No abstract

By changing the three-dimensional structure, a protein can attain new functions, distinct from those of the native protein. Amyloid-forming proteins are one example, in which conformational change may lead to fibril formation and, in many cases, neurodegenerative disease. We have proposed that partial unfolding provides a mechanism to generate new and useful functional variants from a given polypeptide chain. Here we present HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) as an example where partial unfolding and the incorporation of cofactor create a complex with new, beneficial properties. Native alpha-lactalbumin functions as a substrate specifier in lactose synthesis, but when partially unfolded the protein binds oleic acid and forms the tumoricidal HAMLET complex. When the properties of HAMLET were first described they were surprising, as protein folding intermediates and especially amyloid-forming protein intermediates had been regarded as toxic conformations, but since then structural studies have supported functional diversity arising from a change in fold. The properties of HAMLET suggest a mechanism of structure-function variation, which might help the limited number of human protein genes to generate sufficient structural diversity to meet the diverse functional demands of complex organisms.

In addition to its function as an intermediate in nitrogen metabolism, the small molecule urea plays an important role in the homeostasis of osmolarity and fluid volume in many organisms, including mammals, which concentrate urea in the kidney to produce the osmotic gradient necessary for water re-absorption. Because urea is highly polar and consequently poorly permeable through lipid bilayers, specialized urea transporters have evolved to increase its rate of diffusion across cell membranes. To better understand how urea transporters achieve the rapid and selective transport of urea, we have solved the 2.3 A ˚structure of a urea transporter from the bacterium Desulfovibrio vulgaris (dvUT), which has significant homology to mammalian urea transporters. The dvUT fold contains two homologous domains related by a two-fold pseudosymmetry axis perpendicular to the plane of the membrane. Each protomer contains a continuous membrane-spanning pore, suggesting that the protein operates by a channel-like rather than transporter-like mechanism. The constricted selectivity filter at the center of pore can accommodate dehydrated urea molecules passing in single file. Urea is stabilized by backbone and side chain oxygen atoms that provide continuous coordination as it progresses through the filter, and by well-positioned a-helix dipoles. We are now using a variety of functional assays to probe the physical and chemical interactions involved in the transport mechanism, as well as interactions between dvUT and various high-affinity UT blockers.