Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils:  Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

Abstract

We have de novo designed a heterodimeric coiled-coil formed by two peptides as a capture/ delivery system that can be used in applications such as affinity tag purification, immobilization in biosensors, etc. The two strands are designated as K coil (KVSALKE heptad sequence) and E coil (EVSALEK heptad sequence), where positively charged or negatively charged residues occupy positions e and g of the heptad repeat. In this study, for each E coil or K coil, three peptides were synthesized with lengths varying from three to five heptads. The effect of the chain length of each partner upon the kinetic and thermodynamic constants of interaction were determined using a surface plasmon resonance-based biosensor. Global fitting of the interactions revealed that the E5 coil interacted with the K5 coil according to a simple binding model. All the other interactions involving shorter coils were better described by a more complex kinetic model involving a rate-limiting reorganization of the coiled-coil structure. The affinities of these de novo designed coiled-coil interactions were found to range from 60 pM (E5/K5) to 30 µM (E3/K3). From these K d values, we were able to determine the free energy contribution of each heptad, depending on its relative position within the coiled-coils. We found that the free energy contribution of a heptad occupying a central position was 3-fold higher than that of a heptad at either end of the coiled-coil. The wide range of stabilities and affinities for the E/K coil system provides considerable flexibility for protein engineering and biotechnological applications.The R-helical coiled-coil is an oligomerization domain that is naturally present in a wide variety of proteins such as transcription factors, cytoskeletal proteins, motor proteins, and viral fusion proteins (1-3). The structural properties of coiled-coils have been extensively characterized from numerous high-resolution crystal and NMR structures (3-5). The formation of coiled-coils requires the wrapping of two or more amphipathic R-helices around each other in a lefthanded supercoil fashion; the helices may be aligned in either a parallel or antiparallel manner. The R-helices composing the coiled-coil structure are defined by a heptad repeat (denoted abcdefg) in which hydrophobic residues occupy the positions a and d and pack in a characteristic "knobs-intoholes" manner (6), forming the hydrophobic core ( Figure 1). Many studies (1,(7)(8)(9)(10)(11)(12) emphasized the role of charged residues (typically in the e and g positions) in controlling the specificity of oligomerization and opened up the way to the de novo design of heterodimeric coiled-coils (13-17). Their relatively small size, in addition to their well-defined structure, make coiled-coils a very attractive system for protein engineering and biotechnological applications (5, 18) such as the construction of miniaturized antibodies, the control of signaling protein domain oligomerization, and the specific targeting of GFP to cytoskeletal proteins (see ref 4 for ...

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

“…The de novo designed E/K heterodimerizing coiled-coil system as previously described [7] is composed of two distinct peptides, designated E-and K-coil. Each peptide has five repeats of a seven amino acid sequence that contains both charged and hydrophobic residues.…”

“…Individually, the E5-and K5-coil are highly soluble due to the large number of charged residues. When interacting, the E5-and K5-coil form a high affinity coiled-coil dimer [7,8] that has been proven to be useful for several applications, e.g. as a tag for purification purposes or for induced protein-protein interaction studies.…”

Enhancement of the Antagonistic Potency of Transforming Growth Factor-β Receptor Extracellular Domains by Coiled Coil-induced Homo- and Heterodimerization

“…In this report, we use a de novo designed heterodimerizing coiled-coil system (the E5/K5 coiled coil) that we have characterized (21) in order to rapidly produce and evaluate the effect of receptor ectodomain homo-and heterodimerization on their ability to bind to TGF-␤1 and to antagonize signaling. This coiled-coil system is an ideal dimerization domain since the coils are small (35 amino acids) and specifically heterodimerize, with the resulting non-covalent coiled-coil complex being stable.…”

Helix-Helix Interactions between Homo- and Heterodimeric γ-Carboxyglutamate-containing Conantokin Peptides and Their Derivatives

“…The Cys-containing peptides were immobilized on CM5 sensor chips using the standard ligand thiol coupling protocol outlined by the manufacturer (18). The carboxymethylated dextran surface was activated by the injection of 45 l of a solution containing 0.2 M N-ethyl-NЈ-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.05 M N-hydroxysuccinimide, and modified by injection of 40 l of 80 mM 2-(2-pyridinyldithio)ethaneamine in 0.1 M borate buffer, pH 8.5.…”