The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti-/pro-apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N-terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake-1 and mitochondrial calcium uptake-2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca2+ uptake in a stable MCU knockdown HeLa cell line and exerted dominant-negative effects in the wild-type MCU-expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.

Calumenin is a multiple EF-handSarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA) 2 is a major player in muscle relaxation of mammalian heart (2). Tight regulation of SERCA activity is important for normal Ca 2ϩ homeostasis in heart, and altered activity could lead to impaired excitation-contraction (E-C) coupling and cardiac diseases (3). SERCA2a is the predominant isoform in mouse heart, compared with SERCA2b (4). SERCA2a and SERCA2b are identical up to 994 amino acids. However, the C terminus of SERCA2b contains additional 50 amino acids, as compared with only 4 amino acids in SERCA2a (5). Structural and biochemical studies have shown that SERCA2 contains 10 transmembrane segments (M1-10), and the large globular cytoplasmic part is composed of three domains: the ␤ strand domain between M2 and M3, the phosphorylation domain attached to M4 at one end, and the nucleotide binding domain at the other end. The nucleotide binding domain contains a hinge domain, which is connected to the M5 segment (6). The luminal side contains five luminal domains connecting the following segments M1 and M2, M3 and M4, M5 and M6, M7 and M8, and M9 and M10, respectively (6 -8).Recent studies have shown that a number of proteins interact with SERCA2 and regulate its stability and activity. Among them phospholamban (PLN) is the most extensively studied molecule (9). PLN interacts with SERCA2 and decreases apparent affinity of SERCA2 for Ca 2ϩ , and this inhibition can be disrupted by phosphorylation of PLN or by elevation of cytosolic Ca 2ϩ , which leads to reversal of the inhibition (10). PLN is the important regulator of SERCA activity and contractility in heart (11). Other proteins related to the apoptotic pathway such as Bcl2 (12) and Hax1(13) interact with SERCA2 in the cytosolic side of SERCA2 and regulate SERCA2 protein level and stability. EF-hand protein S100A1 interacts with SERCA2 in the cytosolic side and regulates contractility in heart (14). Recent studies have suggested that SR luminal proteins such as calreticulin (15), ER protein 57 (16), sarcalumenin (17), histidine-rich Ca 2ϩ -binding protein (HRC) (18), and calumenin (1) interact with SERCA2. HRC binds to SERCA2 in a Ca 2ϩ -dependent manner, and its overexpression could inhibit SERCA2 activity and Ca 2ϩ cycling in cardiomyocytes (18,19). Sarcalumenin also interacts with SERCA2, which may consequently increase the tendency of its retention in the SR lumen and increase the SERCA2 protein stability (17).Calumenin is a multiple EF-hand Ca 2ϩ -binding protein and is found to have unique C-terminal SR retention signal HDEF (20,21). Calumenin is associated with the ryanodine receptor (RyR) in rabbit skeletal muscle, and its overexpression shows decreased depolarization-induced Ca 2ϩ release in C2C12 myotubes (22

EFhd2/Swiprosin-1 is a cytoskeletal Ca2+-binding protein implicated in Ca2+-dependent cell spreading and migration in epithelial cells. EFhd2 domain architecture includes an N-terminal disordered region, a PxxP motif, two EF-hands, a ligand mimic helix and a C-terminal coiled-coil domain. We reported previously that EFhd2 displays F-actin bundling activity in the presence of Ca2+ and this activity depends on the coiled-coil domain and direct interaction of the EFhd2 core region. However, the molecular mechanism for the regulation of F-actin binding and bundling by EFhd2 is unknown. Here, the Ca2+-bound crystal structure of the EFhd2 core region is presented and structures of mutants defective for Ca2+-binding are also described. These structures and biochemical analyses reveal that the F-actin bundling activity of EFhd2 depends on the structural rigidity of F-actin binding sites conferred by binding of the EF-hands to Ca2+. In the absence of Ca2+, the EFhd2 core region exhibits local conformational flexibility around the EF-hand domain and C-terminal linker, which retains F-actin binding activity but loses the ability to bundle F-actin. In addition, we establish that dimerisation of EFhd2 via the C-terminal coiled-coil domain, which is necessary for F-actin bundling, occurs through the parallel coiled-coil interaction.

RHBDL4 is an active rhomboid that specifically recognizes and cleaves atypical, positively charged transmembrane endoplasmic reticulum-associated degradation (ERAD) substrates. Interaction of valosin-containing protein (p97/VCP) and RHBDL4 is crucial to retrotranslocate polyubiquitinated substrates for ERAD pathway. Here, we report the first complex structure of VCP-binding motif (VBM) with p97 N-terminal domain (p97N) at 1.88 Å resolution. Consistent with p97 adaptor proteins including p47-ubiquitin regulatory X (UBX), gp78-VCP-interacting motif (VIM), OTU1-UBX-like element, and FAF1-UBX, RHBDL4 VBM also binds at the interface between the two lobes of p97N. Notably, the RF residues in VBM are involved in the interaction with p97N, showing a similar interaction pattern with that of FPR signature motif in the UBX domain, although the directionality is opposite. Comparison of VBM interaction with VIM of gp78, another α-helical motif that interacts with p97N, revealed that the helix direction is inversed. Nevertheless, the conserved arginine residues in both motifs participate in the majority of the interface via extensive hydrogen bonds and ionic interactions with p97N. We identified novel VBM-binding mode to p97N that involves a combination of two types of p97-cofactor specificities observed in the UBX and VIM interactions. This highlights the induced fit model of p97N interdomain cleft upon cofactor binding to form stable p97-cofactor complexes. Our mutational and biochemical analyses in defining the specific interaction between VBM and p97N have elucidated the importance of the highly conserved VBM, applicable to other VBM-containing proteins. We also showed that RHBDL4, ubiquitins, and p97 co-operate for efficient substrate dislocation.

The interaction of the rhomboid pseudoprotease Derlin-1 and p97 is crucial for the retrotranslocation of polyubiquitinated substrates in the endoplasmic reticulum-associated degradation pathway. We report a 2.25 Å resolution structure of the p97 N-terminal domain (p97N) in complex with the Derlin-1 SHP motif. Remarkably, the SHP motif adopts a short, antiparallel β-strand that interacts with the β-sheet of p97N-a site distinct from that to which most p97 adaptor proteins bind. Mutational and biochemical analyses contributed to defining the specific interaction, demonstrating the importance of a highly conserved binding pocket on p97N and a signature motif on SHP. Our findings may also provide insights into the interactions between other SHP-containing proteins and p97N.