Katja M. Arndt

Twist and shout: Coiled-coil forming α-helices are of great significance in understanding tertiary structural formation (the figure shows GCN4; an example of a parallel dimeric coiled coil), the design of new proteins, and the control of the oligomeric state. The apparent simplicity of coiled coils is misleading, and rules governing such features are far from fully established. Requirements for the specific pairings of helices during coiled-coil formation are discussed, as are the peculiarities within such domains that give proteins their unique function. By taking advantage of our increasing understanding of this structural class, a growing number of biological and therapeutic applications are being sought.

Antibody single-chain Fv (scFv) fragments are able to form dimers under certain conditions, and the extent of dimerization appears to depend on linker length, antibody sequence, and external factors. We analyzed the factors influencing dimer-monomer equilibrium as well as the rate of interconversion, using the scFv McPC603 as a model system. In this molecule, the stability of the VH-VL interaction can be conveniently varied by adjusting the ionic strength (because of its influence on the hydrophobic effect), by pH (presumably because of the presence of titratable groups in the interface), and by the presence or absence of the antigen phosphorylcholine, which can be rapidly removed due to its very fast off-rate. It was found that the monomer is the thermodynamically stable form with linkers of 15 and 25 amino acids length under all conditions tested (35 microM or less). The dimer is initially formed in periplasmic expression, presumably by domain swapping, and can be trapped by all factors which stabilize the VH-VL interface, such as the presence of the antigen, high ionic strength, and pH below 7.5. Under all other conditions, it converts to the monomer. Predominantly monomer is obtained during in vitro folding. Monomer is stabilized against dimerization at very high concentrations by the same factors which stabilize the VH-VL interaction. These results should be helpful in producing molecules with defined oligomerization states.

Activator protein-1 (AP-1) is a crucial transcription factor implicated in numerous cancers. For this reason, nine homologues of the AP-1 leucine zipper region have been characterized: Fos (c-Fos, FosB, Fra1, and Fra2), Jun (c-Jun, JunB, and JunD), and semirational library-designed winning peptides FosW and JunW. The latter two were designed to specifically target c-Fos or c-Jun. They have been identified by using protein-fragment complementation assays combined with growth competition. This assay removes nonspecific, unstable, and protease susceptible library members from the pool, leaving winners with excellent drug potential. Thermal melts of all 45 possible dimeric interactions have been surveyed, with the FosW-c-Jun complex displaying a melting temperature (T(m)) of 63 degrees C, compared to only 16 degrees C for wild-type c-Fos-c-Jun interaction. This impressive 70,000-fold K(D) decrease is largely due to optimized core packing, alpha-helical propensity, and electrostatics. Contrastingly, due to a poor c-Fos core, c-Fos-JunW dimerizes with lower affinity. However the T(m) far exceeds wild-type c-Fos-c-Jun and averaged JunW and c-Fos, indicating a preference over either homodimer. Finally, and with wider implications, we have compiled a method for predicting interaction of parallel, dimeric coiled coils, using our T(m) data as a training set, and applying it to 59 bZIP proteins previously reported. Our algorithm, unlike others to date, accounts for helix propensity, which is found to be integral in coiled coil stability. Indeed, in applying the algorithm to these 59(2) bZIP interactions, we were able to correctly identify 92% of all strong interactions and 92% of all noninteracting pairs.