Due to Its multiple biological activities, coumarin (a main ingredient of Cinnamon extracts) has gained attention as potentially useful therapeutics for various diseases. However, the efficacy of coumarin for the use of dermatological health has not been fully explored. To clarify the action mechanism of the skin protecting property of coumarin, we firstly investigated the molecular docking property of coumarin on the mammalian adenylyl cyclase, which is the key enzyme of cAMP-induced melanogenesis in the skin cells. In binding study, the benzopyran moiety of coumarin occupies dual sites of the hydrophobic cleft at the interface of two subunits of adenylyl cyclase. We also examined the involvement of coumarin in alpha-MSH and forskolin induced cAMP signaling within a cell based assay. In addition, we inquired into the inhibitory effect of coumarin on melanogenesis and found that the pretreatment with coumarin inhibited the forskolin-induced melanin contents significantly without annihilating the cell viability. Our results strongly suggest that coumarin directly inhibits the activity of adenylyl cyclase, downregulates forskolin-induced cAMP-production pathway, consequently inhibiting melanogenesis. Thus, coumarin may also be used as an effective inhibitor of hyperpigmentation.

Due to its multiple biological activities, 5,7-dihydroxyflavone (chrysin) in propolis has gained attention as potentially useful therapeutics for various diseases. However, the efficacy of chrysin for the use of dermatological health has not been fully explored. To clarify the action mechanism of the skin protecting property of chrysin, we firstly investigated the molecular docking property of chrysin on the mammalian adenylyl cyclase, which is the key enzyme of cAMP-induced melanogenesis. We also examined the involvement of chrysin in alpha-MSH and forskolin-induced cAMP signaling within a cell based assay. In addition, we inquired into the inhibitory effect of chrysin on melanogenesis and found that the pretreatment with chrysin inhibited the forskolin-induced melanin contents significantly without annihilating the cell viability. These results strongly suggest that chrysin directly inhibits the activity of adenylyl cyclase, downregulates forskolin-induced cAMP-production pathway, consequently inhibiting melanogenesis. Thus, chrysin may also be used as an effective inhibitor of hyperpigmentation.

Background: Polyglutamine (polyQ)-induced protein aggregation is the hallmark of a group of neurodegenerative diseases, including Huntington's disease. We hypothesized that a protease that could cleave polyQ stretches would intervene in the initial events leading to pathogenesis in these diseases. To prove this concept, we aimed to generate a protease possessing substrate specificity for polyQ stretches.Methodology/Principal Findings: Hepatitis A virus (HAV) 3C protease (3CP) was subjected to engineering using a yeast-based method known as the Genetic Assay for Site-specific Proteolysis (GASP). Analysis of the substrate specificity revealed that 3CP can cleave substrates containing glutamine at positions P5, P4, P3, P1, P2', or P3', but not substrates containing glutamine at the P2 or P1' positions. To accommodate glutamine at P2 and P1', key residues comprising the active sites of the S2 or S1' pockets were separately randomized and screened. The resulting sets of variants were combined by shuffling and further subjected to two rounds of randomization and screening using a substrate containing glutamines from positions P5 through P3'. One of the selected variants (Var26) reduced the expression level and aggregation of a huntingtin exon1-GFP fusion protein containing a pathogenic polyQ stretch (HttEx1(97Q)-GFP) in the neuroblastoma cell line SH-SY5Y. Var26 also prevented cell death and caspase 3 activation induced by HttEx1(97Q)-GFP. These protective effects of Var26 were proteolytic activity-dependent.Conclusions/Significance: These data provide a proof-of-concept that proteolytic cleavage of polyQ stretches could be an effective modality for the treatment of polyQ diseases.

Exploring biological systems from highly complex datasets is an important task for systems biology. The present study examined co-expression dynamics of mouse heart transcriptome by spectral graph clustering (SGC) to identify a heart transcriptomic network. SGC of microarray data produced 17 classified biological conditions (called condition spectrum, CS) and co-expression patterns by generating bi-clusters. The results showed dynamic co-expression patterns with a modular structure enriched in heart-related CS (CS-1 and -13) containing abundant heart-related microarray data. Consequently, a mouse heart transcriptomic network was constructed by clique analysis from the gene clusters exclusively present in the heart-related CS; 31 cliques were used for constructing the network. The participating genes in the network were closely associated with important cardiac functions ( e. g. , development, lipid and glycogen metabolisms). Online Mendelian Inheritance in Man (OMIM) database indicates that mutations of the genes in the network induced serious heart diseases. Many of the tested genes in the network showed significantly altered gene expression in an animal model of hypertrophy. The results suggest that the present approach is critical for constructing a heart-related transcriptomic network and for deducing important genes involved in the pathogenesis of various heart diseases.

Proteins: Structure, Function, and BioinformaticsVolume 71, Issue 2 p. 1020-1026 Structure NoteFree Access Crystal structure of Bacillus subtilis CodW, a noncanonical HslV-like peptidase with an impaired catalytic apparatus Seong-Hwan Rho, Seong-Hwan Rho Department of Life Science, Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea Seong-Hwan Rho and Hyun Ho Park contributed equally to this work.Search for more papers by this authorHyun Ho Park, Hyun Ho Park Department of Life Science, Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea Seong-Hwan Rho and Hyun Ho Park contributed equally to this work.Search for more papers by this authorGil Bu Kang, Gil Bu Kang Department of Life Science, Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, KoreaSearch for more papers by this authorYoung Jun Im, Young Jun Im Department of Life Science, Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, KoreaSearch for more papers by this authorMin Suk Kang, Min Suk Kang School of Biological Sciences, Seoul National University, Seoul 151-742, KoreaSearch for more papers by this authorByung Kook Lim, Byung Kook Lim School of Biological Sciences, Seoul National University, Seoul 151-742, KoreaSearch for more papers by this authorIhn Sik Seong, Ihn Sik Seong School of Biological Sciences, Seoul National University, Seoul 151-742, KoreaSearch for more papers by this authorJaehong Seol, Jaehong Seol School of Biological Sciences, Seoul National University, Seoul 151-742, KoreaSearch for more papers by this authorChin Ha Chung, Chin Ha Chung School of Biological Sciences, Seoul National University, Seoul 151-742, KoreaSearch for more papers by this authorJimin Wang, Jimin Wang Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520Search for more papers by this authorSoo Hyun Eom, Corresponding Author Soo Hyun Eom eom@gist.ac.kr Department of Life Science, Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, KoreaDepartment of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea===Search for more papers by this author Seong-Hwan Rho, Seong-Hwan Rho Department of Life Science, Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea Seong-Hwan Rho and Hyun Ho Park contributed equally to this work.Search for more papers by this authorHyun Ho Park, Hyun Ho Park Department of Life Science, Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea Seong-Hwan Rho and Hyun Ho Park contributed equally to this work.Search for more papers by this authorGil Bu Kang, Gil Bu Kang Department of Life Science, Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, KoreaSearch for more papers by this authorYoung Jun Im, Young Jun Im Department of Life Science, Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, KoreaSearch for more papers by this authorMin Suk Kang, Min Suk Kang School of Biological Sciences, Seoul National University, Seoul 151-742, KoreaSearch for more papers by this authorByung Kook Lim, Byung Kook Lim School of Biological Sciences, Seoul National University, Seoul 151-742, KoreaSearch for more papers by this authorIhn Sik Seong, Ihn Sik Seong School of Biological Sciences, Seoul National University, Seoul 151-742, KoreaSearch for more papers by this authorJaehong Seol, Jaehong Seol School of Biological Sciences, Seoul National University, Seoul 151-742, KoreaSearch for more papers by this authorChin Ha Chung, Chin Ha Chung School of Biological Sciences, Seoul National University, Seoul 151-742, KoreaSearch for more papers by this authorJimin Wang, Jimin Wang Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520Search for more papers by this authorSoo Hyun Eom, Corresponding Author Soo Hyun Eom eom@gist.ac.kr Department of Life Science, Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, KoreaDepartment of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea===Search for more papers by this author First published: 02 November 2007 https://doi.org/10.1002/prot.21758Citations: 5 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat INTRODUCTION ATP-dependent proteases play vital roles in protein quality control and in regulating the levels of certain cellular proteins.1-3 Escherichia coli and other bacteria, including Bacillus subtilis, contain at least three types of multimeric ATP-dependent proteases homologous to the eukaryotic 26S proteasome: Lon, Clp and HslVU.1, 2, 4, 5 Of these, HslVU has been extensively studied as the simplest proteasome ancestor.6-10 Like the 26S proteasome, HslVU is comprised of two multimeric components: the ATPase HslU, which belongs to the AAA superfamily of ATPases,11 and the peptidase HslV, which shares common structural features with the catalytic β-type subunits of the 20S proteasome. Despite a sequence identity of only about 20%, they share the amino acids crucial for proteolysis and auto-cleavage [Fig. 1(A)]. Moreover, both use a threonine residue at the N-terminus as a catalytic nucleophile exposed by the processing of a methionine residue or a prosegment upon assembly, and are thus members of the N-terminal nucleophile (Ntn)-hydrolase family.14-16 Figure 1Open in figure viewerPowerPoint Crystal structure of CodW. A: Partial sequence alignment of the precursors of 20S proteasome core particle-related proteins accomplished using the program CLUSTALX.12 The residues considered to be crucial for proteolysis are indicated (▴): Thr-1, Asp-17 and Lys-33 in E. coli HslV; Thr-6, Asp-22 and Lys-39 in CodW.13 A downward arrow (↓) indicates the position where autolysis occurs. The N-terminal pro-segments preceding the cleavage site are highly divergent in their lengths and sequences. The aligned sequences are as follows: CodW from B. subtilis (labeled as CodW); HslVs from E.coli, H. influenzae and T. maritima (HslV_Ec, HslV_Hi and HslV_Tm, respectively); 20S poteasome β-subunit from T. acidophilum (PTB_Ta); 20S proteasome β1, β2, β5 and β7 from S. cerevisae (PTB1_Sc, PTB2_Sc, PTB5_Sc and PTB7_Sc, respectively); 20S proteasome βX, βY and βZ form H. sapiens (PTBX_Hs, PTBY_Hs and PTBZ_Hs, respectively). B: Side view of one dodecamer generated by applying a crystallographic twofold operation perpendicular to the cylindrical axis of the half-dodecamer (Mol A) in the asymmetric unit. One protomer and its symmetry mate are colored green. C: View of the half-dodecamer (W6) looking parallel to the molecular sixfold axis. An unbiased composite omit map contoured at 1σ (blue mesh) shows the ordered N-terminal pro-segment in each protomer. The N-terminus is located at the interface between two protomers (red asterisk). D: Close-up view of the pro-segment. The number in parenthesis indicates the position of the residue relative to Thr-6. CodW is the proteolytic component of CodWX, which is highly homologous to HslVU17 and has sequence identities of 52 and 55% with E. coli and H. influenzae HslV, respectively. A previous electron microscopic analysis revealed that CodW and HslV have similar molecular architectures with strict sixfold symmetry and similar dimensions (11 nm in diameter and 10 nm in height).18 In addition, both can form a hybrid protease capable of degrading SulA, suggesting CodWX and HslVU are close relatives.15 Nonetheless, recent biochemical studies suggest that CodW is distinct from HslV in several respects.15, 18, 19 First, although CodW is synthesized as a precursor with additional residues at its N-terminus, just like proteasomal β and other HslV proteases, unlike those others CodW is incapable of autolysis before the nucleophile Thr-6 upon assembly.15 It may be, therefore, that CodW utilizes its N-terminal serine as a catalytic nucleophile. Second, CodWX is an alkaline protease that is maximally active at pH 9.5, unlike HslUV which has a maximal activity at pH 8. Third, CodW by itself has no peptidase activity, even towards small peptides; it requires hydrolysis of ATP by CodX, which in turn leads to formation of a CodWX complex, for digestion of peptide and protein substrates. By contrast, uncomplexed HslV does show weak peptidase activity toward small peptides, and the binding of ATP is sufficient for the formation of HslVU, though ATP hydrolysis is required for the digestion of a protein substrate. This dissimilarity in the behaviors of CodW and HslV is extraordinary, since all of the catalytically important residues in HslV are well conserved in CodW. One possibility is that the observed disparities reflect sequence differences between HslV and CodW away from the conserved active site. If so, comparison of the structures of CodW and HslV should clarify the structural basis of the unique functional properties of CodW. Currently, a structure of CodW in complex with E. coli HslU is available.20 This, however, diffracted poorly due to lattice translocation defects in the crystal and lacks the information necessary for comparison with other HslV structures. Here, we describe the crystal structures of Bacillus subtilis CodW determined in two different space groups, P21 and C2, at pH 5.5 and 7.5, respectively. In both structures, the five extra N-terminal residues remain intact. This lack of autolysis can be explained by the impaired geometry of the canonical catalytic apparatus. MATERIALS AND METHODS Protein purification and crystallization The protein was expressed and purified as described previously.15 CodW protein concentrated to 5 mg/mL was crystallized at 293 K using the sitting drop vapor diffusion method. P21 crystals were obtained using a well solution of 55% (v/v) MPD, 0.1M sodium citrate (pH 5.5), and 8% glycerol. C2 crystals were obtained using a well solution of 30% (v/v) MPD and 0.1M HEPES-NaOH (pH 7.5) with 0.2M sodium citrate as a salt additive. X-ray diffraction data were collected on beam line 6B of the Pohang Accelerator Laboratory (PAL) in Korea. Taking advantage of the high concentration of MPD, crystals were directly frozen and cryo-cooled in a liquid nitrogen stream at 110 K. Data were processed using MOSFILM and SCALA software.21 Diffraction data were collected to a maximum resolution of 2.5 Å for C2 crystals and 3.0 Å for P21 crystals. Data collection, structure determination, and refinement Crystallographic computations were mostly carried out using the CCP421 and CNS22 program packages, and model building was performed using O.23 Molecular replacement searches using the P21 data were carried out to obtain the initial phases. The search model used was a dodecamer generated from the 1.9 Å structure of H. influenzae free HslV (PDB # 1G3K).24 After 12-fold noncrystallographic symmetry (NCS) averaging and prime-and-switch phasing using RESOLVE,25 it was evident that the Ser-1 to Ala-5 prosegment was intact in the structure. An improved electron density map around Ser-1 was computed from the C2 crystal data, which diffracted to a higher resolution of 2.5 Å. Combinations of protomers from the dodecameric structure determined using the P21 data set were used as search models. As a result, one half-dodecamer with a sixfold axis parallel to the c-axis was detected using a hexameric ring model (W6). The other half-dodecamer molecule (W3W3) parallel to the b-axis was then manually positioned by examining the 2Fo − Fc Fourier map computed using the calculated phases from the first W6 molecule. The two half-dodecameric CodW molecules in the asymmetric unit and the crystallographic dyad operation yielded the biological unit. Structural refinements with NCS restraints using CNS were conducted until convergence was obtained (Rcryst = 0.255, Rfree = 0.298). A large difference in isotropic B factors was observed between the two half-dodecameric molecules: 50 Å2 for W6 and 100 Å2 for W3W3. Because this implies the existence of large crystal anisotropy around a sixfold axis coincident with the crystallographic twofold-axis, 12 TLS groups assigned to each monomer were refined using REFMAC.26 As a result, the displacement parameters, which differed significantly for the two half-dodecamers, were well accounted for by the TLS parameterization, leaving residual local displacements that were very similar between protomers and to which NCS restraints could be applied. The TLS refinement gave improvements in crystallographic R and free R factors up to 3% (Rcryst = 0.226, Rfree = 0.265), and the resultant density map was also improved across the entire asymmetric unit. Solvent molecules including a Na+ ion at the conserved cation-binding site in each CodW protomer became apparent during the later stages of refinement and were added to the model. After the structural refinement was completed using C2 data at a resolution of 2.5 Å, the refined structure was transported back to the P21 data at a lower resolution of 3.0 Å and further refined until no further reduction in Rfree was observed (Rcryst = 0.245, Rfree = 0.266). The data collection and refinement statistics are summarized in Table 1. Table 1. Data Collection and Model Refinement Statistics Crystal data Native form I, pH 7.5 Native form II, pH 5.5 Data collection Space group C2 P21 Unit cell dimensions a (Å) 185.1 78.1 b (Å) 106.8 164.4 c (Å) 152.7 95.9 β (°) 112.0 111.5 Resolution range (last shell) (Å) 30.0–2.5 (2.54–2.50) 44.7–3.0 (3.16–3.00) No. of observations 280,849 169,982 No. of unique reflections 90,904 44,182 Completeness (%) 95.5 (88.5) 98.1 (97.6) Average I/σ(I) 17.2 (2.8) 7.1 (2.3) Rsymaa Rsym = Σ|Ihkl − |/Σ, where Ihkl is the scaled intensity of the hkl reflection, and is the mean value of the hkl reflection. (last shell) 0.055 (0.345) 0.089 (0.322) Refinement Rcrystbb Rcryst = Σ|Fobs − Fcalc|/ΣFobs>, where Fobs and Fcalc are the observed and calculated structure factor amplitudes of the hkl reflection. 0.226 0.245 Rfreecc Rfree was calculated in the same manner as Rcryst, but using the test set of reflections. 0.265 0.266 No. of reflections (working set) 86,324 39,712 No. of reflections (test set) 4576 4438 No. of protein atoms 16,571 16,308 No. of solvent/hetero atoms 251 106 rmsd bond lengths (Å) 0.017 0.011 rmsd bond angles (°) 1.5 1.4 a Rsym = Σ|Ihkl − |/Σ, where Ihkl is the scaled intensity of the hkl reflection, and is the mean value of the hkl reflection. b Rcryst = Σ|Fobs − Fcalc|/ΣFobs>, where Fobs and Fcalc are the observed and calculated structure factor amplitudes of the hkl reflection. c Rfree was calculated in the same manner as Rcryst, but using the test set of reflections. Validation and deposition The main chain angles, calculated using PROCHECK,27 showed that all residues fell into the most favored or additionally favored region of the Ramachandran plot. The atomic coordinates and structure factors of the C2 and P21 crystals have been deposited with the PDB codes 2Z3B and 2Z3A, respectively. Figures were produced using PyMOL.28 RESULTS AND DISCUSSION The structure of CodW was determined in two different space groups, P21 and C2, at pH 5.5 and 7.5, respectively. The C2 structure was refined to 2.5 Å and the P21 structure to 3.0 Å. Both structures had good geometry and nearly identical quaternary arrangements: the root-mean-square deviation for the main chain atom pairs was 0.67 Å for subunit comparisons and 0.91 Å for dodecamer comparisons, with the pore-lining residues 91–93 exhibiting the largest deviations, which were greater than 1 Å. The overall architecture of the CodW molecule is a double-doughnut-shaped dodecamer very similar to that of HslV, as was expected from the high degree of sequence conservation [Fig. 1(B,C)]. The rms deviation was 1.2 Å for protomer comparisons and 1.9 Å for dodecamer comparisons between CodW and H. influenzae HslV (PDB #1G4A). The protomers share the typical four-layered fold exhibited by HslV and all 20S proteosome subunits; two sheets of antiparallel β-strands are sandwiched between two layers of α-helices. Each protomer includes all 180 residues and a structural Na+ ion at the conserved cation-binding site formed by three main chain carbonyl oxygen atoms from residues 164, 167, and 170 [Fig. 2(A)]. The mean distance between the metal ion and oxygen atoms in our structure was 2.43 Å, which is nearly identical to the value observed in the T. maritima HslV structure.10, 30 Figure 2Open in figure viewerPowerPoint Structural basis for CodW's lack of autolysis. A: Superposition of monomeric CodW (yellow) with the NLVS-complex (grey) and the free form (cyan) of H. influenza HslV (PDB #1OFI). The ordered portion of the covalently bound inhibitor 4-iodo-3-nitrophenyl-acetyl-leucinyl-leucinyl-leucinyl-vinylsulfone (NLVS) to HslV is shown as grey lines. The red sphere indicates a sodium ion bound at the conserved cation-binding site consisting of three carbonyl groups and an adjacent water molecule. B: Superposition of the conserved active site residues in mature proteasome β/HslV-like proteases. The active site residues from the free form (PDB #1OFH), the HslU-bound form (PDB #1OFH) of H. influenzae HslV, the free T. maritime HslV (PDB #1M4Y) and the β2-subunit of the yeast 20S proteasome (PDB #1RYP) are shown. The residues are numbered as in H. influenzae. Thr-1 and Thr-2 are colored in orange for comparison. C: The active site of the T1A mutant of the β1-subunit of the yeast 20S proteasome, which lacks autolysis, is shown as balls-and-sticks (PDB #1RYP). The modeled side chain of Thr-1 is drawn in green lines overlapping the side chain of Ala-1. The Leu-(-2) to Thr-1 segment shows a typical γ-turn, which ensures the addition of Oγ to Gly-(-1)-C.29 The S1 substrate binding pocket is indicated in grey. D: The impaired geometry of the canonical catalytic apparatus in CodW is evident. The composite omit map shows a clear density consistent with an N-terminal prosegment containing Ser-1, Ser-2, Phe-3, His-4, and Ala-5 [Fig. 1(C,D)]. The backbone atoms of the Ser-2 to His-4 segment form antiparallel β-sheet hydrogen bonds with strand β3. The prosegment appears to be intact in every protomer in the asymmetric unit, unlike in the quintuple mutant of the β6-subunit of yeast 20S proteasome or T. maritima HslV, which was found to be partially processed.31 In CodW, the average main chain B-factor of the pro-segment (45Å2) was lower than that of the whole chain (51 Å2). Moreover, the CodW protein was eluted in fractions with a size of ∼240 kDa on gel filtration chromatography, which corresponds to the size of the dodecamer, and was detected as a single band on SDS-PAGE.15 The prosegment occupies the cleft corresponding to the substrate-binding site in HslV and the catalytic proteasomal β-subunits [Fig. 2(A)]. CodW has all the conserved residues known to be crucial for proteolysis, including Thr-6, Asp-22, Lys-39, Ser-130, and Gly-131 [Fig. 2(D)]. Within the 20S proteasome, every β-subunit contains these conserved residues and is processed through autolysis, which leads to activation.13, 31, 32 Indeed, the mechanism of autolysis is highly correlated with that of proteolysis. In this context, CodW's lack of autolysis is unusual, since at the sequence level CodW appears to meet all the requirements for proteolysis. On the other hand, a comparison of the geometries of the conserved active sites in CodW and the 20S proteasome/HslV-like proteases provides a clue as to why CodW does not self-cleave at the CN bond between Ala-5 and Thr-6. In yeast proteasomal β-subunits, the catalytic apparatus includes Thr-1, Asp-17, and Lys-33 (Thr-1, Asp-17, and Lys-33 in H. influenzae HslV). Although this is analogous to the catalytic triad seen in serine proteases, it differs in that the N-terminal amino group and a catalytic water molecule act as a proton acceptor in proteolysis and autolysis, respectively.8, 14, 31 Superposition of the active site residues in HslV and those in the mature yeast proteasomal β2-subunit shows that the geometry of the active site is maintained precisely [Fig. 2(B)]. Even in H. influenzae HslV, whose activation processing by HslU is accompanied by a conformational change around the substrate binding cleft,33 the positions of the active site residues are almost invariant during activation. The conserved hydrogen-bonding pattern between Thr-1 and the Ser-125/Gly-126 segment appears to serve as a stabilizing factor for Thr-1 at this position, since the turn segment containing the conserved Ser and Gly residues (Ser-130 and Gly-131 in CodW) is the most rigid part of the structure with the lowest average B-factors. In that context, the crystal structures reveal that CodW has an impaired catalytic apparatus. Most importantly, Thr-6 is shifted by ∼3 Å from its expected position, which displaces the side chain of Lys-39 into an outwardly oriented position [Fig. 2(D)]. The same type of impaired geometry of the catalytic apparatus also was seen in the quintuple reactivation mutant of the yeast proteasomal β6-subunit,31, 34 where deviation of Thr-1 from its usual position in the active subunit prevents the Lys-33-Nζ and a catalytic water molecule from staying in the vicinity of Thr-1. Interestingly, this mutant was still capable of partial autolysis, perhaps because the active site residues could spontaneously recover the functional geometry. By contrast, CodW exhibits no autolytic activity, implying the impaired catalytic apparatus of CodW is not spontaneously recoverable. In their description of the structure of the T1A mutant of the yeast proteasomal β1-subunit, Ditzel et al. suggested that the formation of a γ-turn by the Leu-(-2) to Thr-1 segment is one of the major conformational constraints on self-cleavage.29 In their model, Thr-1-Oγ would be centrally positioned over the three-residue γ-turn and attack the carbonyl carbon of Gly-(-1) [Fig. 2(C)], which means that the right conformation of the prosegment with respect to Thr-1-Oγ is also essential for autolysis. In the structure of the proteasomal β1-subunit T1A mutant, the S1 specificity pocket accommodates the hydrophobic side chain of Leu-(-2) and helps to form a bulge at the Leu-(-2) to Thr-1 segment. The corresponding pocket within the structure of CodW is occupied by Lys-39, which is displaced from the active site by Thr-6 [Fig. 2(D)]. Consequently, His-4 at position -2 is displaced from the pocket and stacked against Tyr-169. Apparently, the dislocation of His-4 brings about the extension of the prosegment and prevents the formation of a γ-turn at the His-4 to Thr-6 segment. It is noteworthy that the β6-subunit reactivation mutant shows blurred electron density at residues Asn-(-2) and Gly-(-1), which implies an inherent conformational flexibility in this region. We suggest that autolysis would take place in the reactivation β6-subunit mutant whenever the conformation of the Asn-(-2) to Thr-1 segment meets the structural constraints for autolysis, that is, when it forms a γ-turn. However, the crystal structure of CodW shows neither a γ-turn nor a sign of conformational flexibility at the His-4(-2) to Thr-6 segment [Fig. 2(D)]. Therefore, the spontaneous formation of a functional canonical active site via autolysis must overcome a much higher energy barrier in CodW. Several lines of biochemical evidence imply that Ser-1 is the N-terminal catalytic nucleophile in CodW.15, 18 In the present study, however, the unambiguous density denoting Ser-1-Oγ is located at the cleft formed between two protomers and is close to the entrance of the proteolytic chamber, but it shows no relevant interaction with neighboring residues. This may be attributable to the low pH of the crystallization buffer. In fact, CodWX becomes inactive at pH 5.5. Otherwise, a significant conformational rearrangement around the active site would be inevitable upon activation by CodX. The interiors of the catalytic chambers of the proteolytic components of HslV, ClpP, and the 20S proteasome are hydrophobic and favor denatured polypeptide chains (data not shown).35 By contrast, the catalytic chamber of CodW is strongly basic, but it is apparent that the chamber will become less polar at pH 9.5, where CodW has its maximal activity. The entrance to the proteolytic chamber of CodW is formed by a hydrogen-bonded turn and a short 310-helix and contains a cluster of conserved positively charged residues, Lys-92, Arg-95, and Lys-96 (Arg-86, Arg-89, and Arg-90 in E. coli HslV), whose functions are not yet clear. The average diameter of the pore, which measures 20.5 Å between Cα atoms, is narrow enough to prevent folded proteins from reaching the proteolytic core. The average diameter of the pore is nearly same in the E. coli, H. influenzae and T. maritime enzymes, but it changes slightly upon HslU binding.24, 36 It has been suggested that this "gating" motion is an allosteric activation/inactivation mechanism of HslV by HslU and is analogous the activation mechanism in yeast proteasomes. However, the conformational change at the pore entrance reportedly has no relation to the activation of HslV, and thus its function remains to be discovered.37 Although the pore leading the proteolytic core of CodW remains open in the P21 structure, in the C2 structure it is blocked by the presence of a poly-anion interacting with Lys-92 in each protomer [Fig. 3(A,B)]. Though presumed to be a citrate ion, the identity of the poly-anion remains unidentifiable because of the rotational averaging of sixfold symmetry. It is also unclear how many of the Lys-92 residues in the six protomers have an extended conformation to simultaneously coordinate the anion. It is noteworthy that this type of interaction has not been seen in any other HslV structure. Figure 3Open in figure viewerPowerPoint Obstruction of the pore leading to the proteolytic core by Lys-92. A: A 2Fo − Fc map contoured at 0.8σ within 2 Å from the pore-lining residues is overlaid on the structure (grey mesh). A Fo − Fc map contoured at 3σ shows an unidentified poly-anion from either the co-purification or crystallization buffer at the sixfold symmetry axial pore (blue mesh). B: Disordered side chains of the pore-lining residues in the P21 crystal. In HslUV/proteasome-like proteases, unfolding and translocation of the polypeptide are mediated by the ATPase subunit, and the unfolded chain is transported to the protease subunit in a vectorial fashion.38 A denatured polypeptide chain traveling from the outlet of the ATPase subunit to the active site in the proteolytic chamber can be regarded as a random flight chain with a free end. If some structural elements were to bias the random walk of the polypeptide chain, the efficiency of the translocation of the substrate to the active site cleft would be greatly improved. In fact, the possible existence of a ratcheting mechanism directing substrates to the proteolytic chamber has been anticipated.39 The antechambers of the 20S proteasome, which each have a volume of ∼59 nm3, may provide an example, as they must be able to maintain the polypeptide in an unfolded form as it passes through them. Prokaryotic HslV proteins do not have these antechambers. Instead, they have a flexible insertion containing a cluster of conserved positively charged residues. Moreover, the removal of a positive charge or the introduction of a negative charge at Arg-86 of E. coli HslV inactivates the enzyme,40 which implies that this residue interacts with the substrate polypeptide and that its electrostatic nature is important in the translocation of the substrate. Interestingly, the Δ86–91 mutant was found to be functionally indistinguishable from wild-type enzyme,40 which means the pore-lining segment likely facilitates substrate translocation in a passive manner. In CodW, the Lys-92 residues in some of the six protomers likely constitute a binding site for the C-terminal carboxylate of the substrate. The study presented here revealed the intact structure of the N-terminal prosegment of CodW and showed that its lack of autolysis can be attributable to the impaired geometry of its catalytic apparatus. However, the structures failed to clarify the catalytic mechanism of CodW. Although Ser-1 was previously proposed to be an N-terminalnucleophile, the local structure around it does not provide all the required features of the active site of a protease. Furthermore, restoration of the canonical active site and subsequent autolysis will require input of additional energy to overcome the conformational constraints observed in the current structures. The activation of CodW thus appears to be accompanied by a conformational change either in the canonical catalytic apparatus or Ser-1. It therefore seems plausible that the binding of CodX is critical to the activation of CodW. In summary, the formation of a functionally

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.

The cardiac system has been a major target for intensive studies in the multi-scale modeling field for many years. Reproduction of the action potential and the ionic currents of single cardiomyocytes, as well as the construction of a whole organ model is well established. Still, there are major hurdles to overcome in creating a realistic and predictive functional cardiac model due to the lack of a profound understanding of the complex molecular interactions and their outcomes controlling both normal and pathological cardiophysiology. The recent advent of systems biology offers the conceptual and practical frameworks to tackle such biological complexities. This review provides an overview of major themes in the developing field of cardiac systems biology, summarizing some of the high-throughput experiments and strategies used to integrate the datasets, and various types of computational approaches used for developing useful quantitative models capable of predicting complex biological behavior.

Dual roles of calsequestrin (CSQ-1) being the Ca2+ donor and Ca2+ acceptor make it an excellent Ca2+-buffering protein within the sarcoplasmic reticulum (SR). We have isolated and characterized a calsequestrin (csq-1)-null mutant in Caenorhabditis elegans. To our surprise, this mutant csq-1(jh109) showed no gross defects in muscle development or function but, however, is highly sensitive to perturbation of Ca2+ homeostasis. By taking advantage of the viable null mutant, we investigated the domains of CSQ-1 that are important for polymerization and cellular localization, and required for its correct buffering functions. In transgenic animals rescued with various CSQ-1 constructs, the in vivo patterns of polymerization and localization of several mutated calsequestrins were observed to correlate with the structure-function relationship. Our results suggest that polymerization of CSQ-1 is essential but not sufficient for correct cellular localization and function of CSQ-1. In addition, direct interaction between CSQ-1 and the ryanodine receptor (RyR) was found for the first time, suggesting that the cellular localization of CSQ-1 in C. elegans is indeed modulated by RyR through a physical interaction.

Calcium-dependent gating of the large-conductance Ca2+-activated K+ (BKCa) channel is conferred by the large cytosolic carboxyl terminus containing two domains of the regulator of K+ conductance (RCK) and the high-affinity Ca2+-binding site (the Ca2+-bowl). In our previous study, we located the putative second RCK domain (RCK2) and demonstrated that it interacts directly with RCK1 via a hydrophobic “assembly interface”. In this study, we tested the structural model of the other interface, the “flexible interface”, by strategically positioning charge pairs across the putative interface. Several charge mutations on RCK2 affected the voltage-dependent activation of the channel. In particular, the Gly-to-Asp substitution at position 803 profoundly affected channel activation by stabilizing the open conformation of the channel with minimal effects on its Ca2+ affinity and voltage sensitivity. Various mutations at Gly-803 shifted the channel's conductance-voltage curve either left or right over a 145-mV range. Since this residue is predicted to be in the first loop of RCK2 these results strongly suggest that this loop plays a critical role in determining the intrinsic equilibrium constant for channel opening, and they support the hypothesis that this loop is part of an interface that mediates conformational coupling between RCK1 and RCK2.

Coxsackievirus B3 (CVB3) 3C protease (3CP) plays essential roles in the viral replication cycle, and therefore, provides an attractive therapeutic target for treatment of human diseases caused by CVB3 infection. CVB3 3CP and human rhinovirus (HRV) 3CP have a high degree of amino acid sequence similarity. Comparative modeling of these two 3CPs revealed one prominent distinction; an Asn residue delineating the S2′ pocket in HRV 3CP is replaced by a Tyr residue in CVB3 3CP. AG7088, a potent inhibitor of HRV 3CP, was modified by substitution of the ethyl group at the P2′ position with various hydrophobic aromatic rings that are predicted to interact preferentially with the Tyr residue in the S2′ pocket of CVB3 3CP. The resulting derivatives showed dramatically increased inhibitory activities against CVB3 3CP. In addition, one of the derivatives effectively inhibited the CVB3 proliferation in vitro.

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.

SUMMARY:The Heart and Calcium functional Network (HCNet) database is a collection of functional gene modules calculated from the microarray data compendium available from the GEO database. It is a specialized database designed to assist experimentalists for cardiac calcium signaling research by providing the pre-calculated gene clusters and their potential correlation network in heart. In the current release of HCNet, 57 functional modules from 786 target genes obtained by a bi-clustering analysis of 381 microarray datasets are available. Detailed information of the clusters such as expression profiles, network diagrams is provided in two categories, heart-specific genes and heart-specific genes along with calcium toolkit genes. Overrepresented gene ontological categories and transcription factors in each cluster are also provided to infer the biological implications of the detected functional modules. AVAILABILITY:HCNet is available at http://sbrg2.gist.ac.kr/hcnet.

It has been suggested that the large conductance Ca(2)+-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 Ca(2+)-dependent gating of the large conductance Ca(2+)-activated K(+) channel.

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.

Engineering of secreted protease variants exhibiting altered substrate specificity is a challenging task because effective screening methods for the desired property are not available yet. In this study, we sought to obtain variants of Kex2, a yeast Golgi protease, which exhibit altered P2 specificity. We first randomly mutated three Asp residues (D176, D210, and D211) that constitute the S2 pocket of Kex2 and then isolated from the resulting library Kex2 variants that preferred substrates with Met (poorly preferred by wild type Kex2) at the P2 position using a yeast-based screening method. The Kex2 variants isolated from this initial screening were further tested against various substrate sequences. Four out of the 16 isolated Kex2 variants showed greater preference for Met than for Lys (preferred by the wild-type Kex2) at the P2 position. We therefore suggest that our method might serve as an efficient tool for engineering and directing the evolution of secreted proteases.

Quinolinic acid phosphoribosyltransferase (NadC; EC 2.4.2.19) is the key enzyme of NAD + biosynthesis in both prokaryotes and eukaryotes. NadC catalyzes the decarboxylation of quinolinic acid (QA) to produce nicotinic acid mononucleotide (NAMN), an intermediate in NAD synthesis. NadCs of Helicobacter pylori appeared to be a hexamer during the purification procedure. Three different complexes of NadC, with QA, NAMN and phthalic acid (PA), an analogue of QA, were crystallized at 294 ± 1 K using the hanging-drop vapour-diffusion method. The QA complex crystal was found to belong to space group P 4 1 2 1 2, with unit-cell parameters a  =  b  = 148.8, c = 145.7 Å, α = β = γ = 90°. Diffraction data were collected from the NadC–substrate and NadC–substrate analogue complexes to resolutions of 2.3 Å (QA), 2.8 Å (PA) and 3.3 Å (NAMN) using synchrotron X-ray radiation.

Cytochrome c oxidase, a terminal respiratory enzyme complex, accepts electrons from cytochrome c and reduces dioxygens to waters coupling with pumping protons across the membrane.X-ray structures of bovine heart enzyme at the fully oxidized, reduced and several ligand-binding states at 280 K have been determined (1-4).The enzyme consists of two copies of 13 different subunits.The transmembrane part of each monomer consists of 28 αhelices, including metal centers of hemes a and a3 and CuB at the same level in the membrane.Another metal center, CuA, is located in the intermembrane part.Possible electron and chemical proton transfer pathways, as well as a proton pumping mechanism, have been proposed (3).The crystals diffracting X-rays up to 1.65 Å were recently obtained by using ethyleneglycole as a cryoprotectant.X-ray diffraction data were collected on the image plate of DIP2040 (MAC SCIENCE) at BL44XU of the SPring-8.Structures were determined by the molecular replacement method.Now, we have 1.8 Å oxidized and 1.9 Å reduced structures at 100 K, as well as 2.3 Å oxidized and 2.35 Å reduced enzyme structures at 280 K. Several novel water arrangements functioning in proton translocation were detected by inspecting these structures at both 100 K and 280 K

Relatively little is known about the signaling mechanism of ginseng saponins (ginsenosides), active ingredients of ginseng, in non-neuronal cells. Here, we describe that ginsenosides utilize a common pathway of receptor-mediated signaling pathway in Xenopus oocytes: increase in intracellular concentration via phospholipase C (PLC) and mobilization. Ginsenosides induced a marked and robust artivation of -activated Cl- channels in Xenopus oocytes. The effect of ginsenosides was completely reversible, in a dose-dependent manner with EC of 4.4 /mi, and specifically blocked by niflumic acid, an inhibitor of -activated Cl- channel. Intracellular injection of BAPIA abolished the effect of ginsenosides. Intracellular injection of GTPS also abolished the effect of ginsenosides. The effect of gin senosides on -activated Cl- currents was greatly reduced by the intracellular injection of heparin, an IP receptorantagonist or the pretreatment of PLC inhibitor. These results indicate that ginsenosides activate endogenous -activated Cl- channels via the activation of PLC and the release of from the IP-sensitive intracellular store following the initial interaction with membrane component(s) from extracellular side. This signaling pathway of ginsenosides may be one of the action mechanisms for the pharmacological effects of ginseng.ts of ginseng.