Seong-Hwan Rho

Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism.

BACKGROUND The bacterial heat shock locus HslU ATPase and HslV peptidase together form an ATP-dependent HslVU protease. Bacterial HslVU is a homolog of the eukaryotic 26S proteasome. Crystallographic studies of HslVU should provide an understanding of ATP-dependent protein unfolding, translocation, and proteolysis by this and other ATP-dependent proteases. RESULTS We present a 3.0 A resolution crystal structure of HslVU with an HslU hexamer bound at one end of an HslV dodecamer. The structure shows that the central pores of the ATPase and peptidase are next to each other and aligned. The central pore of HslU consists of a GYVG motif, which is conserved among protease-associated ATPases. The binding of one HslU hexamer to one end of an HslV dodecamer in the 3.0 A resolution structure opens both HslV central pores and induces asymmetric changes in HslV. CONCLUSIONS Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.

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.

An intramolecular interaction between the FHA domain and a coiled coil negatively regulates the kinesin motor KIF1A

Motor proteins not actively involved in transporting cargoes should remain inactive at sites of cargo loading to save energy and remain available for loading. KIF1A/Unc104 is a monomeric kinesin known to dimerize into a processive motor at high protein concentrations. However, the molecular mechanisms underlying monomer stabilization and monomer‐to‐dimer transition are not well understood. Here, we report an intramolecular interaction in KIF1A between the forkhead‐associated (FHA) domain and a coiled‐coil domain (CC2) immediately following the FHA domain. Disrupting this interaction by point mutations in the FHA or CC2 domains leads to a dramatic accumulation of KIF1A in the periphery of living cultured neurons and an enhancement of the microtubule (MT) binding and self‐multimerization of KIF1A. In addition, point mutations causing rigidity in the predicted flexible hinge disrupt the intramolecular FHA–CC2 interaction and increase MT binding and peripheral accumulation of KIF1A. These results suggest that the intramolecular FHA–CC2 interaction negatively regulates KIF1A activity by inhibiting MT binding and dimerization of KIF1A, and point to a novel role of the FHA domain in the regulation of kinesin motors.

A novel activation of Ca2+-activated Cl− channel in Xenopus oocytes by Ginseng saponins: evidence for the involvement of phospholipase C and intracellular Ca2+ mobilization

The signal transduction mechanism of ginsenosides, the active ingredients of ginseng, was studied in Xenopus oocytes using two‐electrode voltage‐clamp technique. Ginseng total saponin (GTS), i.e., an unfractionated mixture of ginsenosides produced a large outward current at membrane potentials more positive than −20 mV when it was applied to the exterior of oocytes, but not when injected intracellularly. The effect of GTS was concentration‐dependent (EC50: 4.4 μg ml−1) and reversible. Certain fractionated ginsenosides (Rb1, Rb2, Rc, Rf, Rg2 and Ro) also produced an outward current in a concentration‐dependent manner with the order of potency of Rf>Ro>Rb1=Rb2>Rg2>Rc. Other ginsenosides (Rd, Re and Rg1) had little or no effect. The GTS effect was completely blocked by bath application of the Ca2+‐activated Cl− channel blocker niflumic acid and by intracellular injection of the calcium chelator BAPTA or the IP3 receptor antagonist heparin. Also, the effect was partially blocked by bath‐applied U‐73122, a phospholipase C (PLC) inhibitor and by intracellularly injected GTPγS, a non‐hydrolyzable GTP analogue. Whereas, it was not altered by pertussin toxin (PTX) pretreatment. These results indicate that: (1) interaction of ginsenosides with membrane component(s) at the extracellular side leads to Ca2+‐activated Cl− channel opening in Xenopus oocyte membrane; and (2) this process involves PLC activation, the release of Ca2+ from the IP3‐sensitive intracellular store and PTX‐insensitive G protein activation.

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.

Hydrophobic Interface between Two Regulators of K+ Conductance Domains Critical for Calcium-dependent Activation of Large Conductance Ca2+-activated K+ Channels

It has been suggested that the large conductance Ca2+-activated K+ channel contains one or more domains known as regulators of K+ conductance (RCK) in its cytosolic C terminus. Here, we show that the second RCK domain (RCK2) is functionally important and that it forms a heterodimer with RCK1 via a hydrophobic interface. Mutant channels lacking RCK2 are nonfunctional despite their tetramerization and surface expression. The hydrophobic residues that are expected to form an interface between RCK1 and RCK2, based on the crystal structure of the bacterial MthK channel, are well conserved, and the interactions of these residues were confirmed by mutant cycle analysis. The hydrophobic interaction appears to be critical for the Ca2+-dependent gating of the large conductance Ca2+-activated K+ channel.

Effects of mutation at a conserved N‐glycosylation site in the bovine retinal cyclic nucleotide‐gated ion channel

Bovine retinal cyclic nucleotide‐gated (CNG) ion channel contains an evolutionary conserved N‐glycosylation site in the external loop between the fifth transmembrane segment and the pore‐forming region. The effect of tunicamycin treatment and the site‐specific mutation suggested that the channel is glycosylated when expressed in Xenopus oocytes. To test the role of glycosylation in this channel, N‐glycosylation was abolished by mutation, and the detailed permeation and the gating characteristics of the mutant channel were investigated. The charge contribution turned out to be detectable, although the mutation of the N‐glycosylation site did not affect expression and functionality of the CNG channel in oocytes.

Engineering of protease variants exhibiting altered substrate specificity

By using an improved genetic screening system, variants of the HAV 3CP protease which exhibit altered P2 specificity were obtained. We randomly mutated the His145, Lys146, Lys147, and Leu155 residues that constitute the S2 pocket of 3CP and then isolated variants that preferred substrates with Gln over the original Thr at the P2 position using a yeast-based screening method. One of the isolated variants cleaved the Gln-containing peptide substrate more efficiently in vitro, proving the efficiency of our method in isolating engineered proteases with desired substrate selectivity.

Crystal structure of a cyanobacterial phytochrome response regulator

The two‐component signal transduction pathway widespread in prokaryotes, fungi, molds, and some plants involves an elaborate phosphorelay cascade. Rcp1 is the phosphate receiver module in a two‐component system controlling the light response of cyanobacteria Synechocystis sp. via cyanobacterial phytochrome Cph1, which recognizes Rcp1 and transfers its phosphoryl group to an aspartate residue in response to light. Here we describe the crystal structure of Rcp1 refined to a crystallographic R‐factor of 18.8% at a resolution of 1.9 Å. The structure reveals a tightly associated homodimer with monomers comprised of doubly wound five‐stranded parallel β‐sheets forming a single‐domain protein homologous with the N‐terminal activator domain of other response regulators (e.g., chemotaxis protein CheY). The three‐dimensional structure of Rcp1 appears consistent with the conserved activation mechanism of phosphate receiver proteins, although in this case, the C‐terminal half of its regulatory domain, which undergoes structural changes upon phosphorylation, contributes to the dimerization interface. The involvement of the residues undergoing phosphorylation‐induced conformational changes at the dimeric interface suggests that dimerization of Rcp1 may be regulated by phosphorylation, which could affect the interaction of Rcp1 with downstream target molecules.

Crystal structure of the apo form of D‐alanine: D‐alanine ligase (Ddl) from Thermus caldophilus: A basis for the substrate‐induced conformational changes

Introduction. D-alanine:D-alanine ligase (Ddl) catalyses the dimerization of D-alanine before its incorporation in peptidoglycan precursors. The synthesis of D-alanine:Dalanine begins with an attack on the first D-alanine by the -phosphate of adenosine triphosphate (ATP) to yield an acylphosphate. That is followed by attack by the amino group of the second D-alanine, which eliminates the phosphate and produces the D-alanine: D-alanine dipeptide. Peptidoglycan biosynthesis has long been an attractive target for antibacterial drugs, such as D-cycloserine, glycopeptide antibiotics (vancomycin and teicoplanins), and -lactams (penicillin and cephalosporins). Vancomycintype antibiotics, for example, bind directly to the D-alanine: D-alanine terminus, thereby inhibiting crosslinking by the transpeptidase. Notably, bacteria that show vancomycin resistance, which develops after prolonged clinical treatment with vancomycin, possess an inactive Ddl and rely on another ligase, D-alanine:D-lactate ligase (Van), which produces D-alanine:D-lactate rather than D-alanine:Dalanine for cell wall synthesis. The switch from D-alanine: D-alanine peptidoglycan termini to D-alanine:D-lactate results in the loss of crucial hydrogen bonding interactions that causes a 1000-fold reduction in vancomycin binding affinity. X-ray crystallographic studies of Ddl and Van have contributed significantly to our understanding of the ligand specificity these two enzymes and suggest that a His residue in Van plays a critical role. A positive charge on the side chain imidazole nitrogen of His would attract the negatively charged lactate to the second substrate binding site at pH values less than 7, but at higher pH values Van would predominantly synthesize D-alanine:D-alanine. In Ddl, a Tyr residue [Tyr216 in Escherichia coli (Eco) DdlB, Tyr232 in Thermus caldophilus (Tca) Ddl] occupies the same spatial position as the His residue, and the hydroxyl group of the Tyr interacts with the COOH-terminal of the second D-alanine substrate. The structure of Eco DdlB complexed with ADP/ phosphorylated phosphinate (PDB ID: 2DLN) or with ADP/phosphorylated phosphonate (PDB ID: 1IOV) has been determined, as have the structures of Leuconostoc mesenteroides (Lme) D-Alanine:D-Lactate ligase complexed with ADP and phosphinophosphate (PDB ID:1EHI) and Enterococcus faecium (Efa) VanA complexed with ADP and phosphinophosphate (PDB ID:1E4E). However, to analyze the reaction mechanisms of these enzymes and their associated conformational changes, it is necessary to know the structures of both the substrate-bound and substrate-free forms of these enzymes. Our aim in the present study, therefore, was to grow crystals of Ddl that diffracted to high resolution in the absence of substrates. Here we report the X-ray structure of TcaDdl resolved to a resolution of 1.9 A and describe the conformational differences of the apo structure, comparing it with the structures of the previously described transition state analogue complex.