Jung-Gyu Lee

Structure and function of the N-terminal domain of the human mitochondrial calcium uniporter.

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.

Characterization of Calumenin-SERCA2 Interaction in Mouse Cardiac Sarcoplasmic Reticulum *

Calumenin is a multiple EF-hand Ca2+-binding protein localized in the sarcoplasmic reticulum (SR) with C-terminal SR retention signal HDEF. Recently, we showed evidence that calumenin interacts with SERCA2 in rat cardiac SR (Sahoo, S. K., and Kim, D. H. (2008) Mol. Cells 26, 265–269). The present study was undertaken to further characterize the association of calumenin with SERCA2 in mouse heart by various gene manipulation approaches. Immunocytochemical analysis showed that calumenin and SERCA2 were partially co-localized in HL-1 cells. Knockdown (KD) of calumenin was conducted in HL-1 cells and 80% reduction of calumenin did not induce any expressional changes of other Ca2+-cycling proteins. But it enhanced Ca2+ transient amplitude and showed shortened time to reach peak and decreased time to reach 50% of baseline. Oxalate-supported Ca2+ uptake showed increased Ca2+ sensitivity of SERCA2 in calumenin KD HL-1 cells. Calumenin and SERCA2 interaction was significantly lower in the presence of thapsigargin, vanadate, or ATP, as compared with 1.3 μm Ca2+, suggesting that the interaction is favored in the E1 state of SERCA2. A glutathione S-transferase-pulldown assay of calumenin deletion fragments and SERCA2 luminal domains suggested that regions of 132–222 amino acids of calumenin and 853–892 amino acids of SERCA2-L4 are the major binding partners. On the basis of our in vitro binding data and available information on three-dimensional structure of Ca2+-ATPases, a molecular model was proposed for the interaction between calumenin and SERCA2. Taken together, the present results suggest that calumenin is a novel regulator of SERCA2, and its expressional changes are tightly coupled with Ca2+-cycling of cardiomyocytes.

High-Field High-Repetition-Rate Sources for the Coherent THz Control of Matter

Ultrashort flashes of THz light with low photon energies of a few meV, but strong electric or magnetic field transients have recently been employed to prepare various fascinating nonequilibrium states in matter. Here we present a new class of sources based on superradiant enhancement of radiation from relativistic electron bunches in a compact electron accelerator that we believe will revolutionize experiments in this field. Our prototype source generates high-field THz pulses at unprecedented quasi-continuous-wave repetition rates up to the MHz regime. We demonstrate parameters that exceed state-of-the-art laser-based sources by more than 2 orders of magnitude. The peak fields and the repetition rates are highly scalable and once fully operational this type of sources will routinely provide 1 MV/cm electric fields and 0.3 T magnetic fields at repetition rates of few 100 kHz. We benchmark the unique properties by performing a resonant coherent THz control experiment with few 10 fs resolution.

Crystal structure of Helicobacter pylori MinE, a cell division topological specificity factor

In Gram‐negative bacteria, proper placement of the FtsZ ring, mediated by nucleoid occlusion and the activities of the dynamic oscillating Min proteins MinC, MinD and MinE, is required for correct positioning of the cell division septum. MinE is a topological specificity factor that counters the activity of MinCD division inhibitor at the mid‐cell division site. Its structure consists of an anti‐MinCD domain and a topology specificity domain (TSD). Previous NMR analysis of truncated Escherichia coli MinE showed that the TSD domain contains a long α‐helix and two anti‐parallel β‐strands, which mediate formation of a homodimeric α/β structure. Here we report the crystal structure of full‐length Helicobacter pylori MinE and redefine its TSD based on that structure. The N‐terminal region of the TSD (residues 19–26), previously defined as part of the anti‐MinCD domain, forms a β‐strand (βA) and participates in TSD folding. In addition, H. pylori MinE forms a dimer through the interaction of anti‐parallel βA‐strands. Moreover, we observed serial dimer–dimer interactions within the crystal packing, resulting in the formation of a multimeric structure. We therefore redefine the functional domain of MinE and propose that a multimeric filamentous structure is formed through anti‐parallel β‐strand interactions.

Ryanodine receptor assembly: a novel systems biology approach to 3D mapping.

Ryanodine receptors (RyRs) are intracellular Ca(2+) release channels (CRCs) that play a pivotal role in cellular Ca(2+) signaling. In striated muscles, RyR-mediated Ca(2+) release from the sarcoplasmic reticulum (SR) induces elevation of cytosolic Ca(2+) concentration and subsequent muscle contraction. Evidence from various sources suggests that RyRs in homo-tetrameric conformation form a large conductance Ca(2+) permeable channel in the central pore and large cytoplasmic domains. RyRs form a large assembly with various cytosolic and luminal proteins. A number of papers have been published concerning the functions of RyRs and the regulation of the associated proteins, but the three dimensional (3D) structure of the assembly has not been addressed in detail. In this paper, we have attempted to establish a 3D-map for the assembly of RyRs by considering published cryo-EM data, available X-ray crystallographic information and molecular modeling methods.

Structural implications of Ca(2+)-dependent actin-bundling function of human EFhd2/Swiprosin-1.

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.

Structural insights into the interaction of p97 N-terminus domain and VBM in rhomboid protease, RHBDL4.

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.

Crystallization and preliminary X-ray crystallographic analysis of human quinolinate phosphoribosyltransferase

Quinolinate phosphoribosyltransferase (QPRTase) is a key NAD-biosynthetic enzyme which catalyzes the transfer of quinolinic acid to 5-phosphoribosyl-1-pyrophosphate, yielding nicotinic acid mononucleotide. Homo sapiens QPRTase (Hs-QPRTase) appeared as a hexamer during purification and the protein was crystallized. Diffraction data were collected and processed at 2.8 Å resolution. Native Hs-QPRTase crystals belonged to space group P2(1), with unit-cell parameters a=76.2, b=137.1, c=92.7 Å, β=103.8°. Assuming the presence of six molecules in the asymmetric unit, the calculated Matthews coefficient is 2.46 Å3 Da(-1), which corresponds to a solvent content of 49.9%.

Structural mechanism underlying regulation of human EFhd2/Swiprosin-1 actin-bundling activity by Ser183 phosphorylation

EF-hand domain-containing protein D2/Swiprosin-1 (EFhd2) is an actin-binding protein mainly expressed in the central nervous and the immune systems of mammals. Intracellular events linked to EFhd2, such as membrane protrusion formation, cell adhesion, and BCR signaling, are triggered by the association of EFhd2 and F-actin. We previously reported that Ca2+ enhances the F-actin-bundling ability of EFhd2 through maintaining a rigid parallel EFhd2-homodimer structure. It was also reported that the F-actin-bundling ability of EFhd2 is regulated by a phosphorylation-dependent mechanism. EGF-induced phosphorylation at Ser183 of EFhd2 has been shown to inhibit F-actin-bundling, leading to irregular actin dynamics at the leading edges of cells. However, the underlying mechanism of this inhibition has remained elusive. Here, we report the crystal structure of a phospho-mimicking mutant (S183E) of the EFhd2 core domain, where the actin-binding sites are located. Although the overall structure of the phospho-mimicking mutant is similar to the one of the unphosphorylated form, we observed a conformational transition from ordered to disordered structure in the linker region at the C-terminus of the mutant. Based on our structural and biochemical analyses, we suggest that phosphorylation at Ser183 of EFhd2 causes changes in the local conformational dynamics and the surface charge distribution of the actin-binding site, resulting in a re-coordination of the actin-binding sites in the dimer structure and a reduction of F-actin-bundling activity without affecting the F-actin-binding capacity.

Structural Insights into the Quaternary Catalytic Mechanism of Hexameric Human Quinolinate Phosphoribosyltransferase, a Key Enzyme in de novo NAD Biosynthesis.

Quinolinate phosphoribosyltransferase (QPRT) catalyses the production of nicotinic acid mononucleotide, a precursor of de novo biosynthesis of the ubiquitous coenzyme nicotinamide adenine dinucleotide. QPRT is also essential for maintaining the homeostasis of quinolinic acid in the brain, a possible neurotoxin causing various neurodegenerative diseases. Although QPRT has been extensively analysed, the molecular basis of the reaction catalysed by human QPRT remains unclear. Here, we present the crystal structures of hexameric human QPRT in the apo form and its complexes with reactant or product. We found that the interaction between dimeric subunits was dramatically altered during the reaction process by conformational changes of two flexible loops in the active site at the dimer-dimer interface. In addition, the N-terminal short helix α1 was identified as a critical hexamer stabilizer. The structural features, size distribution, heat aggregation and ITC studies of the full-length enzyme and the enzyme lacking helix α1 strongly suggest that human QPRT acts as a hexamer for cooperative reactant binding via three dimeric subunits and maintaining stability. Based on our comparison of human QPRT structures in the apo and complex forms, we propose a drug design strategy targeting malignant glioma.