Gil Bu Kang

Mutations in CHD7, Encoding a Chromatin-Remodeling Protein, Cause Idiopathic Hypogonadotropic Hypogonadism and Kallmann Syndrome

CHARGE syndrome and Kallmann syndrome (KS) are two distinct developmental disorders sharing overlapping features of impaired olfaction and hypogonadism. KS is a genetically heterogeneous disorder consisting of idiopathic hypogonadotropic hypogonadism (IHH) and anosmia, and is most commonly due to KAL1 or FGFR1 mutations. CHARGE syndrome, a multisystem autosomal-dominant disorder, is caused by CHD7 mutations. We hypothesized that CHD7 would be involved in the pathogenesis of IHH and KS (IHH/KS) without the CHARGE phenotype and that IHH/KS represents a milder allelic variant of CHARGE syndrome. Mutation screening of the 37 protein-coding exons of CHD7 was performed in 101 IHH/KS patients without a CHARGE phenotype. In an additional 96 IHH/KS patients, exons 6-10, encoding the conserved chromodomains, were sequenced. RT-PCR, SIFT, protein-structure analysis, and in situ hybridization were performed for additional supportive evidence. Seven heterozygous mutations, two splice and five missense, which were absent in > or = 180 controls, were identified in three sporadic KS and four sporadic normosmic IHH patients. Three mutations affect chromodomains critical for proper CHD7 function in chromatin remodeling and transcriptional regulation, whereas the other four affect conserved residues, suggesting that they are deleterious. CHD7s role is further corroborated by specific expression in IHH/KS-relevant tissues and appropriate developmental expression. Sporadic CHD7 mutations occur in 6% of IHH/KS patients. CHD7 represents the first identified chromatin-remodeling protein with a role in human puberty and the second gene to cause both normosmic IHH and KS in humans. Our findings indicate that both normosmic IHH and KS are mild allelic variants of CHARGE syndrome and are caused by CHD7 mutations.

Crystal structure of the Shank PDZ-ligand complex reveals a class I PDZ interaction and a novel PDZ-PDZ dimerization

The Shank/proline-rich synapse-associated protein family of multidomain proteins is known to play an important role in the organization of synaptic multiprotein complexes. For instance, the Shank PDZ domain binds to the C termini of guanylate kinase-associated proteins, which in turn interact with the guanylate kinase domain of postsynaptic density-95 scaffolding proteins. Here we describe the crystal structures of Shank1 PDZ in its peptide free form and in complex with the C-terminal hexapeptide (EAQTRL) of guanylate kinase-associated protein (GKAP1a) determined at 1.8- and 2.25-Å resolutions, respectively. The structure shows the typical class I PDZ interaction of PDZ-peptide complex with the consensus sequence -X-(Thr/Ser)-X-Leu. In addition, Asp-634 within the Shank1 PDZ domain recognizes the positively charged Arg at –1 position and hydrogen bonds, and salt bridges between Arg-607 and the side chains of the ligand at –3 and –5 positions contribute further to the recognition of the peptide ligand. Remarkably, whether free or complexed, Shank1 PDZ domains form dimers with a conserved βB/βC loop and N-terminal βA strands, suggesting a novel model of PDZ-PDZ homodimerization. This implies that antiparallel dimerization through the N-terminal βA strands could be a common configuration among PDZ dimers. Within the dimeric structure, the two-peptide binding sites are arranged so that the N termini of the bound peptide ligands are in close proximity and oriented toward the 2-fold axis of the dimer. This configuration may provide a means of facilitating dimeric organization of PDZ-target assemblies.

The Active Site of a Lon Protease from Methanococcus jannaschii Distinctly Differs from the Canonical Catalytic Dyad of Lon Proteases

ATP-dependent Lon proteases catalyze the degradation of various regulatory proteins and abnormal proteins within cells. Methanococcus jannaschii Lon (Mj-Lon) is a homologue of Escherichia coli Lon (Ec-Lon) but has two transmembrane helices within its N-terminal ATPase domain. We solved the crystal structure of the proteolytic domain of Mj-Lon using multiwavelength anomalous dispersion, refining it to 1.9-Å resolution. The structure displays an overall fold conserved in the proteolytic domain of Ec-Lon; however, the active site shows uniquely configured catalytic Ser-Lys-Asp residues that are not seen in Ec-Lon, which contains a catalytic dyad. In Mj-Lon, the C-terminal half of the β4-α2 segment is an α-helix, whereas it is a β-strand in Ec-Lon. Consequently, the configurations of the active sites differ due to the formation of a salt bridge between Asp-547 and Lys-593 in Mj-Lon. Moreover, unlike Ec-Lon, Mj-Lon has a buried cavity in the region of the active site containing three water molecules, one of which is hydrogen-bonded to catalytic Ser-550. The geometry and environment of the active site residues in Mj-Lon suggest that the charged Lys-593 assists in lowering the pKa of the Ser-550 hydroxyl group via its electrostatic potential, and the water in the cavity acts as a proton acceptor during catalysis. Extensive sequence alignment and comparison of the structures of the proteolytic domains clearly indicate that Lon proteases can be classified into two groups depending on active site configuration and the presence of DGPSA or (D/E)GDSA consensus sequences, as represented by Ec-Lon and Mj-Lon.

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.

Structural basis of E2-25K/UBB+1 interaction leading to proteasome inhibition and neurotoxicity.

E2–25K/Hip2 is an unusual ubiquitin-conjugating enzyme that interacts with the frameshift mutant of ubiquitin B (UBB+1) and has been identified as a crucial factor regulating amyloid-β neurotoxicity. To study the structural basis of the neurotoxicity mediated by the E2–25K-UBB+1 interaction, we determined the three-dimensional structures of UBB+1, E2–25K and the E2–25K/ubiquitin, and E2–25K/UBB+1 complex. The structures revealed that ubiquitin or UBB+1 is bound to E2–25K via the enzyme MGF motif and residues in α9 of the enzyme. Polyubiquitylation assays together with analyses of various E2–25K mutants showed that disrupting UBB+1 binding markedly diminishes synthesis of neurotoxic UBB+1-anchored polyubiquitin. These results suggest that the interaction between E2–25K and UBB+1 is critical for the synthesis and accumulation of UBB+1-anchored polyubiquitin, which results in proteasomal inhibition and neuronal cell death.

Preso, A Novel PSD-95-Interacting FERM and PDZ Domain Protein That Regulates Dendritic Spine Morphogenesis

PSD-95 is an abundant postsynaptic density (PSD) protein involved in the formation and regulation of excitatory synapses and dendritic spines, but the underlying mechanisms are not comprehensively understood. Here we report a novel PSD-95-interacting protein Preso that regulates spine morphogenesis. Preso is mainly expressed in the brain and contains WW (domain with two conserved Trp residues), PDZ (PSD-95/Dlg/ZO-1), FERM (4.1, ezrin, radixin, and moesin), and C-terminal PDZ-binding domains. These domains associate with actin filaments, the Rac1/Cdc42 guanine nucleotide exchange factor βPix, phosphatidylinositol-4,5-bisphosphate, and the postsynaptic scaffolding protein PSD-95, respectively. Preso overexpression increases the density of dendritic spines in a manner requiring WW, PDZ, FERM, and PDZ-binding domains. Conversely, knockdown or dominant-negative inhibition of Preso decreases spine density, excitatory synaptic transmission, and the spine level of filamentous actin. These results suggest that Preso positively regulates spine density through its interaction with the synaptic plasma membrane, actin filaments, PSD-95, and the βPix-based Rac1 signaling pathway.

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.

Crystal structure of Rattus norvegicus Visfatin/PBEF/Nampt in complex with an FK866-based inhibitor

Visfatin (Nampt/PBEF) plays a pivotal role in the salvage pathway for NAD+ biosynthesis. Its potent inhibitor, FK866, causes cellular NAD+ levels to decline, thereby inducing apoptosis in tumor cells. In an effort to improve the solubility and binding interactions of FK866, we designed and synthesized IS001, in which a ribose group is attached to the FK866 pyridyl ring. Here, we report the crystal structure of rat visfatin in complex with IS001. Like FK866, IS001 is positioned at the dimer interface, and all of the residues that interact with IS001 are involved in hydrophobic or π-π-stacking interactions. However, we were unable to detect any strong interactions between the added ribose ring of IS001 and visfatin, which implies that a bulkier modifying group is necessary for a tight interaction. This study provides additional structure-based information needed to optimize the design of visfatin inhibitors.

Structural Basis for Asymmetric Association of the βPIX Coiled Coil and Shank PDZ

betaPIX (p21-activated kinase interacting exchange factor) and Shank/ProSAP protein form a complex acting as a protein scaffold that integrates signaling pathways and regulates postsynaptic structure. Complex formation is mediated by the C-terminal PDZ binding motif of betaPIX and the Shank PDZ domain. The coiled-coil (CC) domain upstream of the PDZ binding motif allows multimerization of betaPIX, which is important for its physiological functions. We have solved the crystal structure of the betaPIX CC-Shank PDZ complex and determined the stoichiometry of complex formation. The betaPIX CC forms a 76-A-long parallel CC trimer. Despite the fact that the betaPIX CC exposes three PDZ binding motifs in the C-termini, the betaPIX trimer associates with a single Shank PDZ. One of the C-terminal ends of the CC forms an extensive beta-sheet interaction with the Shank PDZ, while the other two ends are not involved in ligand binding and form random coils. The two C-terminal ends of betaPIX have significantly lower affinity than the first PDZ binding motif due to the steric hindrance in the C-terminal tails, which results in binding of a single PDZ domain to the betaPIX trimer. The structure shows canonical class I PDZ binding with a beta-sheet interaction extending to position -6 of betaPIX. The betaB-betaC loop of Shank PDZ undergoes a conformational change upon ligand binding to form the beta-sheet interaction and to accommodate the bulky side chain of Trp -5. This structural study provides a clear picture of the molecular recognition of the PDZ ligand and the asymmetric association of betaPIX CC and Shank PDZ.

Crystal structure of the leucine zipper domain of small‐conductance Ca2+‐activated K+ (SKCa) channel from Rattus norvegicus

The leucine zipper (LZ) is a typical member of the coiled coil family, the most common and extensively investigated of structural motifs.1 In terms of their biological functions, LZs participate in a variety of cellular processes, supplying unique protein–protein interactions via leucine–leucine zipping. The LZ domain is defined as an a-helix comprised of heptad repeats (abcdefg)n in which the residues at positions a and d are hydrophobic and mediate critical interhelical interactions, while b, c, e, f, and g are hydrophilic and form the solvent-exposed part of the coiled coil.2–4 Moreover, two or more LZ domains can intertwine in parallel or antiparallel to form a bundle of a-helices that interact via hydrophobic interactions at the inner face of the coiled coils.5–7 LZ domains were originally identified as highly conserved motifs mediating the interaction between transcriptional factors, but are now known to also be present in a number of ion channels.8 The sequences of ion channel LZ domains contain features common to the transitional LZs of transcriptional factors,2,4 but the canonical leucine at position d is often replaced by other residues such as isoluecine, valine, or even a nonhydrophobic residue, thereby forming a modified LZ.9 Recent reports on the function of ion channel LZ domains have suggested that they participate in regulating the channel’s activity by targeting modulator proteins to the channel, promoting formation of ‘‘macromolecular signaling complexes.’’8–16 For instance, the LZ domains of the RyR2 channel recruit adaptor molecules for PKA, PP1, and PP2A, which regulate channel activity through phosphorylation of certain residues.9–11 They also facilitate correct folding and plasma membrane trafficking of ion channels.17–19 By substituting leucine residues, it was confirmed that ‘‘leucine– leucine zipping’’ between the LZs of ion channels and partner proteins is required for assembly of these signal complexes.11,15,17 For instance, the targeting of modulator molecules to the RyR2 channel was impaired when alanine was substituted for the leucine at position d of the LZ.10 In other words, ion channels and signaling complexes form specific LZ heteromers within the macromolecular signaling complexes.20 Thus LZ domains appear to be the major binding motif mediating the interaction between intracellular proteins involved in signal transduction and ion channels. Here we describe our analysis of the 2.1 Å X-ray crystal structure of the LZ domain of the small conductance voltage-independent Ca2þ-activated Kþ channel (SKCa) from rat (Rattus norvegicus). Intriguingly the structure of the LZ domain is characterized by a parallel trimeric ahelix bundle that is incompatible with the tetrameric state of the functional SKCa channel.