Choline kinase alpha is a 457-residue protein that catalyzes the reaction between ATP and choline to yield ADP and phosphocholine. This metabolic action has been well studied because of choline kinase’s link to cancer malignancy and poor patient prognosis. As the myriad of x-ray crystal structures available for this enzyme show, chemotherapeutic drug design has centered on stopping the catalytic activity of choline kinase and reducing the downstream metabolites it produces. Furthermore, these crystal structures only reveal the catalytic domain of the protein, residues 80–457. However, recent studies provide evidence for a non-catalytic protein-binding role for choline kinase alpha. Here, we show that choline kinase alpha interacts with the SH3 domain of c-Src. Co-precipitation assays, surface plasmon resonance, and crystallographic analysis of a 1.5 Å structure demonstrate that this interaction is specific and is mediated by the poly-proline region found N-terminal to the catalytic domain of choline kinase. Taken together, these data offer strong evidence that choline kinase alpha has a heretofore underappreciated role in protein-protein interactions, which offers an exciting new way to approach drug development against this cancer-enhancing protein.

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Human immunodeficiency virus-1 Nef is a myristoylated accessory protein essential for viral replication, immune evasion of infected cells, and AIDS progression. Nef functions in a membrane-bound state through interactions with numerous host cell proteins involved in signal transduction and endocytic trafficking. These include members of the Src and Tec kinase families, which are constitutively activated by interaction with Nef. Nef dimerizes at the plasma membrane in cell-based model systems, and dimerization has an important organizational role in Nef:kinase complex formation and activation. To characterize the biophysical aspects of this key Nef function, we aim to reconstitute complexes of Nef with Tec kinases at a membrane interface utilizing model membranes. Here we report an initial structure of myristoylated, recombinant Nef bound to fluid lipid bilayers determined using neutron scattering on solidsupported sparsely-tethered lipid bilayer membranes (stBLMs). As expected, myristoylation is essential for high affinity membrane interactions with anionic stBLMs. The folded Nef core is displaced from the bilayer in a position that is presumably amenable to engagement with membrane-bound kinases while Nterminal lipidation and a cluster of basic residues anchor the protein on the membrane. The distance of the Nef core from the membrane depends on surface concentration of the protein and appears to differ slightly for wild-type Nef and a dimerization-deficient Nef mutant (D123N). MD simulations constrained by the experimental neutron scattering results are currently exploring the conformational flexibility of the membrane-bound protein. These studies will releval the molecular conformation of Nef in its natural membrane environment, and address the impact of dimerization on Nef structure at the lipid bilayer.