Mun-Kyoung Kim

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.

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.

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.

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.

Crystal structure of quinolinic acid phosphoribosyltransferase from Helicobacter pylori

Introduction. The HP1355 gene from Helicobacter pylori (nadc) encodes a type II quinolinic acid phosphoribosyltransferase (Hp-QAPRTase), which is an essential enzyme in the NAD biosynthetic pathway. This enzyme catalyzes the transfer of a phosphoribosyl moiety from 5-phosphoribosyl-1-pyrophosphate (PRPP) to quinolinic acid (QA), yielding nicotinic acid mononucleotide (NAMN), pyrophosphate and CO2, the last resulting from decarboxylation at position 2 of the quinolinate ring. QA is the first intermediate in the de novo synthesis of NAD that is common to all organisms; it is produced via degradation of tryptophan in most eukaryotes, and from dihydroxyacetone phosphate and L-aspartate in prokaryotes. H. pylori is a spiralshaped bacterium that lives in the stomach and duodenum and is an important human pathogen responsible for most peptic ulcer disease, gastritis, and gastric malignancies. It is adapted to the harsh environment of the stomach and is the only bacterium strongly associated with cancer. The step catalyzed by Hp-QAPRTase is central and indispensable to NAD biosynthesis and cell survival in prokaryotes, and thus represents an attractive target for antibacterial drugs. Here we report the crystal structure of HpQAPRTase with bound QA, NAMN, and phthalic acid (PA). The structure of the Hp-QAPRTase complex was determined to a resolution of 2.4 Å using the multiwavelength anomalous dispersion (MAD) method with selenomethionine labeling. The data collection, model, and refinement statistics are summarized in Table I. The crystal shows the symmetry of space group P41212 and contains three molecules related by threefold noncrystallographic symmetry (NCS). A single subunit of Hp-QAPRTase, which has a molecular weight of 30.8 kDa, is comprised of 12 -strands and 11 -helices arranged into two structural domains, the N-terminal open-face -sandwich domain (residues 1–116, 258–273) and the C-terminal / -barrel domain (residue 117–257) [Fig. 1(A)]. The N-terminal domain contains four -helices and two antiparallel -sheets. The N-terminal 1 helix stacks on top of helices 3 and 4, which marks the start of the C-terminal / -barrel. The C-terminal domain is an open / -barrel with six -strands and eight -helices. A structural similarity search, carried out with the coordinates of Hp-QAPRTase using the DALI server, revealed that its closest structural homolog is the QAPRTase from Mycobacterium tuberculosis (Mt-QAPRTase, PDB: 1QPN). The RMSD between Hp-QAPRTase and Mt-QAPRTase was 1.30 Å over 221 aligned residues with 29% sequence identity. The structural difference arises from the 1, 2, and 7 helices and the 7– 8 loop, all of which are far from the active site. Structural studies with QAPRTase from several species, including M. tuberculosis, Salmonella typhimurium, and Thermotoga maritima showed that the active enzyme exists as a dimer. This dimerization is thought to be essential for increasing substrate specificity and for the proper functioning of the enzyme, as the two active sites are situated at the dimer interface and composed of residues from both subunits. In the crystal, the HpQAPRTase dimer is formed by a twofold crystallographic symmetry that places the N-terminal domain of one subunit next to the C-terminal domain of the other [Fig. 1(B)]. The dimer interface buries 2901 Å of protein surface, which represents approximately 20% of the total surface area of each subunit. The active sites are located at the interfaces between the / -barrel of one subunit and the -sandwich of the other. All active site residues essential for catalysis are highly conserved with respect to both sequence and structure. The QA binding site of HpQAPRTase is a solvent-accessible pocket located at the center of the / -barrel and has a highly positive electrostatic surface [Fig. 1(C)]. The pocket is composed of conserved basic residues (Arg125, Arg148, and His147), hydrophobic residues (Met156 and Met207), and one threonine residue (Thr124). The side chains of the basic residues recognize the anionic carboxylate groups of QA. The binding site and the conformation of NAMN within the structure of Hp-QAPRTase are nearly identical to those seen within the structures of Mtand St-QA-

Crystal structure of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from Thermus caldophilus

Introduction. Peptidoglycan biosynthesis is initiated with the formation of UDP-N-acetylmuramic acid via the stepwise action of two enzymes, UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and UDP-N-acetylenolpyruvylglucosamine reductase (MurB), which has proven to be attractive targets for the development of antimicrobial agents. MurA catalyzes the first stage of the reaction, transferal of the enolpyruvate moiety of phosphoenolpyruvate to the 30-hydroxyl of UDP-N-acetylglucosamine with the release of inorganic phosphate. The resulting intermediate, enolpyruvyl-UDP-N-acetylglucosamine (EP-UDPGlcNAc), then undergoes a reduction catalyzed by MurB, which utilizes one equivalent of nicotinamide adenine dinucleotide phosphate and a solvent-derived proton. The reduced product, UDP-N-acetlymuramic acid, can then serve as the locus of attachment for the peptide portion of the cell wall. The resulting pentapeptide then participates in the crosslinking that gives the cell wall its rigidity. Mur enzymes have been well-studied, and the structures of MurA-G have been solved. Moreover, the structures of MurB have been classified into two types based on the presence or absence of various secondary structural elements and on how these structural elements interact with substrates. For instance, Escherichia coli MurB is classified as Type I and contains a Tyr loop and split babb fold, whereas Staphylococcus aureus MurB is classified as Type II and lacks these secondary elements. Here, we report the crystal structures of Thermus caldophilus MurB, which is also classified as Type II. This is the first report describing the substrate bound structure of a Type II MurB.

Overexpression and Purification of the RyR1 Pore-Forming Region

Ryanodine receptor 1 (RyR1) is a large homotetrameric calcium channel that plays a pivotal role in skeletal muscle contraction. Sequence comparison and mutagenesis studies indicate that the pore architecture of RyR1, including the last two transmembrane helices and the luminal loop linking them, is similar to that of the bacterial KcsA K(+) channel. Here, we describe the overexpression and purification of the C-terminal polyhistidine-tagged RyR1 pore-forming region. The nonionic detergent lauryldimethylamine oxide (LDAO) was selected for solubilization of the protein based on its ability to extract the protein from the membrane and to maintain it in a monodisperse state. The protein was then purified using nickel-affinity chromatography and gel filtration. Gel filtration analysis confirmed that the RyR1 fragment containing the pore-forming region (amino acids 4829-5037) is sufficient to form a tetramer.

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%.

Crystallization and preliminary X-ray crystallographic analysis of the GluR0 ligand-binding core from Nostoc punctiforme.

GluR0 from Nostoc punctiforme (NpGluR0) is a bacterial homologue of the ionotropic glutamate receptor. The ligand-binding core of NpGluR0 was crystallized at 294 K using the hanging-drop vapour-diffusion method. The L-glutamate-complexed crystal belongs to space group C222(1), with unit-cell parameters a = 78.0, b = 145.1, c = 132.1 A. The crystals contain three subunits in the asymmetric unit, with a VM value of 2.49 A3 Da(-1). The diffraction limit of the L-glutamate complex data set was 2.1 A using synchrotron X-ray radiation at beamline BL-4A of the Pohang Accelerator Laboratory (Pohang, Korea).