HAMLET (Human
Alpha-lactalbumin Made LEthal to
Tumor cells) is the first member in a new family of
protein-
lipid complexes with broad tumoricidal activity. Elucidating the molecular structure and the domains crucial for HAMLET formation is fundamental for understanding its tumoricidal function. Here we present the low-resolution
solution structure of the complex of
oleic acid bound HAMLET, derived from small angle X-ray scattering data. HAMLET shows a two-domain conformation with a large globular domain and an extended part of about 2.22 nm in length and 1.29 nm width. The structure has been superimposed into the related crystallographic structure of human α-
lactalbumin, revealing that the major part of α-
lactalbumin accommodates well in the shape of HAMLET. However, the C-terminal residues from
L105 to L123 of the crystal structure of the human α-
lactalbumin do not fit well into the HAMLET structure, resulting in an extended conformation in HAMLET, proposed to be required to form the tumoricidal active HAMLET complex with
oleic acid. Consistent with this low resolution structure, we identified biologically active
peptide epitopes in the globular as well as the extended domains of HAMLET.
Peptides covering the alpha1 and alpha2 domains of the
protein triggered rapid ion fluxes in the presence of
sodium oleate and were internalized by
tumor cells, causing rapid and sustained changes in cell morphology. The alpha
peptide-
oleate bound forms also triggered
tumor cell death with comparable efficiency as HAMLET. In addition, shorter
peptides corresponding to those domains are biologically active. These findings provide novel insights into the structural prerequisites for the dramatic effects of HAMLET on
tumor cells.