In human cells, cytosolic citrate is a major precursor for the synthesis of fatty acids, triacylglycerols, cholesterol and low-density lipoprotein. Cytosolic citrate further regulates the cell’s energy balance by activating the fatty acid synthesis pathway while down-regulating both the glycolysis and fatty acid β-oxidation pathways (Supplementary Fig. 1) 1–4. The rate of fatty acid synthesis in liver and adipose cells, the two major tissue types for such synthesis, correlates directly with the concentration of citrate in the cytosol 2–5. The cytosolic citrate concentration partially depends on direct import across the plasma membrane via the Na+-dependent citrate transporter (NaCT) 6,7. Mutations of the homologous fly gene (INDY, I’m Not Dead Yet) result in reduced fat storage through calorie restriction 8. More recently, NaCT-knockout mice have been found to have increased hepatic mitochondrial biogenesis, higher lipid oxidation and energy expenditure, and reduced lipogenesis, which taken together protect the mice from obesity and insulin resistance 9. To understand the transport mechanism of NaCT/INDY proteins, here we report the 3.2 Å crystal structure of a bacterial INDY homolog. One citrate molecule and one sodium ion are bound per protein, and their binding sites are defined by conserved amino acid motifs, forming the structural basis for understanding the transporters’ specificity. Comparison of the structures of the two symmetrical halves of the transporter suggests conformational changes that propel substrate translocation.

Interplay among four genes--egl-1, ced-9, ced-4 and ced-3--controls the onset of programmed cell death in the nematode Caenorhabditis elegans. Activation of the cell-killing protease CED-3 requires CED-4. However, CED-4 is constitutively inhibited by CED-9 until its release by EGL-1. Here we report the crystal structure of the CED-4-CED-9 complex at 2.6 A resolution, and a complete reconstitution of the CED-3 activation pathway using homogeneous proteins of CED-4, CED-9 and EGL-1. One molecule of CED-9 binds to an asymmetric dimer of CED-4, but specifically recognizes only one of the two CED-4 molecules. This specific interaction prevents CED-4 from activating CED-3. EGL-1 binding induces pronounced conformational changes in CED-9 that result in the dissociation of the CED-4 dimer from CED-9. The released CED-4 dimer further dimerizes to form a tetramer, which facilitates the autoactivation of CED-3. Together, our studies provide important insights into the regulation of cell death activation in C. elegans.

KChIPs coassemble with pore-forming Kv4 alpha subunits to form a native complex in the brain and heart and regulate the expression and gating properties of Kv4 K(+) channels, but the mechanisms underlying these processes are unknown. Here we report a co-crystal structure of the complex of human Kv4.3 N-terminus and KChIP1 at a 3.2-A resolution. The structure reveals a unique clamping action of the complex, in which a single KChIP1 molecule, as a monomer, laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner, forming an octamer. The proximal N-terminal peptide of Kv4.3 is sequestered by its binding to an elongated groove on the surface of KChIP1, which is indispensable for the modulation of Kv4.3 by KChIP1, and the same KChIP1 molecule binds to an adjacent T1 domain to stabilize the tetrameric Kv4.3 channels. Taken together with biochemical and functional data, our findings provide a structural basis for the modulation of Kv4 by KChIPs.

Crystal structure analyses for biological macromolecules without known structural relatives entail solving the crystallographic phase problem. Typical de novo phase evaluations depend on incorporating heavier atoms than those found natively; most commonly, multi- or single-wavelength anomalous diffraction (MAD or SAD) experiments exploit selenomethionyl proteins. Here we realize routine structure determination using intrinsic anomalous scattering from native macromolecules. We devised robust procedures for enhancing signal-to-noise in the slight anomalous scattering from generic native structures by combining data measured from multiple crystals at lower-than-usual x-ray energy. Using this multi-crystal SAD method (5–13 equivalent crystals), we determined structures at modest resolution (2.8Å-2.3Å) for native proteins varying in size (127–1148 unique residues) and number of sulfur sites (3–28). With no requirement for heavy-atom incorporation, such experiments provide an attractive alternative to selenomethionyl SAD experiments.