Controlled formation of model homo- and heterodimer coiled coil polypeptides

Abstract

Sequence-simplified coiled coil polypeptides were synthesized and their folding properties characterized in order to define the role of charged border residues at the coiled coil interface for the controlled formation of homodimer and heterodimer structures. Three peptides were designed to form parallel coiled coils with valine and leucine occupying the hydrophobic interface positions a and d, respectively, of the heptad repeat abcdefg. The polypeptide designated E/K42, with the heptad repeat sequence VSSLESK, contained glutamate and lysine in the interface border positions e and g, respectively, and was designed to form a coiled coil homodimer at neutral pH. Two other polypeptides, designated E/E35 and K/K35, have the heptad repeats VSSLESE and VSSLKSK, respectively. E/E35 contains only glutamic acid at both e and g positions; K/K35, only lysine, E/E35 and K/K35 were designed to form a stable coiled coil heterodimer when combined at neutral pH. All three polypeptides were prepared by solid-phase synthesis and purified by reverse-phase high-performance liquid chromatography followed by size-exclusion chromatography. E/K42 formed a stable dimeric coiled coil structure as determined by circular dichroism and size-exclusion chromatography. The alpha-helical content of E/K42 was highest at neutral pH and decreased at extremes of pH. The alpha-helical structure of E/K42 at micromolar concentrations had a Tm of 62-65 degrees C and exhibited a concentration dependence of thermal denaturation consistent with dimer formation. In contrast to results with E/K42, a mixture of E/E35 and K/K35, but neither alone, forms alpha-helix at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)

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

“…We have designed a heterodimeric coiledcoil ( Figure 1 and Table 1), based on principles revealed by more than 15 years of investigation (13,16,17,22). The primary stabilizing interactions are the hydrophobic interactions in the core (34,37), electrostatic attractions across the interface (5,7,9,16), and helical propensity (16,(38)(39)(40)(41).…”

“…This demonstrated that the contribution of Leu-Leu hydrophobic interactions to the stability of the coiled-coil structure were less important at the ends of the coiled-coil, and that the ends of the coiled-coil were more flexible. More recently, Holtzer et al (65) clearly demonstrated by 13 C R chemical shifts that such an end-fraying also occurred in a GCN4-like leucine zipper. All these studies suggest that the different heptads which make up a dimeric coiled-coil structure contribute differently to its stability; the heptads located at the ends of the coiled-coil are able to fray whereas the ones composing the core of the coiled-coil are more stable due to the tighter packing of the hydrophobic residues.…”

“…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)(14)(15)(16)(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 review).…”

Oligomerization properties of GCN4 leucine zipper e and g position mutants

“…The failure of putative repulsive interactions to prevent heterodimer formation presumably reflects differences in the sensitivities of the homodimerization and heterodimerization assays in vivo. It should be noted that, because our genetic test currently requires that the mutants we tested be able to form fusion proteins with strong enough dimerization to be detected as functional repressors, our data set includes no examples of pairs where the formation of heterodimers leads to a net increase in the number of attractive ion pairs, as would be observed with Fos-Jun heterodimers and in the designed heterodimers with acidic and basic monomers (O'Shea et al, 1992(O'Shea et al, , 1993Graddis et al, 1993;Zhou et al, 1994a). In these cases, the pH dependence of homodimer and heterodimer formation suggests that charge-charge interactions play a role in determining specificity.…”

Complementary Interhelical Interactions between Three Buried Glu-Lys Pairs within Three Heptad Repeats Are Essential for Hec1-Nuf2 Heterodimerization and Mitotic Progression

“…Hydrophobic amino acids are often present at the a and d "buried" positions every 3.6 residues, and polar/charged residues are at the adjacent flanking e and g "surface" positions (11)(12)(13)(14)(15). At this interval, a hydrophobic dimerization interface (core) is created, surrounded by interhelical electrostatic interactions that may dictate protein interaction specificity.…”