Characterisation of fission yeast alp11 mutants defines three functional domains within tubulin-folding cofactor B

The proper folding of tubulins prior to their incorporation into microtubules requires a group of conserved proteins called cofactors A to E. In fission yeast, homologues of these cofactors (at least B, D and E) are necessary for the biogenesis of microtubules and for cell viability. Here we show that the temperature-sensitive alp11-924 mutant, which is defective in the cofactor B homologue, contains an opal nonsense mutation, which results in the production of a truncated Alp11^B protein (Alp11_1–118). We isolated a tRNA^Trp gene as a multicopy suppressor of this mutation, which rescues alp11-924 by read-through of the nonsense codon. The truncated Alp11_1–118 protein lacks the C-terminal half of Alp11^B, consisting of a central coiled-coil region and the distal CLIP-170 domain found in a number of proteins involved in microtubule functions. Both of these domains are required for the maintenance of microtubule architecture in vivo. Detailed functional analyses lead us to propose that Alp11^B comprises three functional domains: the N-terminal half executes the essential function, the central coiled-coil region is necessary for satisfactory maintenance of cellular α-tubulin levels, and the C-terminal CLIP-170 domain is required for efficient binding to α-tubulin. Received: 29 November 1999 / Accepted: 18 April 2000     

Cloning,Overexpression, Purification and Preliminary Characterization of Human Septin 8

Mammalian septins comprise a family of 14 genes that encode GTP-binding proteins involved in important cellular processes such as cytokinesis and exocytosis. Expression of three different constructs encoding human septin 8 were analyzed and the results show that SEPT8GC, a clone expressing the conserved domain plus C-terminal domain of human septin 8 yields the highest amount of recombinant protein. This protein was purified by affinity chromatography followed by a gel filtration chromatography. CD spectrum of SEPT8GC is characteristic of folded proteins and it presents a transition profile with a T _m of 54 °C. Fluorescence emission spectra, analytic gel filtration and DLS reflect the sample oligomeric heterogeneity with the predominance of dimers in solution. Homology models indicate clearly that the preferred dimer interface is the one comprising the GTP binding site.     

The SH3 domain of UNC-89 (obscurin) interacts with paramyosin,a coiled-coil protein,in Caenorhabditis elegans muscle

UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC-89’s SH3 domain and residues 294–376 of paramyosin and has a K_D of ∼1.1 μM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89’s SH3 is α-helical and lacks prolines. Homology modeling of UNC-89’s SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a “skip residue,” which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity.     

Comparative characterization of deletion derivatives of the modular xylanase XynA of Thermotoga maritima

The modular Xylanase XynA from Thermotoga maritima consists of five domains (A1-A2-B-C1-C2). Two similar N-terminal domains (A1-A2-) are family 22 carbohydrate-binding modules (CBMs), followed by the catalytic domain (-B-) belonging to glycoside hydrolase family 10, and the C-terminal domains (-C1-C2), which are members of family 9 of CBMs. The gradual deletion of the non-catalytic domains resulted in deletion derivatives (XynAΔC; XynAΔA1C and XynAΔNC) with increased maximum activities (V _max) at 75°C, pH 6.2. Furthermore, these deletions led to a shift of the optimal NaCl concentration for xylan hydrolysis from 0.25 (XynA) to 0.5 M (XynAΔNC). In the presence of the family 22 CBMs, the catalytic domain retained more activity in the acidic range of the pH spectrum than without these domains. In addition to the deletion derivatives of XynA, the N-terminal domains A1 and A2 were produced recombinantly, purified, and investigated in binding studies. For soluble xylan preparations, linear β-1,4-glucans and mixed-linkage β-1,3-1,4-glucans, only the A2 domain mediated binding, not the A1 domain, in accordance with previous observations. The XynA deletion enzymes lacking the C domains displayed low affinity also to hydroxyethylcellulose and carboxymethylcellulose. With insoluble oat spelt xylan and birchwood xylan as the binding substrates, the highest affinity was observed with XynAΔC and the lowest affinity with XynAΔNC. Although the domain A1 did not bind to soluble xylan preparations, the insoluble oat spelt xylan-binding data suggest that this domain does play a role in substrate binding in that it improves the binding to insoluble xylans.     

Crystal structure of a coiled-coil domain from human ROCK I

The small GTPase Rho and one of its targets, Rho-associated kinase (ROCK), participate in a variety of actin-based cellular processes including smooth muscle contraction, cell migration, and stress fiber formation. The ROCK protein consists of an N-terminal kinase domain, a central coiled-coil domain containing a Rho binding site, and a C-terminal pleckstrin homology domain. Here we present the crystal structure of a large section of the central coiled-coil domain of human ROCK I (amino acids 535-700). The structure forms a parallel α-helical coiled-coil dimer that is structurally similar to tropomyosin, an actin filament binding protein. There is an unusual discontinuity in the coiled-coil; three charged residues (E613, R617 and D620) are positioned at what is normally the hydrophobic core of coiled-coil packing. We speculate that this conserved irregularity could function as a hinge that allows ROCK to adopt its autoinhibited conformation.     

Dissection of a human septin: definition and characterization of distinct domains within human SEPT4

The septins are a conserved family of guanosine-5'-triphosphate (GTP)-binding proteins. In mammals they are involved in a variety of cellular processes, such as cytokinesis, exocytosis, and vesicle trafficking. Specifically, SEPT4 has also been shown to be expressed in both human colorectal cancer and malignant melanoma, as well as being involved in neurodegenerative disorders. However, many of the details of the modes of action of septins in general remain unclear, and little is known of their detailed molecular architecture. Here, we define explicitly and characterize the domains of human SEPT4. Regions corresponding to the N-terminal, GTPase, and C-terminal domains as well as the latter two together were successfully expressed in Escherichia coli in soluble form and purified by affinity and size-exclusion chromatographies. The purified domains were analyzed by circular dichroism spectroscopy, fluorescence spectroscopy, dynamic light scattering, and small-angle X-ray scattering, as well as with bioinformatics tools. Of the three major domains that comprise SEPT4, the N-terminal domain contains little regular secondary structure and may be intrinsically unstructured. The central GTPase domain is a mixed alpha/beta structure, probably based on an open beta sheet. As defined here, it is catalytically active and forms stable homodimers in vitro. The C-terminal domain also forms homodimers and can be divided into two regions, the second of which is alpha-helical and consistent with a coiled-coil structure. These studies should provide a useful basis for future biophysical studies of SEPT4, including the structural basis for their involvement in diseases such as cancer and neurodegenerative disorders.     

Analysis of the major domains of the F fertility inhibition protein, FinO

The mechanism of fertility inhibition of conjugation by the F plasmid depends on the presence of both the FinO protein and an antisense RNA, FinP, which together control the expression of the positive regulator of the transfer operon TraJ. FinO both prevents the degradation of FinP, allowing its intracellular concentration to rise, and promotes duplex formation with its target, the traJ mRNA. In this study, deletions in finO were constructed and fused to gst, encoded by the pGEX-2T expression vector, to give GST-FinO fusions of varying lengths. These fusions were then tested for their ability to bind FinP and traJ mRNA, and to promote duplex formation. Our results suggest that the predicted basic N-terminal α-helix is involved in RNA binding, while the central domain is involved in duplex formation. The presence of the acidic C-terminal domain protects FinP from ribonucleolase degradation and might enhance binding of the N-terminal α-helical domain in a manner reminiscent of the Rom protein of ColE1. Received: 4 May 1998 / Accepted: 15 June 1998     

Molecular characterization of the modular chitin binding protein Cbp50 from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> serovar <Emphasis Type="Italic">konkukian</Emphasis>

Bacillus thuringiensis is an insecticidal bacterium whose chitinolytic system may be exploited to improve the insecticidal system of Bt-crops. A nucleotide fragment of 1368 bp from B. thuringiensis serovar konkukian S4, containing the complete coding sequence of the chitin binding protein Cbp50, was cloned and sequenced. Analyses have shown the protein to contain a modular structure consisting of an N-terminal CBM33 domain, two copies of a fibronectin-like domain and a C-terminal chitin binding domain classified as CBM5. The Cbp50 protein was heterologously expressed in Escherichia coli, purified and assessed for chitin binding activity. A deletion mutant (CBD-N; containing only the N-terminal CBM33 domain) of Cbp50 was produced to determine the role of C-terminal domains in the binding activity of the protein. The full-length Cbp50 was shown to bind β-chitin most efficiently followed by α-chitin, colloidal chitin and cellulose. The polysaccharide binding activity of CBD-N was drastically decreased. The data demonstrate that both the N-terminal and C-terminal domains of Cbp50 are essential for the efficient binding of chitin. The purified Cbp50 showed antifungal activity against the phytopathogenic fungus Fusarium oxysporum and the opportunistic human pathogen Aspergillus niger. This is the first report of a modular chitin binding protein in bacteria.     

A critical evaluation of the conformational requirements of fusogenic peptides in membranes

It is generally assumed that fusogenic peptides would require a certain conformation, which triggers or participates in the rate-determining step of membrane fusion. Previous structure analyses of the viral fusion peptide from gp41 of HIV-1 have yielded contradictory results, showing either an α-helical or a β-stranded conformation under different conditions. To find out whether either of these conformations is relevant in the actual fusion process, we have placed sterically demanding substitutions into the fusion peptide FP23 to prevent or partially inhibit folding and self-assembly. A single substitution of either D- or L-CF₃-phenylglycine was introduced in different positions of the sequence, and the capability of these peptide analogues to fuse large unilamellar vesicles was monitored by lipid mixing and dynamic light scattering. If fusion proceeds via a β-stranded oligomer, then the D- and L-epimers are expected to differ systematically in their activity, since the D-epimers should be unable to form β-structures due to sterical hindrance. If an α-helical conformation is relevant for fusion, then the D-epimers would be slightly disfavoured compared to the L-forms, hence a small systematic difference in fusion activity should be observed. Interestingly, we find that (1) all D- and L-epimers are fusogenically active, though to different extents compared to the wild type, and – most importantly – (ii) there is no systematic preference for either the D- or L-forms. We therefore suggest that a well-structured α-helical peptide conformation or a β-stranded oligomeric assembly can be excluded as the rate-determining state. Instead, fusion appears to involve conformationally disordered peptides with a pronounced structural plasticity. Dedicated to Prof. K. Arnold on the occasion of this 65th birthday.     

A single residue mutation abolishes attachment of the CBM26 starch-binding domain from <Emphasis Type="Italic">Lactobacillus amylovorus</Emphasis> α-amylase

Starch is degraded by amylases that frequently have a modular structure composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. The C-terminal domain from the Lactobacillus amylovorus α-amylase has an unusual architecture composed of five tandem starch-binding domains (SBDs). These domains belong to family 26 in the carbohydrate-binding modules (CBM) classification. It has been reported that members of this family have only one site for starch binding, where aromatic amino acids perform the binding function. In SBDs, fold similarities are better conserved than sequences; nevertheless, it is possible to identify in CBM26 members at least two aromatic residues highly conserved. We attempt to explain polysaccharide recognition for the L. amylovorus α–amylase SBD through site-directed mutagenesis of aromatic amino acids. Three amino acids were identified as essential for binding, two tyrosines and one tryptophan. Y18L and Y20L mutations were found to decrease the SBD binding capacity, but unexpectedly, the mutation at W32L led to a total loss of affinity, either with linear or ramified substrates. The critical role of Trp 32 in substrate binding confirms the presence of just one binding site in each α-amylase SBD.     

Design and biological testing of peptidic dimerization inhibitors of human Hsp90 that target the C-terminal domain

BackgroundSmall molecules targeting the dimerization interface of the C-terminal domain of Hsp90, a validated target for cancer treatment, have yet to be identified.MethodsThree peptides were designed with the aim to inhibit the dimerization of Hsp90. Computational and biophysical methods examined the α-helical structure for the three peptides. Based on the Autodisplay technology, a novel flow cytometer dimerization assay was developed to test inhibition of Hsp90 dimerization. Microscale thermophoresis was used to determine the K_D of the peptides towards the C-terminal domain of Hsp90.ResultsMD simulations and CD spectroscopy indicated an α-helical structure for two of the three peptides. By flow cytometer analysis, IC₅₀ values of 2.08 μM for peptide H2 and 8.96 μM for peptide H3 were determined. Dimer formation of the C-terminal dimerization domain was analyzed by microscale thermophoresis, and a K_D of 1.29 nM was determined. Furthermore, microscale thermophoresis studies demonstrated a high affinity binding of H2 and H3 to the C-terminal domain, with a K_D of 1.02 μM and 1.46 μM, respectively.ConclusionsThese results revealed the first peptidic inhibitors of Hsp90 dimerization targeting the C-terminal domain. Furthermore, it has been shown that these peptides bind to the C-terminal domain with a low micromolar affinity.General significanceThese results can be used to design and screen for small molecules that inhibit the dimerization of the C-terminal domain of Hsp90, which could open a new route for cancer therapy.     

A statistical investigation of amphiphilic properties of C-terminally anchored peptidases

A number of DD-peptidases have been reported to interact with the membrane via C-terminal amphiphilic α-helices, but experimental support for this rests with a few well-characterized cases. These show the C-terminal interactions of DD-carboxypeptidases to involve high levels of membrane penetration, DD-endopeptidases to involve membrane surface binding and class C penicillin-binding proteins to involve membrane binding with intermediate properties. Here, we have characterized C-terminal α-helices from each of these peptidase groups according to their amphiphilicity, as measured by mean <μH>, and the corresponding mean hydrophobicity, <H>. Regression and statistical analyses showed these properties to exhibit parallel negative linear relationships, which resulted from the spatial ordering of α-helix amino acid residues. Taken with the results of compositional and graphical analyses, our results suggest that the use of C-terminal α-helices may be a universal feature of the membrane anchoring for each of these groups of DD-peptidases. Moreover, to accommodate differences between these mechanisms, each group of C-terminal α-helices optimizes its structural amphiphilicity and hydrophobicity to fulfil its individual membrane-anchoring function. Our results also show that each anchor type analysed requires a similar overall balance between amphiphilicity for membrane interaction, which we propose is necessary to stabilize their initial membrane associations. In addition, we present a methodology for the prediction of C-terminal α-helical anchors from the classes of DD-peptidases analysed, based on a parallel linear model.     

Structural and Thermodynamic Properties of Septin 3 Investigated by Small-Angle X-Ray Scattering

Septins comprise a family of proteins involved in a variety of cellular processes and related to several human pathologies. They are constituted by three structural domains: the N- and C-terminal domains, highly variable in length and composition, and the central domain, involved in the guanine nucleotide (GTP) binding. Thirteen different human septins are known to form heterogeneous complexes or homofilaments, which are stabilized by specific interactions between the different interfaces present in the domains. In this work, we have investigated by in-solution small-angle x-ray scattering the structural and thermodynamic properties of a human septin 3 construct, SEPT3-GC, which contains both of both interfaces (G and NC) responsible for septin-septin interactions. In order to shed light on the role of these interactions, small-angle x-ray scattering measurements were performed in a wide range of temperatures, from 2 up to 56°C, both with and without a nonhydrolysable form of GTP (GTP_γS). The acquired data show a temperature-dependent coexistence of monomers, dimers, and higher-order aggregates that were analyzed using a global fitting approach, taking into account the crystallographic structure of the recently reported SEPT3 dimer, PDB:3SOP. As a result, the enthalpy, entropy, and heat capacity variations that control the dimer-monomer dissociation equilibrium in solution were derived and GTPγS was detected to increase the enthalpic stability of the dimeric species. Moreover, a temperature increase was observed to induce dissociation of SEPT3-GC dimers into monomers just preceding their reassembling into amyloid aggregates, as revealed by the Thioflavin-T fluorescence assays.     

α-Helical coiled-coil oligomerization domains in extracellular proteins

Subunit oligomerization of many proteins is mediated by α-helical coiled-coil domains. 3,4-Hydrophobic heptad repeat sequences, the characteristic feature of the coiled-coil protein folding motif, have been found in a wide variety of gene products including cytoskeletal, nuclear, muscle, cell surface, extracellular, plasma, bacterial, and viral proteins. Whereas the majority of coiled-coil structures is represented by intracellular α-helical bundles that contain two polypeptide chains, examples of extracellular coiled-coil proteins are fewer in number. Most proteins located in the extracellular space form three-stranded α-helical assemblies. Recently, five-stranded coiled coils have been identified in thrombospondins 3 and 4 in cartilage oligomeric matrix protein, and the formation of a heterotetramer has been observed in in vitro studies with the recombinant asialoglycoprotein receptor oligomerization domain. Coiled-coil domains in laminins and probably also in tenascins and thrombospondins are responsible for the formation of tissue-specific isoforms by selective oligomerization of different polypeptide chains.     

Backbone and side chain NMR assignments for the ribosome assembly factor Nop6 from Saccharomyces cerevisiae

The Saccharomyces cerevisiae Nop6 protein is involved in the maturation of the small ribosomal subunit. It contains a central RNA binding domain and a predicted C-terminal coiled-coil domain. Here we report the almost complete (>90 %) ¹H,¹³C,¹⁵N backbone and side chain NMR assignment of a 15 kDa Nop6 construct comprising the RNA binding and coiled-coil domains.     

Proteolysis of <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Saccharomycopsis fibuligera</Emphasis>: characterization of digestion products

α-Amylase from Saccharomycopsis fibuligera R-64 was successfully purified by butyl Toyopearl hydrophobic interaction chromatography, followed by Sephadex G-25 size exclusion and DEAE Toyopearl anion exchange chromatography. The enzyme has a molecular mass of 54 kDa, as judged by SDS PAGE analysis. Upon tryptic digestion, two major fragments with relative molecular masses of 39 kDa and 10 kDa, which resemble the A/B and C-terminal domains in the homologous Taka-amylase, were obtained and were successfully separated with the Sephadex G-50 size exclusion column. The 39-kDa fragment demonstrated a similar amylolytic activity to that of the undigested enzyme. However, it was found that the K _m value of the 39-kDa fragment was about two-times higher than that of the undigested enzyme. Moreover, thermostability studies showed a lower half-life time for the 39-kDa fragment. These findings suggest that the 39-kDa fragment is the catalytic domain, while the 10-kDa fragment is the C-terminal one, which plays a role in thermostability and starch binding. Although the undigested enzyme is able to act on raw starches at room temperature, with maize starches as the best substrate, neither the undigested enzyme nor the fragments adsorb the tested raw starches. These results propose Saccharomycopsis fibuligera α-amylase as a raw starch-digesting but not adsorbing amylase, with a similar domain organization to that of Taka-amylase A.     

Troponin Regulatory Function and Dynamics Revealed by H/D Exchange-Mass Spectrometry

Muscle contraction is tightly regulated by Ca²⁺ binding to the thin filament protein troponin. The mechanism of this regulation was investigated by detailed mapping of the dynamic properties of cardiac troponin using amide hydrogen exchange-mass spectrometry. Results were obtained in the presence of either saturation or non-saturation of the regulatory Ca²⁺ binding site in the NH₂ domain of subunit TnC. Troponin was found to be highly dynamic, with 60% of amides exchanging H for D within seconds of exposure to D₂O. In contrast, portions of the TnT-TnI coiled-coil exhibited high protection from exchange, despite 6 h in D₂O. The data indicate that the most stable portion of the trimeric troponin complex is the coiled-coil. Regulatory site Ca²⁺ binding altered dynamic properties (i.e. H/D exchange protection) locally, near the binding site and in the TnI switch helix that attaches to the Ca²⁺-saturated TnC NH₂ domain. More notably, Ca²⁺ also altered the dynamic properties of other parts of troponin: the TnI inhibitory peptide region that binds to actin, the TnT-TnI coiled-coil, and the TnC COOH domain that contains the regulatory Ca²⁺ sites in many invertebrate as opposed to vertebrate troponins. Mapping of these affected regions onto the troponin highly extended structure suggests that cardiac troponin switches between alternative sets of intramolecular interactions, similar to previous intermediate resolution x-ray data of skeletal muscle troponin.     

Clustering of OB-fold domains of the partner protease complexed with trimeric stomatin from Thermococcales

The C-terminal soluble domain of stomatin operon partner protein (STOPP) of the hyperthermophilic archaeon Pyrococcus horikoshii has an oligonucleotide binding-fold (OB-fold). STOPP lacks the conserved surface residues necessary for binding to DNA/RNA. A tryptophan (W) residue is conserved instead at the molecular surface. Solvent-accessible W residues are often found at interfaces of protein–protein complexes, which suggested the possibility of self-assembling of STOPP. Protein–protein interactions among the C-terminal soluble domains of STOPP PH1510 (1510-C) were then analyzed by chemical linking and blue native polyacrylamide gel electrophoresis (BN-PAGE) methods. These results suggest that the soluble domains of STOPP could assemble into homo-oligomers. Since hexameric subcomplex I from archaeal proteasome consists of coiled-coil segments and OB-fold domains, molecular modeling of 1510-C was performed using hexameric subcomplex I as a template. Although 1510-C is a comparatively small polypeptide consisting of approximately 60 residues, numerous salt bridges and hydrophobic interactions were observed in the predicted hexamer of 1510-C, suggesting the stability of the homo-oligomeric structure. This oligomeric property of STOPP may be favorable for triplicate proteolysis of the trimer of prokaryotic stomatin.