A hydrophobic segment within the C-terminal domain is essential for both client-binding and dimer formation of the HSP90-family molecular chaperone.

The alpha isoform of human 90-kDa heat shock protein (HSP90alpha) is composed of three domains: the N-terminal (residues 1-400); middle (residues 401-615) and C-terminal (residues 621-732). The middle domain is simultaneously associated with the N- and C-terminal domains, and the interaction with the latter mediates the dimeric configuration of HSP90. Besides one in the N-terminal domain, an additional client-binding site exists in the C-terminal domain of HSP90. The aim of the present study is to elucidate the regions within the C-terminal domain responsible for the bindings to the middle domain and to a client protein, and to define the relationship between the two functions. A bacterial two-hybrid system revealed that residues 650-697 of HSP90alpha were essential for the binding to the middle domain. An almost identical region (residues 657-720) was required for the suppression of heat-induced aggregation of citrate synthase, a model client protein. Replacement of either Leu665-Leu666 or Leu671-Leu672 to Ser-Ser within the hydrophobic segment (residues 662-678) of the C-terminal domain caused the loss of bindings to both the middle domain and the client protein. The interaction between the middle and C-terminal domains was also found in human 94-kDa glucose-regulated protein. Moreover, Escherichia coli HtpG, a bacterial HSP90 homologue, formed heterodimeric complexes with HSP90alpha and the 94-kDa glucose-regulated protein through their middle-C-terminal domains. Taken together, it is concluded that the identical region including the hydrophobic segment of the C-terminal domain is essential for both the client binding and dimer formation of the HSP90-family molecular chaperone and that the dimeric configuration appears to be similar in the HSP90-family proteins.     

Domain-domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90 molecular chaperone.

In the present study, we investigated the domain structure and domain-domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90. Limited proteolysis of recombinant HtpG, revealed three major tryptic sites, i.e. Arg7-Gly8, Arg336-Glu337 and Lys552-Leu553, of which the latter two were located at the positions equivalent to the major cleavage sites of human HSP90alpha. A similar pattern was obtained by papain treatment under nondenaturing conditions but not under denaturing conditions. Thus, HtpG consists of three domains, i.e. Domain A, Met1-Arg336; domain B, Glu337-Lys552; and domain C, Leu553-Ser624, as does HSP90. The domains of HtpG were expressed and their interactions were estimated on polyacrylamide gel electrophoresis under nondenaturing conditions. As a result, two kinds of domain-domain interactions were revealed: domain B interaction with domain A of the same polypeptide and domain C of one partner with domain B of the other in the dimer. Domain B could be structurally and functionally divided into two subdomains, the N-terminal two-thirds (subdomain BI) that interacted with domain A and the C-terminal one-third (subdomain BII) that interacted with domain C. The C-terminal two-thirds of domain A, i.e. Asp116-Arg336, were sufficient for the binding to domain B. We finally propose the domain organization of an HtpG dimer.     

Calmodulin fragments can not activate target enzymes

Under conditions where nM level of calmodulin was able to show full activation of myosin light chain kinase and cyclic-nucleotide phosphodiesterase, the fragments of calmodulin at concentrations as high as 20 microM failed to activate these enzymes in the presence of Ca2+. The fragments tested were Ala1-Lys75 (F12), Ala1-Arg74 (F12'), Lys75-Lys148 (F34'), Met76-Lys148 (F34'), Asp78-Lys148 (F34), Ala1-Arg106 (F123), and His107-Lys148 (F4). Purification of the proteolytic fragments through HPLC was necessary to remove contaminant calmodulin. Among the fragments, that corresponding to the C-terminal half domain inhibited myosin light chain kinase activity with the inhibition constant of 13 microM. The integrated structure of calmodulin consisting of N-terminal half domain, C-terminal half domain, and the linker peptide was indispensable for the enzyme activation. We discuss the functions of the two structural domains (N-domain and C-domain) in the activation of various enzymes.     

Mapping DNA-binding sites of HIV-1 integrase by protein footprinting.

The HIV-1 integrase protein catalyzes integration of the viral genome into host cell DNA. Whereas the structures of the three domains of integrase have been solved separately, both the structural organization of the full-length protein and its interaction with DNA remain unresolved. A protein footprinting approach was employed to investigate the accessibility of residues in the full-length soluble integrase mutant, INF(185K,C280S), to proteolytic attack in the absence and presence of DNA. The N-terminal and C-terminal domains were relatively more accessible to proteolytic attack than the core domain. The susceptibility to proteolytic attack was specifically affected by DNA at residues Lys34, in the N-terminal domain, Lys111, Lys136, Glu138, Lys156-Lys160, Lys185-Lys188, in the core domain, and Asp207, Lys 215, Glu246, Lys258 and Lys273 in the linker and C-terminal domain, suggesting that these regions are involved in, or shielded by, DNA binding. Lys34 is positioned in a putative dimerization domain, consistent with the notion that DNA stabilizes the dimeric state of integrase.     

Lysine-50 is a likely site for anchoring the plasminogen N-terminal peptide to lysine-binding kringles.

Interactions between the kringle 4 (K4) domain of human plasminogen (Pgn) and segments of the N-terminal Glu1-Lys77 peptide (NTP) have been investigated via 1H-NMR at 500 MHz. NTP peptide stretches devoid of Lys residues but carrying an internal Arg residue show negligible affinity toward K4 (equilibrium association constant Ka < 0.05 mM(-1)). In contrast, while most fragments containing an internal Lys residue exhibit affinities comparable to that shown by the blocked Lys derivative Nalpha-acetyl-L-lysine-methyl ester (Ka approximately 0.2 mM(-1), peptides encompassing Lys50O consistently show higher Ka values. Among the investigated linear peptides, Nalpha-acetyl-Ala-Phe-Tyr-His-Ser-Ser-Lys5O-Glu-Gln-NH2 (AcAFYHSK5OEQ-NH2) exhibits the strongest interaction with K4 (Ka approximately 1.4 mM(-1)), followed by AcYHSK50EQ-NH2 (Ka approximately 0.9 mM(-1)). Relative to the wild-type sequence, mutated hexapeptides exhibit lesser affinity for K4. When a Lys50 --> Ser mutation was introduced (==> AcYHSS50EQ-NH2), binding was abolished. The Ile27-lle56 construct (L-NTP) contains the Lys50 site within a loop constrained by two cystine bridges. The propensity of recombinant Pgn K1 (rK1) and K2 (rK2) modules, and of Pgn fragments encompassing the intact K4 and K5 domains, for binding L-NTP, was investigated. We find that L-NTP interacts with rK1, rK2, K4, and K5-all lysine-binding kringles-in a fashion that closely mimics what has been observed for the Glul-HSer57 N-terminal fragment of Pgn (CB-NTP). Thus, both the constellation of kringle lysine binding site (LBS) aromatic residues that are perturbed upon complexation of L-NTP and magnitudes of kringle-L-NTP binding affinities (rK1, Ka approximately 4.3 mM(-1); rK2, Ka approximately 3.7 mM(-1; K4, Ka approximately 6.4 mM(1); and K5, Ka approximately 2.1 mM(-1)) are essentially the same as for the corresponding kringle-CB-NTP pairs. Molecular modeling studies suggest that the Glu39-Lys50 stretch in NTP generates an area that complements, both topologically and electrostatically, the solvent-exposed kringle LBS surface.     

Crystal structure of aminopeptidase N (proteobacteria alanyl aminopeptidase) from Escherichia coli and conformational change of methionine 260 involved in substrate recognition

Aminopeptidase N from Escherichia coli is a broad specificity zinc exopeptidase belonging to aminopeptidase clan MA, family M1. The structures of the ligand-free form and the enzyme-bestatin complex were determined at 1.5- and 1.6-A resolution, respectively. The enzyme is composed of four domains: an N-terminal beta-domain (Met(1)-Asp(193)), a catalytic domain (Phe(194)-Gly(444)), a middle beta-domain (Thr(445)-Trp(546)), and a C-terminal alpha-domain (Ser(547)-Ala(870)). The structure of the catalytic domain exhibits similarity to thermolysin, and a metal-binding motif (HEXXHX(18)E) is found in the domain. The zinc ion is coordinated by His(297), His(301), Glu(320), and a water molecule. The groove on the catalytic domain that contains the active site is covered by the C-terminal alpha-domain, and a large cavity is formed inside the protein. However, there exists a small hole at the center of the C-terminal alpha-domain. The N terminus of bestatin is recognized by Glu(121) and Glu(264), which are located in the N-terminal and catalytic domains, respectively. Glu(298) and Tyr(381), located near the zinc ion, are considered to be involved in peptide cleavage. A difference revealed between the ligand-free form and the enzyme-bestatin complex indicated that Met(260) functions as a cushion to accept substrates with different N-terminal residue sizes, resulting in the broad substrate specificity of this enzyme.     

Limited proteolysis of streptokinase and properties of some fragments.

Limited proteolysis of streptokinase (Sk) by trypsin and thermolysin was performed under various incubation conditions and analysed by polyacrylamide gel electrophoresis. Several fragments (Sk1, Tr27, Tr17, Th26, and Th16) were isolated and characterized further. The N-terminal sequences of Tr27, Tr17, Th26, Th16 and the C-terminal sequences of Tr27 and Th26 were determined by partial sequencing. The evidence available allows the positioning of these fragments within the Sk sequence. Fragment Sk1 is obtained by carefully standardized tryptic digestion of Sk and gel chromatography under non-denaturing conditions. Sk1 is formed by a large polypeptide Ser60-Lys293 and non-covalently bonded smaller polypeptides composed of amino acids from the N-terminal region Ile1-Lys59 of Sk. Fragment Tr27 consists of the large polypeptide Ser60-Lys293 of Sk1, and can be obtained from Sk1 by removal of the smaller N-terminal polypeptides under denaturing conditions. Fragment Th26 is composed of amino acids Phe63-His291. The N-termini of fragments Tr17 and Th16 start with Glu148 and Ile151. From their electrophoretically-determined sizes it can be concluded that they most probably have the same C-terminal amino acids, Lys293 and His291, as fragments Tr27 and Th26, respectively. Secondary structure elements of similar composition were found in all the fragments studied using circular dichroism (c.d.) and infrared (i.r.) measurements. Differential scanning calorimetric (d.s.c.) measurements were performed in order to correlate the sequence regions of Sk to energetic folding units of the protein. Fragments Sk1, Tr27, Th26, Tr17, and Th16 show one melting peak in the temperature range from 42.8 to 46.1 degrees C (thermal unfolding stage). For fragment Sk1, this melting peak can be separated by deconvolution into two transitions at T1 = 46.1 degree C and T2 = 47.3 degrees C with delta H1 = 450 kJ/mol and delta H2 = 219 kJ/mol, respectively. Fragments Tr17 and Th16 show one two-state transition at T = 42.8 degrees C with delta H = 326 kJ/mol.     

Recognition of RhoA by Clostridium botulinum C3 exoenzyme

The C3-like ADP-ribosyltransferases exhibit a very confined substrate specificity compared with other Rho-modifying bacterial toxins; they selectively modify the RhoA, -B, and -C isoforms but not other members of the Rho or Ras subfamilies. In this study, the amino acid residues involved in the RhoA substrate recognition by C3 from Clostridium botulinum are identified by applying mutational analyses of the nonsubstrate Rac. First, the minimum domain responsible for the recognition by C3 was identified as the N-terminal 90 residues. Second, the combination of the N-terminal basic amino acids ((Rho)Arg(5)-Lys(6)), the acid residues (Rho)Glu(47) and (Rho)Glu(54) only slightly increases ADP-ribosylation but fully restores the binding of the respective mutant Rac to C3. Third, the residues (Rho)Glu(40) and (Rho)Val(43) also participate in binding to C3 but they are mainly involved in the correct formation of the ternary complex between Rho, C3, and NAD(+). Thus, these six residues (Arg(5), Lys(6), Glu(40), Val(43), Glu(47), and Glu(54)) distributed over the N-terminal part of Rho are involved in the correct binding of Rho to C3. Mutant Rac harboring these residues shows a kinetic property with regard to ADP-ribosylation, which is identical with that of RhoA. Differences in the conformation of Rho given by the nucleotide occupancy have only minor effects on ADP-ribosylation.     

The molecular chaperone gp96/GRP94 interacts with Toll-like receptors and integrins via its C-terminal hydrophobic domain

The structural basis for molecular chaperones to discern misfolded proteins has long been an enigma. As the endoplasmic reticulum paralogue of the cytosolic HSP90, gp96 (GRP94, HSP90b1) is an essential molecular chaperone for Toll-like receptors (TLRs) and integrins. However, little is known about its client-binding domain (CBD). Herein, we provide genetic and biochemical evidence to definitively demonstrate that a C-terminal loop structure, formed by residues 652-678, is the critical region of CBD for both TLRs and integrins. Deletion of this region affects neither the intrinsic ATPase activity nor the overall conformation of gp96. However, without it, the chaperoning function of gp96 collapses. We also find a critical Met pair (Met(658)-Met(662)) for the folding of integrins but not TLRs. Moreover, we find that the TLR binding to gp96 is also dependent on the C-terminal dimerization domain but not the N-terminal ATP-binding pocket of gp96. Our study has unveiled surprisingly the exquisite specificity of gp96 in substrate binding and suggests a manipulation of its CBD as an alternative strategy for targeted therapy of a variety of diseases.     

Identification of domain-domain docking sites within Clostridium symbiosum pyruvate phosphate dikinase by amino acid replacement

Potential domain-domain docking residues, identified from the x-ray structure of the Clostridium symbiosum apoPPDK, were replaced by site-directed mutagenesis. The steady-state and transient kinetic properties of the mutant enzymes were determined as a way of evaluating docking efficiency. PPDK mutants, in which one of two stringently conserved docking residues located on the N-terminal domain (Arg(219) and Glu(271)) was substituted, displayed largely unimpeded catalysis of the phosphoenolpyruvate partial reaction at the C-terminal domain, but significantly impaired catalysis (>10(4)) of the ATP pyrophosphorylation of His(455) at the N-terminal domain. In contrast, alanine mutants of two potential docking residues located on the N-terminal domain (Ser(262) and Lys(149)), which are not conserved among the PPDKs, exhibited essentially normal catalytic turnover. Arg(219) and Glu(271) were thus proposed to play an important role in guiding the central domain and, hence, the catalytic His(455) into position for catalysis. Substitution of central domain residues Glu(434)/Glu(437) and Thr(453), the respective docking partners of Arg(219) and Glu(271), resulted in mutants impaired in catalysis at the ATP active site. The x-ray crystal structure of the apo-T453A PPDK mutant was determined to test for possible misalignment of residues at the N-terminal domain-central domain interface that might result from loss of the Thr(453)-Glu(271) binding interaction. With the exception of the mutation site, the structure of T453A PPDK was found to be identical to that of the wild-type enzyme. It is hypothesized that the two Glu(271) interfacial binding sites that remain in the T453A PPDK mutant, Thr(453) backbone NH and Met(452) backbone NH, are sufficient to stabilize the native conformation as observed in the crystalline state but may be less effective in populating the reactive conformation in solution.     

Liberation of the intramolecular interaction as the mechanism of heat-induced activation of HSP90 molecular chaperone.

The molecular chaperone function of HSP90 is activated under heat-stress conditions. In the present study, we investigated the role of the interactions in the heat-induced activation of HSP90 molecular chaperone. The preceding paper demonstrated two domain-domain interactions of HtpG, an Escherichia coli homologue of mammalian HSP90, i.e. an intra-molecular interaction between the N-terminal and middle domains and an intermolecular one between the middle and C-terminal domains. A bacterial two-hybrid system revealed that the two interactions also existed in human HSP90alpha. Partners of the interaction between the N-terminal and middle domains of human HSP90alpha could, but those between the middle and C-terminal domains could not, be replaced by the domains of HtpG. Thus, the interface between the N-terminal and middle domains is essentially unvaried from bacterial to human members of the HSP90-family proteins. The citrate synthase-binding activity of HtpG at an elevated temperature was solely localized in the N-terminal domain, but HSP90alpha possessed two sites in the N-terminal and other domains. The citrate-synthase-binding activity of the N-terminal domain was suppressed by the association of the middle domain. The complex between the N-terminal and middle domains is labile at elevated temperatures, but the other is stable even at 70 degrees C. Taken together, we propose the liberation of the N-terminal client-binding domain from the middle suppressor domain is involved in the temperature-dependent activation mechanism of HSP90 molecular chaperone.     

Deduced amino-acid sequence and possible catalytic residues of a novel pectate lyase from an alkaliphilic strain of Bacillus.

The nucleotide sequence of the gene for a highly alkaline, low-molecular-mass pectate lyase (Pel-15) from an alkaliphilic Bacillus isolate was determined. It harbored an open reading frame of 672 bp encoding the mature enzyme of 197 amino acids with a predicted molecular mass of 20 924 Da. The deduced amino-acid sequence of the mature enzyme showed very low homology (< 20.4% identity) to those of known pectinolytic enzymes in the large pectate lyase superfamily (the polysaccharide lyase family 1). In an integrally conserved region designated the BF domain, Pel-15 showed a high degree of identity (40.5% to 79.4%) with pectate lyases in the polysaccharide lyase family 3, such as PelA, PelB, PelC, and PelD from Fusarium solani f. sp. pisi, PelB from Erwinia carotovora ssp. carotovora, PelI from E. chrysanthemi, and PelA from a Bacillus strain. By site-directed mutagenesis of the Pel-15 gene, we replaced Lys20 in the N-terminal region, Glu38, Lys41, Glu47, Asp63, His66, Trp78, Asp80, Glu83, Asp84, Lys89, Asp106, Lys107, Asp126, Lys129, and Arg132 in the BF domain, and Arg152, Tyr174, Lys182, and Lys185 in the C-terminal region of the enzyme individually with Ala and/or other amino acids. Consequently, some carboxylate and basic residues selected from Glu38, Asp63, Glu83, Asp106, Lys107, Lys129, and Arg132 were suggested to be involved in catalysis and/or calcium binding. We constructed a chimeric enzyme composed of Ala1 to Tyr105 of Pel-15 in the N-terminal regions, Asp133 to Arg159 of FsPelB in the internal regions, and Gln133 to Tyr197 of Pel-15 in the C-terminal regions. The substituted PelB segment could also express beta-elimination activity in the chimeric molecule, confirming that Pel-15 and PelB share a similar active-site topology.     

Identification of surface-exposed components of MOMP of Chlamydia trachomatis serovar F

The identification of surface-exposed components of the major outer membrane protein (MOMP) of Chlamydia is critical for modeling its three-dimensional structure, as well as for understanding the role of MOMP in the pathogenesis of Chlamydia-related diseases. MOMP contains four variable domains (VDs). In this study, VDII and VDIV of Chlamydia trachomatis serovar F were proven to be surface-located by immuno-dot blot assay using monoclonal antibodies (MAbs). Two proteases, trypsin and endoproteinase Glu-C, were applied to digest the intact elementary body of serovar F under native conditions to reveal the surface-located amino acids. The resulting peptides were separated by SDS-PAGE and probed with MAbs against these VDs. N-terminal amino acid sequencing revealed: (1) The Glu-C cleavage sites were located within VDI (at Glu61) and VDIII (at Glu225); (2) the trypsin cleavage sites were found at Lys79 in VDI and at Lys224 in VDIII. The tryptic peptides were then isolated by HPLC and analyzed with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer and a quadrupole-orthogonal-TOF mass spectrometer coupled with a capillary liquid chromatograph. Masses and fragmentation patterns that correlated to the peptides cleaved from VDI and VDIII regions, and C-terminal peptides Ser333-Arg358 and Ser333-Lys350 were observed. This result demonstrated that these regions are surface-exposed. Data derived from comparison of nonreduced outer membrane complex proteolytic fragments with their reduced fractions revealed that Cys26, 29, 33, 116, 208, and 337 were involved in disulfide bonds, and Cys26 and 337, and 116 and 208 were paired. Based on these data, a new two-dimensional model is proposed.     

The region adjacent to the highly immunogenic site and shielded by the middle domain is responsible for self-oligomerization/client binding of the HSP90 molecular chaperone

We here investigated the mechanism of self-oligomerization of the 90-kDa heat shock protein (HSP90) molecular chaperone, because it is known that this oligomerization reflects the client-binding activity. The transition temperatures for the self-oligomerization of the full-length forms of human HSP90alpha and HtpG (bacterial HSP90), i.e., 45 and 60 degrees C, respectively, were identical to those for the dissociation of the recombinant N domain (residues 1-400 of human HSP90alpha and residues 1-336 of HtpG in our definition) from the remainder of the molecule. The N domain of human HSP90alpha expressed in Escherichia coli was oligomeric, and the oligomerization activity was localized within residues 311-350, i.e., C-terminally adjacent to the highly immunogenic site (residues 291-304). Particularly, residues 341-350 were critical on oligomerization. On the other hand, residues 289-389 were indispensable for the interaction with the M domain (residues 401-618) of the molecule. Oligomer formation of the N domain was efficiently suppressed by its extension until Lys546, i.e., residues 401-546, which is required for the interaction with the N domain. Among highly conserved amino acids at residues 289-400, Trp297, Pro379, and Phe384 were essential for the interaction with the M domain. With these observations taken together, we propose as the activation mechanism of HSP90 molecular chaperone that heat stress induces the liberation of the oligomerization/client-binding site of residues 311-350 by disrupting the intramolecular interaction between residues 289-389 and 401-546.     

Functional identification of toxin-antitoxin molecules from Helicobacter pylori 26695 and structural elucidation of the molecular interactions

Bacterial toxin-antitoxin (TA) systems are associated with many important cellular processes including antibiotic resistance and microorganism virulence. Here, we identify and structurally characterize TA molecules from the gastric pathogen, Helicobacter pylori. The HP0894 protein had been previously suggested, through our structural genomics approach, to be a putative toxin molecule. In this study, the intrinsic RNase activity and the bacterial cell growth-arresting activity of HP0894 were established. The RNA-binding surface was identified at three residue clusters: (Lys(8) and Ser(9)), (Lys(50)-Lys(54) and Glu(58)), and (Arg(80) and His(84)-Phe(88)). In particular, the -UA- and -CA- sequences in RNA were preferentially cleaved by HP0894, and residues Lys(52), Trp(53), and Ser(85)-Lys(87) were observed to be the main contributors to sequence recognition. The action of HP0894 could be inhibited by the HP0895 protein, and the HP0894-HP0895 complex formed an oligomer with a binding stoichiometry of 1:1. The N and C termini of HP0894 constituted the binding sites to HP0895. In contrast, the unstructured C-terminal region of HP0895 was responsible for binding to HP0894 and underwent a conformational change in the process. Finally, DNA binding activity was observed for both HP0895 and the HP0894-0895 complex but not for HP0894 alone. Taken together, it is concluded that the HP0894-HP0895 protein couple is a TA system in H. pylori, where HP0894 is a toxin with an RNase function, whereas HP0895 is an antitoxin functioning by binding to both the toxin and DNA.     

Variation in 16S-23S rRNA intergenic spacer regions in Photobacterium damselae: a mosaic-like structure

Phenotypically, Photobacterium damselae subsp. piscicida and P. damselae subsp. damselae are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of Photobacterium damselae subsp. piscicida and two strains of P. damselae subsp. damselae to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNA(Glu(UUC)), tRNA(Lys(UUU)), tRNA(Val(UAC)), and tRNA(Ala(GGC)). Five amplicons contained tRNA(Glu(UUC)) combined with two additional tRNA genes, including tRNA(Lys(UUU)), tRNA(Val(UAC)), or tRNA(Ala(UGC)). Five amplicons contained tRNA(Ile(GAU)) and tRNA(Ala(UGC)). Two amplicons contained tRNA(Glu(UUC)) and tRNA(Ala(UGC)). Two different isoacceptor tRNA(Ala) genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA(Glu(UUC))-tRNA(Val(UAC))-tRNA(Ala(UGC)) and tRNA(Glu(UUC))-tRNA(Ala(UGC)) have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (rrn) in the P. damselae chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure.     

Determination of β-glucosidase aggregating factor (BGAF) binding and polymerization regions on the maize β-glucosidase isozyme Glu1

β-Glucosidases (Glu1 and Glu2) in maize specifically interact with a lectin called β-glucosidase aggregating factor (BGAF). We have shown that the N-terminal (Glu⁵⁰–Val¹⁴⁵) and the C-terminal (Phe⁴⁶⁶–Ala⁵¹²) regions of maize Glu1 are involved in binding to BGAF. Sequence comparison between sorghum β-glucosidases (dhurrinases, which do not bind to BGAF) and maize β-glucosidases, and the 3D-structure of Glu1 suggested that the BGAF-binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF-binding site, we constructed additional chimeric β-glucosidases. The results showed that a region spanning 11 amino acids (Ile⁷²–Thr⁸²) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser¹–Thr²⁹, together with C-terminal region Phe⁴⁶⁶–Ala⁵¹², affects the size of Glu1–BGAF complexes. The dissociation constants (K_d) of chimeric β-glucosidase–BGAF interactions also demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile⁷²–Thr⁸² on Glu1 for BGAF binding, we constructed a chimeric sorghum β-glucosidase, Dhr2 (C-11, Dhr2 whose Val⁷²–Glu⁸² region was replaced with the Ile⁷²–Thr⁸² region of Glu1). C-11 binds to BGAF, indicating that the Ile⁷²–Thr⁸² region is indeed a major interaction site on Glu1 involved in BGAF binding.     

On the nucleation of amyloid beta-protein monomer folding

Neurotoxic assemblies of the amyloid beta-protein (Abeta) have been linked strongly to the pathogenesis of Alzheimer's disease (AD). Here, we sought to monitor the earliest step in Abeta assembly, the creation of a folding nucleus, from which oligomeric and fibrillar assemblies emanate. To do so, limited proteolysis/mass spectrometry was used to identify protease-resistant segments within monomeric Abeta(1-40) and Abeta(1-42). The results revealed a 10-residue, protease-resistant segment, Ala21-Ala30, in both peptides. Remarkably, the homologous decapeptide, Abeta(21-30), displayed identical protease resistance, making it amenable to detailed structural study using solution-state NMR. Structure calculations revealed a turn formed by residues Val24-Lys28. Three factors contribute to the stability of the turn, the intrinsic propensities of the Val-Gly-Ser-Asn and Gly-Ser-Asn-Lys sequences to form a beta-turn, long-range Coulombic interactions between Lys28 and either Glu22 or Asp23, and hydrophobic interaction between the isopropyl and butyl side chains of Val24 and Lys28, respectively. We postulate that turn formation within the Val24-Lys28 region of Abeta nucleates the intramolecular folding of Abeta monomer, and from this step, subsequent assembly proceeds. This model provides a mechanistic basis for the pathologic effects of amino acid substitutions at Glu22 and Asp23 that are linked to familial forms of AD or cerebral amyloid angiopathy. Our studies also revealed that common C-terminal peptide segments within Abeta(1-40) and Abeta(1-42) have distinct structures, an observation of relevance for understanding the strong disease association of increased Abeta(1-42) production. Our results suggest that therapeutic approaches targeting the Val24-Lys28 turn or the Abeta(1-42)-specific C-terminal fold may hold promise.     

Purification of limited tryptic fragments of Ca2+,Mg2+-adenosine triphosphatase of the sarcoplasmic reticulum and identification of conformation-sensitive cleavage sites

Sarcoplasmic reticulum membranes were treated with trypsin, and samples enriched with A1a, A1b, and C fragments (Saito, K. et al. (1984) J. Biochem. 95, 1297-1304), respectively, were prepared. A1b and C fragments were purified to apparent homogeneity, and an approximately equimolar mixture of A1(Met1-Arg198), A1a, and A1b fragments free from other contaminants was also obtained through gel permeation and hydroxylapatite chromatography in the presence of sodium dodecyl sulfate. N- and C-terminal amino acid sequence analyses of these peptides were carried out in order to identify the tryptic cleavage sites responsible for the formation of these fragments. Both A1a and A1b fragments had the same C-terminal sequence as A1 fragment. Single cleavage of A1 at T3a (Lys218-Ala219) yielded A1a, while a cleavage between either Lys234-Ile235 or Arg236-Asp237 (collectively designated as T3b) resulted in A1b fragment. Thus, A1a and A1b fragments differed from A1 fragment only by their loss of short stretches corresponding to the N-terminal region of the latter. On the other hand, C fragment represented the C-terminal half of B fragment (Ala506-Gly994). It had the same C-terminal sequence as B fragment and was produced by cleavage at T4 (Lys728-Thr729). Cleavages at T3a and T3b profoundly affected the catalytic properties of SR-ATPase (Imamura, Y. and Kawakita, M. (1986) J. Biochem. 100, 133-141), and it was suggested that the segment of the ATPase molecule including the region between Ala199 and Arg236 is important in mediating the coupling between ATP splitting and Ca2+-transport.     

Electrostatic couplings in OmpA ion-channel gating suggest a mechanism for pore opening

The molecular forces that drive structural transitions between the open and closed states of channels and transporters are not well understood. The gate of the OmpA channel is formed by the central Glu52-Arg138 salt bridge, which can open to form alternate ion pairs with Lys82 and Glu128. To gain deeper insight into the channel-opening mechanism, we measured interaction energies between the relevant side chains by double-mutant cycle analysis and correlated these with the channel activities of corresponding point mutants. The closed central salt bridge has a strong interaction energy of -5.6 kcal mol(-1), which can be broken by forming the open-state salt bridge Glu52-Lys82 (DeltaDeltaG(Inter) = -3.5 kcal mol(-1)) and a weak interaction between Arg138 and Glu128 (DeltaDeltaG(Inter) = -0.6 kcal mol(-1)). A covalent disulfide bond in place of the central salt bridge completely blocks the channel. Growth assays indicate that this gating mechanism could physiologically contribute to the osmoprotection of Escherichia coli cells from environmental stress.