Design of three-dimensional domain-swapped dimers and fibrous oligomers

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 Å 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.

A peptide that inhibits hydroxyapatite growth is in an extended conformation on the crystal surface

Proteins play an important role in the biological mechanisms controlling hard tissue development, but the details of molecular recognition at inorganic crystal interfaces remain poorly characterized. We have applied a recently developed homonuclear dipolar recoupling solid-state NMR technique, dipolar recoupling with a windowless sequence (DRAWS), to directly probe the conformation of an acidic peptide adsorbed to hydroxyapatite (HAP) crystals. The phosphorylated hexapeptide, DpSpSEEK (N6, where pS denotes phosphorylated serine), was derived from the N terminus of the salivary protein statherin. Constant-composition kinetic characterization demonstrated that, like the native statherin, this peptide inhibits the growth of HAP seed crystals when preadsorbed to the crystal surface. The DRAWS technique was used to measure the internuclear distance between two 13C labels at the carbonyl positions of the adjacent phosphoserine residues. Dipolar dephasing measured at short mixing times yielded a mean separation distance of 3.2 ± 0.1 Å. Data obtained by using longer mixing times suggest a broad distribution of conformations about this average distance. Using a more complex model with discrete α-helical and extended conformations did not yield a better fit to the data and was not consistent with chemical shift analysis. These results suggest that the peptide is predominantly in an extended conformation rather than an α-helical state on the HAP surface. Solid-state NMR approaches can thus be used to determine directly the conformation of biologically relevant peptides on HAP surfaces. A better understanding of peptide and protein conformation on biomineral surfaces may provide design principles useful for the modification of orthopedic and dental implants with coatings and biological growth factors that are designed to enhance biocompatibility with surrounding tissue.