To maintain chromosome stability in eukaryotic cells, replication origins must be licensed by loading mini-chromosome maintenance (MCM2-7) complexes once and only once per cell cycle. This licensing control is achieved through the activities of geminin and cyclin-dependent kinases. Geminin binds tightly to Cdt1, an essential component of the replication licensing system, and prevents the inappropriate reinitiation of replication on an already fired origin. The inhibitory effect of geminin is thought to prevent the interaction between Cdt1 and the MCM helicase. Here we describe the crystal structure of the mouse geminin-Cdt1 complex using tGeminin (residues 79-157, truncated geminin) and tCdt1 (residues 172-368, truncated Cdt1). The amino-terminal region of a coiled-coil dimer of tGeminin interacts with both N-terminal and carboxy-terminal parts of tCdt1. The primary interface relies on the steric complementarity between the tGeminin dimer and the hydrophobic face of the two short N-terminal helices of tCdt1 and, in particular, Pro 181, Ala 182, Tyr 183, Phe 186 and Leu 189. The crystal structure, in conjunction with our biochemical data, indicates that the N-terminal region of tGeminin might be required to anchor tCdt1, and the C-terminal region of tGeminin prevents access of the MCM complex to tCdt1 through steric hindrance.

iron-sulfur protein ͉ transcription factor ͉ MerR

[NiFe] hydrogenase maturation proteins HypC, HypD, and HypE catalyze the insertion and cyanation of the iron center of [NiFe] hydrogenases by an unknown mechanism. We have determined the crystal structures of HypC, HypD, and HypE from Thermococcus kodakaraensis KOD1 at 1.8 A, 2.07 A, and 1.55 A resolution, respectively. The structure of HypD reveals its probable iron binding and active sites for cyanation. An extended conformation of each conserved motif of HypC and HypE allows the essential cysteine residues of both proteins to interact with the active site of HypD. Furthermore, the C-terminal tail of HypE is shown to exist in an ATP-dependent dynamic equilibrium between outward and inward conformations. Unexpectedly, the [4Fe-4S] cluster environment of HypD is quite similar to that of ferredoxin:thioredoxin reductase (FTR), indicating the existence of a redox cascade similar to the FTR system. These results suggest a cyanation reaction mechanism via unique thiol redox signaling in the HypCDE complex.

[NiFe] hydrogenase maturation represents one of the most dynamic and sophisticated processes in metallocenter assembly. The Fe(CN)(2)CO moiety of [NiFe] hydrogenases is assembled via unknown transient interactions among specific maturation proteins HypC (metallochaperone), HypD (redox protein), and HypE (cyanide synthesis/donor). Here, we report the structures of the HypC-HypD and HypC-HypD-HypE complexes, providing a view of the transient interactions that take place during the maturation process. HypC binds to the conserved region of HypD through extensive hydrophobic interactions. The ternary complex formation between HypE and the HypCD complex involves both HypC and HypD, rendering the HypE conformation favorable for cyanide transfer. In the complex, the conserved cysteines of HypC and HypD form an Fe binding site. The conserved C-terminal cysteine of HypE can access the thiol redox cascade of HypD. These results provide structural insights into the Fe atom cyanation in the HypCDE complex.

Calcium ion (Ca 2+ ) is an important second messenger that regulates numerous cellular functions. Intracellular Ca 2+ concentration ([Ca 2+ ]i) is strictly controlled by Ca 2+ channels and pumps on the endoplasmic reticulum (ER) and plasma membranes. The ER calcium pump, sarco/endoplasmic reticulum calcium ATPase (SERCA), imports Ca 2+ from the cytosol into the ER in an ATPase activitydependent manner. The activity of SERCA2b, the ubiquitous isoform of SERCA, is negatively regulated by disulfide bond formation between two luminal cysteines. Here, we show that ERdj5, a mammalian ER disulfide reductase, which we reported to be involved in the ER-associated degradation of misfolded proteins, activates the pump function of SERCA2b by reducing its luminal disulfide bond.