and NPFF 2 receptors stably expressed in Chinese Hamster Ovary (CHO) cells. Despite their large differences in affinity and selectivity, all analogues tested behaved as agonists toward NPFF 1 and NPFF 2 receptors. High NaCl concentration in the assay strongly increased the efficacy toward NPFF 2 receptors and augmented differences among agonists. In low sodium conditions, whereas the potencies of agonists correlated with their affinities for NPFF 1 receptors, NPFF 2 receptors exhibited an extraordinary activity since all compounds tested displayed EC 50 values of GTPγS binding lower than their K I values. Comparisons of functional values between NPFF 1 and NPFF 2 receptors revealed unexpected potent selective NPFF 2 agonists especially for the PLRFamide and the VGRFamide sequences. By using blocker peptides, we also show that Gα i3 and Gα s are the main transducers of NPFF 1 receptors while NPFF 2 are probably coupled with Gα i2, Gα i3, Gα o and Gα s proteins. Our data indicate that NPPF 1 and NPFF 2 receptors are differently coupled to G proteins in CHO cells.

Trisaccharide mimetics of the aminoglycoside antibiotic neomycin

A highly convergent approach for the chemical synthesis of eight structurally related trisaccharides that contain 3 to 5 amino groups has been described. Fourier-transformation ion cyclotron resonance mass spectrometry (FT-ICR MS) has been employed to determine the dissociation constants (Kd) for the binding of the trisaccharides to a prototypical fragment of 16S ribosomal RNA. A compound that contained a 4,6-dideoxy-4-amino-beta-D-glucopyranoside moiety at C-3 displayed binding in the low micromolar range. It was found that small structural changes of the saccharides resulted in large differences in affinity. The described structure-activity relationship is expected to be valuable for the development of novel antibiotics that target rRNA.

Diastereomeric Molecular Recognition and Binding Behavior of Bile Acids by l/d-Tryptophan-Modified β-Cyclodextrins

[reaction: see text] Binding behavior of L- and D-tryptophan-modified beta-cyclodextrins (L/D-Trp-beta-CD) (1 and 2) with four bile acids, i.e., cholate (CA), deoxycholate (DCA), glycocholate (GCA), and taurocholate (TCA), has been investigated by fluorescence, circular dichroism, and 2D-NMR spectroscopies and fluorescence lifetime measurement, as well as isothermal titration microcalorimetry. From the induced circular dichroism (ICD) and 2D NMR spectra, it is deduced that the D-Trp moiety of 2 attached to beta-CD is more deeply self-included in the cavity than that of the antipodal L-Trp moiety of 1, indicating appreciably enantioselective binding of the chiral sidearm by beta-CD. Interestingly, the original difference in conformation between 1 and 2 led to quite a large difference in affinity toward DCA, giving 3.3 times higher binding ability for 2 than for 1. Thermodynamically, the inclusion complexation of 1 and 2 with bile acids was entirely driven by favorable enthalpy change (DeltaH degrees) with accompanying negative entropy change (DeltaS degrees). The stronger binding of bile acids by L/D-Trp-beta-CD is attributable to higher enthalpic gains. The combined use of the calorimetric and NMR ROESY spectral examinations revealed the correlation between the thermodynamic parameters and the role of sidearm conformation in modified beta-cyclodextrins.

The variable C-terminus of 14-3-3 proteins mediates isoform-specific interaction with sucrose-phosphate synthase in the yeast two-hybrid system

Sucrose-6-phosphate synthase (SPS) is a target for 14-3-3 protein binding in plants. Because several isoforms of the 14-3-3 protein are expressed in plants, I investigated which isoforms have the ability to bind SPS. Two 14-3-3 isoforms (T14-3d and a novel isoform designated T14-3 g) were found to interact with SPS from tobacco (Nicotiana tabacum L.) in a two-hybrid screen. To further address the question of isoform specificity of 14-3-3s, four additional isoforms were tested for their ability to interact with SPS in the yeast two-hybrid system. The results clearly revealed large differences in affinity between individual 14-3-3 isoforms toward SPS. Deletion analysis suggested that these differences were mediated by the variable C-terminus of 14-3-3s. Site-directed mutagenesis of candidate 14-3-3 binding sites on SPS demonstrated that interaction could be independent of a phosphorylated serine residue within conserved binding motifs in the yeast system. These findings suggest that the large number of 14-3-3 isoforms present in plants reflects functional specificity.

Bisquaternary caracurine V and iso-caracurine V salts as ligands for the muscle type of nicotinic acetylcholine receptors: SAR and QSAR studies

The binding constants (Ki values) of 24 caracurine V and 6 iso-caracurine V analogues for the muscle type of nicotinic ACh receptors (nAChR) from Torpedo californica were determined in a binding assay using (±)-[3H]epibatidine as a radioligand. The allyl alcohol group present in the iso-caracurine V ring system was found to be essential for high binding affinity. The most potent compounds are the dimethyl and di-(4-nitrobenzyl)-iso-caracurinium V salts 29 (18nM), and 31 (79nM), respectively. Compound 29 and the corresponding diallyl analogue 30 (350nM) exhibited similar binding affinities as the equally substituted neuromuscular-blocking agents toxiferine I (14nM) and alcuronium (234nM), respectively. The SAR results were confirmed by QSAR studies, which additionally revealed that the presence of hydrogen-bond acceptor groups close to the quaternary nitrogen, is detrimental for the nicotinic binding affinity. The diallyl- and dimethylcaracurinium V salts 13 and 27, respectively, which are known to be among the most potent allosteric modulators of M2 receptors (EC50=10 and 8nM, respectively), exhibited rather low nicotinic binding affinities for muscle type nAChR (Ki=1.5 and 5.2μM, respectively). Such a large difference in affinity suggests that it is possible to develop compounds with high muscarinic allosteric potency and low or negligible affinities for (α1)2β1γδ nAChR. Additionally, the iso-caracurine V analogues with binding affinities comparable to those of (+)-tubocurarine and alcuronium could become a new class of neuromuscular-blocking agents.

Substantial Energetic Improvement with Minimal Structural Perturbation in a High Affinity Mutant Antibody

Here, we compare an antibody with the highest known engineered affinity (K(d)=270 fM) to its high affinity wild-type (K(d)=700 pM) through thermodynamic, kinetic, structural, and theoretical analyses. The 4M5.3 anti-fluorescein single chain antibody fragment (scFv) contains 14 mutations from the wild-type 4-4-20 scFv and has a 1800-fold increase in fluorescein-binding affinity. The dissociation rate is approximately 16,000 times slower in the mutant; however, this substantial improvement is offset somewhat by the association rate, which is ninefold slower in the mutant. Enthalpic contributions to binding were found by calorimetry to predominate in the differential binding free energy. The crystal structure of the 4M5.3 mutant complexed with antigen was solved to 1.5A resolution and compared with a previously solved structure of an antigen-bound 4-4-20 Fab fragment. Strikingly, the structural comparison shows little difference between the two scFv molecules (backbone RMSD of 0.6A), despite the large difference in affinity. Shape complementarity exhibits a small improvement between the variable light chain and variable heavy chain domains within the antibody, but no significant improvement in shape complementarity of the antibody with the antigen is observed in the mutant over the wild-type. Theoretical modeling calculations show electrostatic contributions to binding account for -1.2 kcal/mol to -3.5 kcal/mol of the binding free energy change, of which -1.1 kcal/mol is directly associated with the mutated residue side-chains. The electrostatic analysis reveals several mechanistic explanations for a portion of the improvement. Collectively, these data provide an example where very high binding affinity is achieved through the cumulative effect of many small structural alterations.