Papain does not cleave operator-bound lambda repressor: structural characterization of the carboxy terminal domain and the hinge

The circular dichroism spectra of three different purified carboxy terminal fragments 93-236, 112-236 and 132-236 of the bacteriophage lambda cI repressor have been measured and compared with those of the intact repressor and the amino terminal fragment 1-92. All three carboxy terminal fragments contain mostly beta-strands and loops, a minor helix content increasing with the size of the fragment, showing that the 93-131 region previously called a hinge is structured. Fourier transformed infrared spectra also showed that fragment 93-236 contains alpha-helices, alpha-sheets and turns but fragment 132-236 contains no detectable alpha-helix, only beta-sheets and turns. Papain is known to cleave the lambda repressor, but it is shown here that it cannot cleave the operator-bound repressor dimer. For the 132-236 fragment, both the wt and the SN228 mutant previously shown to be dimerization defective in the intact, gave similar dimerization properties as investigated by HPLC at 2 to 100 microM protein concentration, with a KD of 13.2 microM and 19.1 microM respectively. The papain cleavage for wt and SN228 proceed at equal rates for the first cleavage at 92-93; however, the subsequent cleavages are faster for SN228. The three Cys residues in the 132-236 fragment were found to be unreactive upon incubation with DTNB, indicating the thiol sulfur atoms are buried in the repressor carboxy terminal domain. Denaturation of the 132-236 fragment studied by tryptophan fluorescence shows two transitions centered at 1.5 M and 4.5 M of urea.     

A comparative three-dimensional model of the carboxy-terminal domain of the lambda repressor and its use to build intact repressor tetramer models bound to adjacent operator sites

A model for residues 93-236 of the lambda repressor (1gfx) was predicted, based on the UmuD(') crystal structure, as part of four intact repressor molecules bound to two adjacent operator sites. The structure of region 136-230 in 1gfx was found to be nearly identical to the independently determined crystal structure of the 132-236 fragment, 1f39, released later by the PDB. Later, two more tetrameric models of the lambda repressor tetramer bound to two adjacent operator sites were constructed by us; in one of these, 1j5g, the N-domain and C-domain coordinates and hence monomer-monomer and dimer-dimer interactions are almost the same as in 1gfx, but the structure of the linker region is partly based on the linker region of the LexA dimer in 1jhe; in the other, 1lwq, the crystalline tetramer for region 140-236 has been coopted from the crystal structure deposited in 1kca, the operator DNA and N-domain coordinates of which are same as those in 1gfx and 1j5g, but the linker region is partly based on the LexA dimer structures 1jhe and 1jhh. Monomer-monomer interactions at the same operator site are stabilized by exposed hydrophobic side chains in beta-strands while cooperative interactions are mostly confined to beta(6) and some adjacent residues in both 1gfx and 1j5g. Mutational data, existence of a twofold axis relating two C-domains within a dimer, and minimization of DNA distortion between adjacent operator sites allow us to roughly position the C-domain with respect to the N-domain for both 1gfx and 1j5g. The study correlates these models with functional, biochemical, biophysical, and immunological data on the repressor in the literature. The oligomerization mode observed in the crystal structure of 132-236 may not exist in the intact repressor bound to the operator since it is shown to contradict several published biochemical data on the intact repressor.     

Conformational prediction and circular dichroism studies on the lac repressor

Using the protein predictive model of Chou & Fasman (1974b), the secondary structure of the lac repressor has been elucidated from its amino acid sequence of 347 residues. The conformation is predicted to contain 37% α-helix and 35% β-sheet for the repressor, and 29% helix and 41% β-sheet for the trypsin-resistant core (residues 60 to 327). Circular dichroism studies indicate that native lac repressor contains 40% helix and 42% β-sheet, while the core has 16% helix and 54% β-sheet, in general agreement with the predicted conformation. The sharp reduction in helicity for the trypsinized lac repressor could be due to the loss of two long helical regions, 26–45 and 328–344, predicted at both terminals. There are extensive β-sheets predicted in the 215–324 region, which may be responsible for tetrameric stabilization found in both the lac repressor and the core. Residues 17 to 33 were previously predicted by Adler et al. (1972) to be helical and were proposed to bind in the major groove of DNA. However, the present analysis shows that there are two anti-parallel β-sheet regions: 4–7 and 17–24 at the N-terminal as well as 315–318 and 321–324 at the C-terminal of the lac repressor. These β-sheet pairs may assume the twisted “polypeptide double helix” conformation (Carter & Kraut, 1974) and bind to complementary regions in the major groove of DNA. The OH groups of Tyr at the N-terminal and those of Thr and Ser side chains, in both β-sheets at the N and C-terminal ends, could form hydrogen bonds to specific sites on the lac operator. There are 23 reverse β-turns predicted that may control the tertiary folding of the lac repressor, which is essential for operator binding. The behavior of several lac repressor mutants can be satisfactorily explained in terms of polar to non-polar group replacements as well as conformational changes in light of the present predicted model.     

Regulation in repressor inactivation by RecA protein

Treatments that damage DNA or inhibit DNA synthesis in E. coli induce the expression of a set of functions called SOS functions that are involved in DNA repair, mutagenesis, arrest of cell division and prophage induction. Induction of SOS functions is triggered by inactivation of the LexA repressor or a phage repressor. Inactivation of these repressors results from their cleavage by the E. coli RecA protein in the presence of single-stranded DNA and a nucleoside triphosphate.We found that these cleavage reactions are controlled by two mechanisms in vitro: one is through the structural change of the RecA protein in the ternary complex, RecA-ssDNA-ATP-γ-S. The active ternary complex is formed by binding of ATP-γ-S to a complex of RecA protein and ssDNA. On the other hand, when the RecA protein binds to ATP-γ-S prior to its binding to ssDNA, the resulting complex has no or only very weak cleavage activity toward the repressor. This structural change is negatively controlled by its C-terminal part. The loss of the 25 amino acid residues from the C-terminal leads the RecA protein to stable binding to dsDNA as well as ssDNA, and the protein takes the activated form for the repressor cleavage constitutively. The other mechanism is through the structural change of the repressor. The cleavage reaction of a ∅80cI repressor is greatly stimulated by the presence of d(G-G), and d(G-G) stimulates the cleavage by binding to the C-terminal half of the ∅80cI repressor. Moreover, the C-terminal fragment of the cleaved products of the 80cI repressor was able to cleave a ∅80cI-λ chimeric repressor. These results strongly suggested that th active site of the repressor cleavage was located in the C-terminal domain of the repressor and that the C-terminal fragment produced by the cleavage could cleave the repressor.     

Differences in the structural stability and cooperativity between monomeric variants of natural and de novo Cro proteins revealed by high-pressure Fourier transform infrared spectroscopy

It is widely accepted that pressure affects the structure and dynamics of proteins; however, the underlying mechanism remains unresolved. Our previous studies have investigated the effects of pressure on fundamental secondary structural elements using model peptides, because these peptides represent a basis for understanding the effects of pressure on more complex structures. This study targeted monomeric variants of naturally occurring bacteriophage λ Cro (natural Cro) and de novo designed λ Cro (SN4m), which are α + β proteins. The sequence of SN4m is 75% different from that of natural Cro, but the structures are almost identical. Consequently, a comparison of the folding properties of these proteins is of interest. Pressure- and temperature-variable Fourier transform infrared spectroscopic analyses revealed that the α-helices and β-sheets of natural Cro are cooperatively and reversibly unfolded by pressure and temperature, whereas those of SN4m are not cooperatively unfolded by pressure; i.e., the α-helices of SN4m unfold at significantly higher pressures than the β-sheets and irreversibly unfold with increases in temperature. The higher unfolding pressure for the α-helices of SN4m indicates the presence of an intermediate structure of SN4m that does not retain β-sheet structure but does preserve the α-helices. These results demonstrate that the α-helices of natural Cro are stabilized by global tertiary contacts among the α-helices and the β-sheets, whereas the α-helices of SN4m are stabilized by local tertiary contacts between the α-helices.     

Evidence for an essential role of intradimer interaction in catalytic function of carnosine dipeptidase II using electrospray‐ionization mass spectrometry

Carnosine dipeptidase II (CN2/CNDP2) is an M20 family metallopeptidase that hydrolyses various dipeptides including β‐alanyl‐l ‐histidine (carnosine). Crystallographic analysis showed that CN2 monomer is composed of one catalytic and one dimerization domains, and likely to form homodimer. In this crystal, H228 residue of the dimerization domain interacts with the substrate analogue bestatin on the active site of the dimer counterpart, indicating that H228 is involved in enzymatic reaction. In the present study, the role of intradimer interaction of CN2 in its catalytic activity was investigated using electrospray‐ionization time‐of‐flight mass spectrometry (ESI‐TOF MS). First, a dimer interface mutant I319K was prepared and shown to be present as a folded monomer in solution as examined by using ESI‐TOF MS. Since the mutant was inactive, it was suggested that dimer formation is essential to its enzymatic activity. Next, we prepared H228A and D132A mutant proteins with different N‐terminal extended sequences, which enabled us to monitor dimer exchange reaction by ESI‐TOF MS. The D132A mutant is a metal ligand mutant and also inactive. But the activity was partially recovered time‐dependently when H228A and D132A mutant proteins were incubated together. In parallel, H228A/D132A heterodimer was formed as detected by ESI‐TOF MS, indicating that interaction of a catalytic center with H228 residue of the other subunit is essential to the enzymatic reaction. These results provide evidence showing that intradimer interaction of H228 with the reaction center of the dimer counterpart is essential to the enzymatic activity of CN2.     

Monte Carlo study of the formation and conformational properties of dimers of Aβ42 variants

Small soluble oligomers, and dimers in particular, of the amyloid β-peptide (Aβ) are believed to play an important pathological role in Alzheimer's disease. Here, we investigate the spontaneous dimerization of Aβ42, with 42 residues, by implicit solvent all-atom Monte Carlo simulations, for the wild-type peptide and the mutants F20E, E22G and E22G/I31E. The observed dimers of these variants share many overall conformational characteristics but differ in several aspects at a detailed level. In all four cases, the most common type of secondary structure is intramolecular antiparallel β-sheets. Parallel, in-register β-sheet structure, as in models for Aβ fibrils, is rare. The primary force driving the formation of dimers is hydrophobic attraction. The conformational differences that we do see involve turns centered in the 20-30 region. The probability of finding turns centered in the 25-30 region, where there is a loop in Aβ fibrils, is found to increase upon dimerization and to correlate with experimentally measured rates of fibril formation for the different Aβ42 variants. Our findings hint at reorganization of this part of the molecule as a potentially critical step in Aβ aggregation.     

NMR study of the reconstitution of the β-sheet of thioredoxin by fragment complementation

The study of complementary protein fragments is thought to be generally useful to identify early folding intermediates. A prerequisite for these studies is the reconstitution of the native-like structure by fragment complementation. Structural analysis of the complementation of the domain-sized proteolytic fragments of E. coli thioredoxin, using a combination of H-exchange and 2D NMR experiments as a fingerprint technique, provide evidence for the extensive reconstitution of a native β-sheet, with local conformational adjustments near the cleavage site. Remarkably, the antiparallel β-strand between the fragments shows a native-like protection of the amide protons to solvent exchange. Our results indicate that these fragments can be useful to study the early events in the still little understood formation of β-sheets. © 1995 Wiley-Liss, Inc.     

Isolation of a Raw Starch-Binding Fragment from Barley α-Amylase

Barley α-amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with β-cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel β-sheets in domain C and the α₇-and α₈-helices of the (α/β)₈ domain.     

Carboxy terminal fragment of human alpha-1-antitrypsin from hydroxylamine cleavage: homology with antithrombin III.

Human α-1-antitrypsin (AT) was reacted with hydroxylamine at pH 9.0 giving cleavage at an Asn-Gly bond. A fragment of molecular weight 8,500 was released and this was isolated and sequenced. The fragment had the same carboxy terminal amino acid sequence as intact AT. The 80 residue polypeptide contained the Z variant mutation site and a portion of sequence identical to that found by others for the reactive site, inferring the presence in AT of two active sites. This sequence combined with prviously published work gives a continuous sequence of 152 amino acid residues from the carboxy terminal end of the AT molecule, including the mutation site of the S variant. The sequence shows strong homology with human antithrombin III.     

Structure of the membrane protein of influenza virus. I. Isolation and characterization of cyanogen bromide cleavage products.

After cleavage of the membrane (M) protein of influenza A/WSN virus by using cyanogen bromide (CNBr), six peptide peaks representing approximate molecular weights of 6,000, 4,000, 2,200, 1,600, 1,200, and 1,000 were resolved by gel filtration on BioGel P6. Analysis by thin-layer chromatography indicates that the first, second, fourth, and fifth peaks contain single-peptide components, whereas the third and sixth peaks contain more than one peptide. By using Whatman CM52 ion-exchange chromatography in 5 M urea, four peptides were resolved from the third BioGel P6 peak. The amino acid composition of each of the purified peptides has been determined, and partial sequences were obtained for several peptides. Based on finding a blocked amino terminal residue, the 6,000-dalton fragment appears to contain the amino terminus of the M protein, whereas the carboxy terminal peptide was identified as a 2,000-dalton peptide.     

Cathepsin B Is Secreted Apically from Xenopus 2F3 Cells and Cleaves the Epithelial Sodium Channel (ENaC) to Increase Its Activity

The epithelial sodium channel (ENaC) plays an important role in regulating sodium balance, extracellular volume, and blood pressure. Evidence suggests the α and γ subunits of ENaC are cleaved during assembly before they are inserted into the apical membranes of epithelial cells, and maximal activity of ENaC depends on cleavage of the extracellular loops of α and γ subunits. Here, we report that Xenopus 2F3 cells apically express the cysteine protease cathepsin B, as indicated by two-dimensional gel electrophoresis and mass spectrometry analysis. Recombinant GST ENaC α, β, and γ subunit fusion proteins were expressed in Escherichia coli and then purified and recovered from bacterial inclusion bodies. In vitro cleavage studies revealed the full-length ENaC α subunit fusion protein was cleaved by active cathepsin B but not the full-length β or γ subunit fusion proteins. Both single channel patch clamp studies and short circuit current experiments show ENaC activity decreases with the application of a cathepsin B inhibitor directly onto the apical side of 2F3 cells. We suggest a role for the proteolytic cleavage of ENaC by cathepsin B, and we suggest two possible mechanisms by which cathepsin B could regulate ENaC. Cathepsin B may cleave ENaC extracellularly after being secreted or intracellularly, while ENaC is present in the Golgi or in recycling endosomes.     

The adenomatous polyposis coli protein and retinoblastoma protein are cleaved early in apoptosis and are potential substrates for caspases

Apoptosis in human monocytic THP.1 tumour cells, induced by diverse stimuli, was accompanied by proteolytic cleavage of the adenomatous polyposis coli gene product (APC) and by sequential cleavage of the retinoblastoma susceptibility gene product (Rb). Cleavage of poly(ADP-ribose) polymerase (PARP), APC and the initial cleavage of Rb at the carboxy terminal region all occurred at a similar time, early in the apoptotic process. Subsequently, Rb underwent a secondary cleavage to 43 kDa and 30 kDa protein fragments. Two caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK) and acetyl-Tyr-Val-Ala-Asp chloromethyl ketone (YVAD.CMK), had markedly different effects on the induction of apoptosis. Z-VAD.FMK inhibited the primary and secondary cleavage of Rb, cleavage of APC and PARP, and apoptosis assessed by flow cytometry. In marked contrast, YVAD.CMK inhibited cleavage of APC and the secondary cleavage of Rb to the 43 kDa and 30 kDa protein fragments but did not inhibit the primary carboxy terminal cleavage of Rb, PARP proteolysis or apoptosis assessed by flow cytometry. These results suggest that different caspases are responsible for the cleavage of different substrates at different stages during the apoptotic process and that a caspase may either cleave APC directly or may be involved in the pathway leading to APC proteolysis. This is the first report suggesting that a cytoplasmic tumour suppressor gene (APC) may be cleaved by a caspase during apoptosis.     

Lambda repressor inactivation: properties of purified ind- proteins in the autodigestion and RecA-mediated cleavage reactions

Under physiological conditions, lambda repressor can be inactivated in vivo or in vitro by RecA-mediated cleavage of the polypeptide chain. The repressor protein is thought to cleave itself, with RecA acting to stimulate autodigestion. ind- repressor mutants are resistant to RecA-mediated inactivation in vivo. In this paper, we report the purification of 15 ind- repressor proteins and the behaviors of these proteins in the RecA-mediated and autodigestion cleavage reactions. None of these proteins undergoes substantial RecA-dependent cleavage. However, eight mutant proteins autodigest at the same rate as wild-type repressor, six mutants do not autodigest or autodigest slower, and one mutant autodigests faster than wild-type. We discuss these results with respect to repressor structure and RecA-binding, and suggest possible roles for the RecA protein in the cleavage reaction.     

Epitope mapping of a monoclonal antibody directed against the α‐subunit of phosphofructokinase‐1 from Saccharomyces cerevisiae by screening phage display libraries

Phosphofructokinase‐1 from Saccharomyces cerevisiae is composed of two types of subunits, α and β. Subunit‐specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, α‐F3, binds to an epitope either at the C‐terminal or at the N‐terminal part of the α‐polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D–A–F and D–S–F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody α‐F3 to native phosphofructokinase‐1. The peptide G–I–K–D–A–F–L inhibited the binding more strongly (IC₅₀ = 1.5 µM) than the peptide A–P–W–H–D–S–F (IC₅₀ = 33.3 µM). Sequence matching revealed the presence of the D–A–F motif in the polypeptide chain of phosphofructokinase‐1 at amino acid position 172–174. As a control, the nonapeptide A–P–T–S–K–D–A–F–L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC₅₀ = 0.3 µM) it was concluded that this nonapeptide represents the continuous epitope of phosphofructokinase‐1 that is recognized by antibody α‐F3. Copyright © 1999 John Wiley & Sons, Ltd.     

Caspase 7 can cleave tumor necrosis factor receptor-I (p60) at a non-consensus motif, in vitro.

Ligand binding to tumor necrosis factor receptor-I (TNFRI) can promote cell survival or activate the apoptotic caspase cascade. Cytoplasmic interaction of TNFRI with TRAF2 and RIP allows for the activation of JNK and NFkappaB pathways. Alternatively, a carboxy terminal death domain protein interaction motif can recruit TRADD, which then recruits FADD/MORT1, and finally procaspase 8. Aggregation of these components form a death inducing signaling complex, leading to the cleavage and activation of caspase 8. We have found that during apoptosis human TNFRI protein is lost in a caspase-dependent manner. The cytoplasmic tail of human TNFRI was found to be susceptible to caspase cleavage but not by caspase 8. Instead, the downstream executioner caspase 7 was the only caspase capable of cleaving TNFRI, in vitro. Identification and characterization of the cleavage site revealed a derivative of the classic EXD motif that incorporates a glutamate (E) in the P1 position. Using several criteria to establish that caspase activity was responsible for cleavage at this site, we confirmed that caspase 7 can cleave at a GELE motif. Mutation of the cleavage site prevented the apoptosis-associated cleavage of TNFRI. This ability of caspase 7 to cleave at a non-EXD or -DXXD motif suggests that the specificity of caspases may be broader than is currently held.     

Clathrin light chain: importance of the conserved carboxy terminal domain to function in living cells

Clathrin triskelions assemble into coats capable of packaging membrane and receptors for transport to intracellular destinations. A triskelion is formed from three heavy chains bound to three light chains. All clathrin light chains (clc) contain an acidic amino terminal domain, a central coiled segment, and a carboxy terminal domain conserved in amino acid sequence. To assess their functional contribution in vivo, we expressed tagged segments of the Dictyostelium clcA in clc-minus Dictyostelium (clc null) cells. We examined the ability of these clcA fragments to rescue clathrin phenotypic deficiencies, to cluster into punctae on membranes, and to bind to the heavy chain. When expressed in clc null cells, a clcA fragment containing the amino terminal domain and the central coiled domain bound heavy chain but was dispensable for clathrin function. Instead, the carboxy terminal domain of clcA was a critical determinant for association with punctae, for clathrin function and for robust binding to the heavy chain. A 70 amino acid carboxy terminal fragment was necessary and sufficient for full function, and for localization into punctae on intracellular membranes. A shorter 49 amino acid carboxy terminal fragment could distribute into punctae but failed to rescue developmental deficiencies. These results reveal the importance of the carboxy terminal domain of the light chain in vivo.     

Molecular dynamics simulation of the nanofibrils formed by amyloid-based peptide amphiphiles

Abstract

Atomistic molecular dynamics simulations have been performed on the peptide amphiphiles (PAs) with four amyloid beta peptide fragments as head groups. The stable structures were monitored by the root mean square deviation with respect to the energy minimised initial structures. Random coil and β-sheet structures with hydrogen bonds along and perpendicular to the long axis of the nanofibre were obtained due to the different nature of the head groups. Influences of pH and capping ends on the nanofibre structures were investigated through variation of the protonation states of the ionic amino acids in the peptides. The peptides with opposite charges on both sides were found to have the fewest β-sheet structures, and the charges on the outer terminal tended to destruct the β-sheets while those at the inner side did not. The isolated charge in the centre of peptides was found to be able to promote the formation of regular β-sheets, while multiple charged residues could not support ordered β-sheet structures. When charge neutralisation occurred between adjacent residues, regular β-sheet laminates might also occur for systems with charges at the outer terminal. With the increase of β-sheet structures formed, the original twisted structures found for random coil structures of the PAs could be diminished by the hydrogen bonds.     

小鼠热休克蛋白gp96羧基端片段的克隆、表达及纯化

热休克蛋白(Heat shock protein)gp96(Grp94)是近年来新发现的一类糖蛋白,除了分子伴侣的功能外,现有越来越多的献报道了它在先天性免疫和获得性免疫中的重要作用。gp96可以促进抗原呈递细胞的成熟以及细胞因子的分泌。热休克蛋白抗原肽复合体可以引起特异性的细胞毒T淋巴细胞效应,应用这个特点可以设计抗病毒及抗肿瘤药物。但是gp96全长分子量大,蛋白在大肠杆菌中表达量低,不稳定,难纯化。组织提取的gp96又受组织来源和样品量的限制。对gp96的结构和功能的研究带来困难。克隆并表达了小鼠热休克蛋白gp96的羧基端560．751aa约四分之一长的功能片段,该段包含gp96的一个肽结合区和二聚化位点。将该功能片段在大肠杆菌中进行融合表达,纯化后将融合的片段切掉,并对目的片段进行了分析,结果表明该段可能是形成二聚体密切相关的片段,为进一步研究其结构和功能打下基础。