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Biological and Environmental Phenomena at the Interface
A Y-Shaped Three-Arm Structure for Probing Bivalent Interactions Between Protein Receptor-Ligand Using AFM and SPR Subhadip Senapati, Sudipta Biswas, Saikat Manna, Robert Ros, Stuart Lindsay, and Peiming Zhang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00735 • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018
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A Y–Shaped Three-Arm Structure for Probing Bivalent Interactions Between Protein ReceptorLigand Using AFM and SPR Subhadip Senapati, † Sudipta Biswas, † Saikat Manna, Robert Ros, Stuart Lindsay, * and a,b
a,b
a,b
a,c,d
a,b,c
Peiming Zhang * a
a
Biodesign Institute, School of Molecular Sciences, Department of Physics, and Center for b
c
d
Biological Physics, Arizona State University, Tempe, AZ 85281 (USA)
ABSTRACT. The goal of this research was to develop linkage chemistry for the study of bivalent interactions between a receptor and its ligand using atomic force microscopy (AFM) and surface plasmon resonance (SPR). We conceived a three-arm structure composed of flexible chains connected to a large rigid core with orthogonal functional groups at their ends for formation and attachment (or immobilization) of bivalent ligands. To demonstrate the principle, we chose the well-known biotin-streptavidin interaction as a model system. Based on a crystal structure of the biotin-streptavidin complex, we designed and synthesized a bis-biotin ligand to have a Y shape with two biotin motifs on its arms for binding and a functional group on its stem for immobilization or attachment, referred as to y-Bisbiotin. First, we found that the y-Bisbiotin ligand stabilized the streptavidin more than its mono-biotin counterpart did in solution, which indicates that the bivalent interaction was synergistic. The y-Bisbiotin was attached to AFM tips
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through a click reaction for the force measurement experiments, which showed that unbinding the bisbiotin from streptavidin needed twice the force of unbinding a monobiotin. For the SPR study, we added a ω-thiolated alkyl chain to y-Bisbiotin for its incorporation to a monolayer. The SPR data indicated that the streptavidin dissociated from a mixed monolayer bearing y-Bisbiotin much slower than from the one bearing monobiotin. This work demonstrates unique chemistry for the study of bivalent interactions using AFM and SPR.
Introduction When two monovalent ligands are connected together through a proper linker, they can bivalently bind to their receptor with an increased apparent affinity (avidity) and specificity. As 1
a typical class of multivalent interactions, the bivalent binding has been exploited in artificial supramolecular systems, medicinal chemistry, 2-4
1, 5, 6
pretargeted radioimmunotherapy, and so on. 7, 8
Though, a “true” bivalent interaction is often obscured by “statistical binding” of a homodimer or “dual action” of a heterodimer connected through a linker of insufficient length,
10, 11
9
both of
which show increases in the binding affinities owing to increased binding probabilities. Conventional analytical techniques, such as radio- or fluorescence-labeled ligand competitive binding assays, are not sufficient to distinguish between these subtle differences.
12
AFM, as well as SPR, can be useful tools for the study of bivalent interactions. SPR monitors biomolecular interaction in a label-free and real-time manner, which has widely been used in drug discovery,
13, 14
but it is limited to those interactions with affinities in a range of 500 µM to 1
nM. For the tighter binding, AFM should be more effective because it can detect biomolecular 15
interactions with the K ’s in a micromolar (µM) to femtomolar (fM) range, with a concentration 16
d
sensitivity limit of 10 M, which measures mechanical forces within a range of 10 – 10 pN at a −17
17
4
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single molecule level. In an AFM measurement, a ligand tethered to an AFM tip is brought 18
close to a surface where a receptor is immobilized to interact with each other. By pulling the tip back, a force-distance rupture curve is generated, from which the unbinding forces can be determined. In addition, the pulling back process could limit the rebinding interference that 19
occurs during the dissociation process. Since both of them are a surface-based technique, SPR 20
requires that either ligand or protein is immobilized on its chip, and AFM requires that the ligand is tethered to its tip and the protein immobilized to a substrate or vice versa for the detection of biomolecular interactions. In the present study, we have developed a three-arm structure as a skeleton that can be used for both formation and immobilization (or attachment) of bivalent ligands, which would prompt the effective bivalent interactions and facilitate the measurement using AFM and SPR. To demonstrate the principle, we chose the interaction of biotin with streptavidin as a model system. First, we designed and synthesized a Y-shaped bis-biotin ligand (hereinafter referred to as yBisbiotin) by tethering the ligand to the three-arm structure, and then studied its interaction with streptavidin in solution by gel electrophoresis as well as on the surfaces by SPR and AFM. Results and Discussion Design of the three-arm structure. A bivalent ligand should have each of its two binding motifs unstrained and independent so that it can achieve a maximum binding enthalpy (DH ) Bi
about twice as much as the one for the corresponding monovalent binding (DH ). Meanwhile, mono
21
the ligand should be managed to have a minimum loss of entropy caused by freezing rotatable bonds as it binds to a receptor. One commonly used approach is prepaying the entropy cost by preorganization. It has been known that even a small change in entropy would result in a 22
substantial difference in the entropic term (𝑇∆S) of Gibbs free energy. We implemented these
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principles in our design of the three-arm structure by (a) making it have sufficient flexibility so that its pendent ligands can bind to their receptors in much the same way as they are in the free state, (b) maximizing its rigidity to reduce the possible loss in conformational entropy upon binding, and (c) reducing its non23
specific interactions. From this aspect, we avoid using charged species, such as DNA,
Figure 1. (A) View of the crystal structure of a
as a linker. It is because the negatively
biotin-streptavidin complex, adopted from the
charged DNA molecule can interact with
Protein Data Bank (PDB ID: 3RY2); (B) Distances
some proteins through electrostatic attraction between biotins in the complex, determined by and release counterions,
24-26
which is an
measuring the distance between the ureido oxygen
entropically favorable process resulting in
atoms, and between oxygen atoms of the
strong nonspecific binding.
carboxylates in the crystal structure; (C) Chemical
We chose the biotin-streptavidin complex
structure of a y-Bisbiotin ligand; (D) A DFT model
as a model system because it has been well
of
studied by AFM and SPR,
combination with a 6-31G* basis set in a vacuum
27-30
and its high-
resolution (0.95Å) crystal structure is
y-Bisbiotin,
calculated
by
B3LYP
in
using software Spartan’14.
available in Protein Data Bank. As far as the 31
streptavidin is concerned, it is a tetrameric protein with a point group symmetry of 222, each subunit bearing a biotin binding site. From the projection shown in Figure 1-A, we can see three 32
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bound biotin molecules clearly, which indicates that the binding sites can be reached by a bi- or tri-valent ligand simultaneously. Taylor et al. have demonstrated that trisbiotinylated oligonucleotides with the length of > 20 nucleotides could occupy three of streptavidin’s four biotin-binding sites. In the crystal structure, the distances between biotin molecules are 33
measured in a range from 19 to 35 Å (Figure 1-B). Accordingly, we proposed a three-arm structure composed of a rigid core 3,5-bis(phenylethynyl)aniline to which three flexible oligo[ethylene glycol] (OEG) chains are connected (Figure 1-C). To form the bivalent ligand yBisbiotin, two biotin molecules are respectively attached to the amino ends of OEGs situated at the para positions of the phenyl ring 1 and 3, and an OEG bearing an orthogonal azido function is placed at the aniline segment for attachment. The design was inspired by the structure of immunoglobulin G (IgG, Figure S1, Supporting Information, hereinafter referred to as SI), making the three-arm structure have a Y-shape. DFT (Density Functional Theory) calculation shows that the rigid core has a width of 17 Å, and the entire molecule can display as a Y-shape with its two biotins separated by a distance of about 34 Å (Figure 1-D), comparable to the one between biotin 1 and biotin 2 in the crystal structure (see Figure 1-B). Besides, y-Bisbiotin has the same dyad axis of symmetry as those in the streptavidin-biotin complex, providing a favorable conformation for the binding. We designate the two branches connecting to biotins as Arm 1 and Arm 2, and the third one as the Stem (Figure 1-D). The OEG chain composed of 6 ethylene glycol units should provide flexibility and length necessary for biotins to reach the binding pockets of streptavidin as well as sufficient solubility for the molecule to dissolve in water. In aqueous solution (a good solvent), the OEG chain likely adapts a helical structure with its –O-C-C-O- backbone folded into a trans-gauche-trans conformation.
34, 35
Given that a 6 unit
OEG chain has an estimated Flory radius (R ) of ~ 8.1 Å, the three-arm structure should provide 36
f
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the width for two biotins of y-Bisbiotin to simultaneously bind to the streptavidin without causing any structural strain. As mentioned above, its third arm was used for both immobilization of y-Bisbiotin on an SPR chip and attachment of it to an AFM tip (vide infra). Synthesis. We synthesized the three-arm structure following a route as shown in Scheme 1. Under the Sonogashira cross-coupling conditions,
37, 38
3,5-diethynylaniline (1) reacted with p39
Reagents and conditions: (i) Pd(PPh3)2Cl2, CuI, THF: Et3N = 1:1, rt, 5 h; (ii) S7a or S7b, pyridine in dichloromethane, rt, 12 h; (iii) trifluoroacetic acid, rt, 10 min; (iv) biotin N-hydroxysuccinimide ester, triethylamine, DMF, rt, 3 h.
Scheme 1. Synthesis of y-Bisbiotin ligands
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iodobenzyloxy-OEG -NHBoc (2, synthesis in S1, SI), giving compound 3 in a 75% yield. In the 6
presence of pyridine, the arylamine of compound 3 reacted with α-OEGylated acetyl chloride S7a and S7b (synthesis in S2, SI) respectively, generating 4a (59%) and 4b (69%), which were subsequently treated with trifluoroacetic acid to remove the Boc protecting groups, resulting in two three-arm linkers 5a (69%) and 5b (70%) distinguishable by the length of their Stems. Thus, y-Bisbiotin 6a and 6b were readily produced by reacting of 5a and 5b with biotin Nhydroxysuccinimide ester in a yield of ~ 62% and 67%, respectively. These two compounds were used for the study of the bivalent interactions in solution and by AFM. For the SPR study, y-Bisbiotin 6a was modified with a ω-thiolated alkyl chain using a reagent triphenyl phosphane 11-(acetylthio)undecanoate (7, synthesis in S3, SI ) we developed, resulting in y-Bisbiotin bearing an acetylated thiol group at its Stem (8, synthesis in S4, SI). The acetyl
Figure 2. Structures of thiolated y-Bisbiotin (8), monobiotin ligands (9 and 10), and the molecular diluent (11) used in mixed monolayers (Ac: acetyl) for SPR study
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group is used to protect the thiol from oxidization during storage and removed in situ before the formation of monolayers on gold substrates. Also, monobiotin 9 was purchased from Quanta Biodesign and 10 synthesized in-house, which were used as monovalent controls for studying 40
the bivalent interaction. Compound 11 was also synthesized in house (S5, SI) as a diluent for the formation of mixed monolayers (Figure 2). Interaction of y-Bisbiotin with streptavidin in solution. We first tested y-Bisbiotin for its interaction with streptavidin in solution. In a PBS buffer (pH 7.4), streptavidin was mixed with y-Bisbiotin 6a in a 1:2 molar ratio and with monobiotin 9 (control) in a 1:4 molar ratio, respectively. Both solutions were incubated at room temperature for 15 minutes, and then aliquoted to a Laemmli sample buffer. To determine the thermal stability of these biotin-streptavidin
Figure 3. SDS-PAGE of (A) streptavidin complexed with monobiotin 9 (1:4) and (B) yBisbiotin 6a (1:2) heated at different temperatures under denaturing conditions.
complexes, we heated individual aliquots for 10 min at the different temperatures, quickly cooled them down, and then loaded them onto a SDS polyacrylamide gel (12%) for electrophoresis using a denaturing condition as reported in the literature. Figure 3 shows two typical images 41
obtained from the gel electrophoresis (also see Figure S2, SI for the image of an original gel). The major bands appear at the position corresponding to 70 kD mass, ascribed to the streptavidin-biotin complexes (Mw of streptavidin from Sigma: ~ 60,000 Da). The apostreptavidin started to break down at 60°C and was completely converted to its monomers above 70°C (Figure S3, SI). However, its stability increases by forming a complex with biotins as a
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result of increased inter-subunit associations. As shown in Figure 3-A, the streptavidin mixed 42
with monobiotin 9 only started to break down at ~ 80°C and was completely broken down at ~ 90°C. Thus, each monobiotin increased the stability of streptavidin by ~ 5°C. When mixed with y-Bisbiotin 6a, the streptavidin was stabilized more, beginning decomposition at ~ 90°C and completing at ~ 100°C (Figure 3-B). From mono-biotin to bis-biotin, the stability of the streptavidin tetramer was further increased by ~ 10 degrees. Thus, a bivalent interaction stabilized the streptavidin by an extra 2.5°C per biotin on average. Besides the major band, we notice that there are light bands showing up above the 70 kD position in the y-Bisbiotin gel, which disappear at ~ 100°C. It indicates that there was polymerization occurring during 6a interacting with streptavidin molecules. Wilbur et al. have synthesized a series of biotin dimers through linear linkers and trimers built on a phenyl ring, respectively, for streptavidin-based pretargeting radioimmunotherapy. Analyzing by size 43
exclusion (SE) HPLC, they found that their biotin dimers interacting with streptavidin resulted in cross-linked products, which increased with the length of the linker and reached a yield as high as 40% when the linker was about 49 Å long. Apparently, the SE-HPLC could not detect the 44
intramolecular binding because of its limited resolution to small changes (
