Design of three-dimensional domain-swapped dimers and fibrous oligomers - PubMed (original) (raw)
Design of three-dimensional domain-swapped dimers and fibrous oligomers
N L Ogihara et al. Proc Natl Acad Sci U S A. 2001.
Abstract
Three-dimensional (3D) domain-swapped proteins are intermolecularly folded analogs of monomeric proteins; both are stabilized by the identical interactions, but the individual domains interact intramolecularly in monomeric proteins, whereas they form intermolecular interactions in 3D domain-swapped structures. The structures and conditions of formation of several domain-swapped dimers and trimers are known, but the formation of higher order 3D domain-swapped oligomers has been less thoroughly studied. Here we contrast the structural consequences of domain swapping from two designed three-helix bundles: one with an up-down-up topology, and the other with an up-down-down topology. The up-down-up topology gives rise to a domain-swapped dimer whose structure has been determined to 1.5 A resolution by x-ray crystallography. In contrast, the domain-swapped protein with an up-down-down topology forms fibrils as shown by electron microscopy and dynamic light scattering. This demonstrates that design principles can predict the oligomeric state of 3D domain-swapped molecules, which should aid in the design of domain-swapped proteins and biomaterials.
Figures
Figure 1
Design of up-down-up and up-down-down three-helix bundles, and their domain-swapped counterparts. (A) Design of a DSD, beginning with Mon1 (up-down-up topology). (B) Design of a domain-swapped open aggregate, starting with Mon2 (up-down-down topology). (C) Helical wheel diagrams and amino acid sequence of DSAg and DSD. Each helical wheel diagram illustrates a single functional unit. The topologies of the functional units of DSAg and DSD differ only with respect to the orientation of helix III′ (antiparallel to helix I in DSAg and parallel in DSD). The positions of the Glu and Lys residues at the helix interfaces have been arranged to differentially stabilize the two different topologies. Notice that the leucine-containing hydrophobic cores are identical. In the amino acid sequences of DSD and DSAg, the leucine core (a and_d_ heptad positions) are the same. Only the charged_e_ and g positions are redistributed to reorient the molecules.
Figure 2
The designed e_–_g salt bridges stabilizing the conformations for DSD and DSAg. + indicates a positively charged Lys side chain; − indicates a negatively charged Glu side chain. Colored cylinders represent the α-helical domains. Notice DSD contains only two functional units, and the DSAg fibril can contain an unlimited number of functional units because the C-terminal domain of each monomer is always available to bind an additional molecule.
Figure 3
Thermal denaturation of DSD. CD data monitoring the loss of helical signal at 222 nm with increasing temperature at varying concentrations of Gdn⋅HCl: 0 M, 2.19 M, 3 M, 3.3 M, 3.6 M, 3.9 M, and 5.6 M, respectively. The smooth lines represent theoretical curves obtained by using parameters from the fitting procedure (see Materials and Methods).
Figure 4
Electron micrographs of the DSAg fibrils. (a) DSAg at neutral conditions (pH 6.5) under ×70,000 magnification. (b) DSAg at acidic conditions (pH 2.4) under ×70,000 magnification. The Inset is shown at ×276,000 magnification. Notice the fibril is composed of several protofibrils.
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