Sedimentation Equilibrium Experiments Research Articles

Investigation of the Dimer Interface and Substrate Specificity of Prolyl Dipeptidase DPP8

DPP8 belongs to the family of prolyl dipeptidases, which are capable of cleaving the peptide bond after a penultimate proline residue. Unlike DPP-IV, a drug target for type II diabetes, no information is available on the crystal structure of DPP8, the regulation of its enzymatic activity, or its substrate specificity. In this study, using analytical ultracentrifugation and native gel electrophoresis, we show that the DPP8 protein is predominantly dimeric when purified or in the cell extracts. Four conserved residues in the C-terminal loop of DPP8 (Phe(822), Val(833), Tyr(844), and His(859)), corresponding to those located at the dimer interface of DPP-IV, were individually mutated to Ala. Surprisingly, unlike DPP-IV, these single-site mutations abolished the enzymatic activity of DPP8 without disrupting its quaternary structure, indicating that dimerization itself is not sufficient for the optimal enzymatic activity of DPP8. Moreover, these mutations not only decreased k(cat), as did the corresponding DPP-IV mutations, but also dramatically increased K(m). We further show that the K(m) effect is independent of the substrate assayed. Finally, we identified the distinctive and strict substrate selectivity of DPP8 for hydrophobic or basic residues at the P2 site, which is in sharp contrast to the much less discriminative substrate specificity of DPP-IV. Our study has identified the residues absolutely required for the optimal activity of DPP8 and its unique substrate specificity. This study extends the functional importance of the C-terminal loop to the whole family of prolyl dipeptidases.

Reversible self-association of recombinant bovine factor B

The recombinant bovine factor B, obtained by a newly developed bacterial expression system, was found to exhibit features characteristic of a reversible self-associating system. Using size-sieving chromatography, distribution of the factor B species ranged from a monomer to a trimer, but not oligomers of higher molecular weights. At high protein concentrations, factor B migrated as a single band in a native gel. Cross-linking with the amino-reactive cross-linking reagent bis (sulfosuccinimidyl) suberate (BS), at a low cross-linker to protein ratio yielded cross-linked products identified as factor B dimer and trimer. The cross-linking pattern was shown to be a function of the protein and cross-linker concentrations. The range of sedimentation coefficients in a sedimentation velocity experiment suggested that the largest particle present in the distribution was more than twice as large as the smallest. The data obtained under multiple conditions in the sedimentation equilibrium experiments are best fit to a model describing a reversible self-association of a monomer–trimer of factor B species, with a dissociation constant Kd 1,3 = 2.48 × 10 − 10 M 2.

The Galactose Switch in Kluyveromyces lactis Depends on Nuclear Competition between Gal4 and Gal1 for Gal80 Binding

The Gal4 protein represents a universally functional transcription activator, which in yeast is regulated by protein-protein interaction of its transcription activation domain with the inhibitor Gal80. Gal80 inhibition is relieved via galactose-mediated Gal80-Gal1-Gal3 interaction. The Gal4-Gal80-Gal1/3 regulatory module is conserved between Saccharomyces cerevisiae and Kluyveromyces lactis. Here we demonstrate that K. lactis Gal80 (KlGal80) is a nuclear protein independent of the Gal4 activity status, whereas KlGal1 is detected throughout the entire cell, which implies that KlGal80 and KlGal1 interact in the nucleus. Consistently KlGal1 accumulates in the nucleus upon KlGAL80 overexpression. Furthermore, we show that the KlGal80-KlGal1 interaction blocks the galactokinase activity of KlGal1 and is incompatible with KlGal80-KlGal4-AD interaction. Thus, we propose that dissociation of KlGal80 from the AD forms the basis of KlGal4 activation in K. lactis. Quantitation of the dissociation constants for the KlGal80 complexes gives a much lower affinity for KlGal1 as compared with Gal4. Mathematical modeling shows that with these affinities a switch based on competition between Gal1 and Gal4 for Gal80 binding is nevertheless efficient provided two monomeric Gal1 molecules interact with dimeric Gal80. Consistent with such a mechanism, analysis of the sedimentation behavior by analytical ultracentrifugation demonstrates the formation of a heterotetrameric KlGal80-KlGal1 complex of 2:2 stoichiometry.

The Periplasmic Domains of Escherichia coli HflKC Oligomerize through Right-Handed Coiled-Coil Interactions

The periplasmic domains of the Escherichia coli HflK and HflC were coexpressed and purified. The two polypeptides copurified in a 1:1 ratio, as determined by quantitative amino acid analysis. Circular dichroism studies showed the complex to have substantial helical/coiled-coil content that melted with midpoints in the range of 26-29 degrees C depending upon the concentration, implying a reversible oligomerization. The average molecular weight of the soluble HflKC determined by sedimentation equilibrium ultracentrifugation using a single-species model varied with rotor speed, providing further evidence of concentration-dependent oligomerization. The data were well-fit by models that specified a protomer to n-mer oligomerization, with the heterodimeric HflKC as the protomer and values of n between 7 and 10. Multiple-sequence alignments of both HflK and HflC revealed regions near the C termini to contain 11-residue hendecad repeats, indicative of right-handed coiled coils, with characteristic small residues in the a, f, h, and j positions. To test the importance of the small size of these positions, two residues in the HflC domain, Ala-262 in a f position and Gly-268 in an a position, were mutated to isoleucine. The HflKC:A262I mutant complex showed lower helicity than the wild type, and its melting was less concentration-dependent. During purification of HflKC:G268I, the mutated HflC subunit precipitated, leaving a preparation of the pure peripheral HflK domain. This polypeptide behaved as a monomer in sedimentation equilibrium experiments and showed low helicity, implying that the protein conformation is largely dependent upon heteromeric subunit interactions. These results demonstrate the importance of right-handed coiled-coil interactions in the oligomerization of HflKC, and a model entailing the formation of a right-handed helical barrel is proposed.

Properties of the C-terminal Domain of Enzyme I of the Escherichia coli Phosphotransferase System

The bacterial phosphoenolpyruvate (PEP):glycose phosphotransferase system (PTS) mediates uptake/phosphorylation of sugars. The transport of all PTS sugars requires Enzyme I (EI) and a phosphocarrier histidine protein of the PTS (HPr). The PTS is stringently regulated, and a potential mechanism is the monomer/dimer transition of EI, because only the dimer accepts the phosphoryl group from PEP. EI monomer consists of two major domains, at the N and C termini (EI-N and EI-C, respectively). EI-N accepts the phosphoryl group from phospho-HPr but not PEP. However, it is phosphorylated by PEP(Mg(2+)) when complemented with EI-C. Here we report that the phosphotransfer rate increases approximately 25-fold when HPr is added to a mixture of EI-N, EI-C, and PEP(Mg(2+)). A model to explain this effect is offered. Sedimentation equilibrium results show that the association constant for dimerization of EI-C monomers is 260-fold greater than the K(a) for native EI. The ligands have no detectable effect on the secondary structure of the dimer (far UV CD) but have profound effects on the tertiary structure as determined by near UV CD spectroscopy, thermal denaturation, sedimentation equilibrium and velocity, and intrinsic fluorescence of the 2 Trp residues. The binding of PEP requires Mg(2+). For example, there is no effect of PEP on the T(m), an increase of 7 degrees C in the presence of Mg(2+), and approximately 14 degrees C when both are present. Interestingly, the dissociation constants for each of the ligands from EI-C are approximately the same as the kinetic (K(m)) constants for the ligands in the complete PTS sugar phosphorylation assays.

Molecular Dissection of Rab11 Binding from Coiled-Coil Formation in the Rab11-FIP2 C-Terminal Domain

The Rab11-family interacting protein (Rab11-FIP) group of effector proteins contain a highly conserved region in their C-termini that bind the GTPase, Rab11. Rab11 belongs to the largest family of small GTPases and is believed to regulate vesicle docking with target membranes and vesicle fusion. The amino acid sequence of the Rab11-FIP proteins predicts coiled-coil formation in the conserved C-terminal domain. In this study on Rab11-FIP2, we found experimental evidence for the coiled-coil and then defined the minimal structured core using limited proteolysis. We also showed that the Rab11-FIP2 coiled-coil domain forms a parallel homodimer in solution using cross-linking and mutagenesis and sedimentation equilibrium experiments. Various constructs representing the C-terminal domain of Rab11-FIP2 were characterized by circular dichroism, and their affinity with Rab11 was measured using isothermal titration calorimetry. The longest construct was both well-structured and bound Rab11. A construct truncated at the N-terminus was poorly structured but retained the same affinity for binding to Rab11. Conformational changes were also demonstrated upon complex formation between Rab11 and Rab11-FIP2. A construct truncated at the C-terminus, which was the minimal coiled-coil domain defined by limited proteolysis, did not retain the ability to interact with Rab11, although it was as well-structured as the longer peptide. These data show that coiled-coil formation and Rab11 binding are separable functions of the C-terminal domain of Rab11-FIP2. The dissection of Rab11 binding from the formation of defined structure in a coiled-coil provides a potential mechanism for regulating Rab11-dependent endosomal trafficking.

Folding is coupled to dimerization of Tctex-1 dynein light chain.

Equilibrium analyses have been performed to elucidate the role of dimerization in folding and stability of dynein light chain Tctex-1. The equilibrium unfolding transition was monitored by intrinsic fluorescence intensity, fluorescence anisotropy, and circular dichroism and was modeled as a two-state mechanism where a folded dimer dissociates to two unfolded monomers without populating thermodynamically stable monomeric or dimeric intermediates. Sedimentation equilibrium and chemical cross-linking experiments performed at increasing concentrations of denaturants show no change in the association state before the unfolding transition and are consistent with the two-state model of dissociation coupled to unfolding. A linear dependence on denaturant concentration is observed by fluorescence intensity and anisotropy before unfolding in the 0-2 M GdnCl, and 0-4 M urea concentration range. This change is not protein concentration-dependent and possibly reflects relief of quenching associated with premelting conformational disorder in the vicinity of Trp 83. The data clearly show that the dissociation-coupled unfolding mechanism of Tctex-1 is different from the three-state denaturation mechanism of its structural homologue light chain LC8. The absence of a stable monomer in Tctex-1 offers insight into its functional differences from LC8.

Structural and Functional Variations in Human Apolipoprotein E3 and E4

There are three major apolipoprotein E (apoE) isoforms. Although APOE-epsilon3 is considered a longevity gene, APOE-epsilon4 is a dual risk factor to atherosclerosis and Alzheimer disease. We have expressed full-length and N- and C-terminal truncated apoE3 and apoE4 tailored to eliminate helix and domain interactions to unveil structural and functional disturbances. The N-terminal truncated apoE4-(72-299) and C-terminal truncated apoE4-(1-231) showed more complicated or aggregated species than those of the corresponding apoE3 counterparts. This isoformic structural variation did not exist in the presence of dihexanoylphosphatidylcholine. The C-terminal truncated apoE-(1-191) and apoE-(1-231) proteins greatly lost lipid binding ability as illustrated by the dimyristoylphosphatidylcholine turbidity clearance. The low density lipoprotein (LDL) receptor binding ability, determined by a competition binding assay of 3H-LDL to the LDL receptor of HepG2 cells, showed that apoE4 proteins with N-terminal (apoE4-(72-299)), C-terminal (apoE4-(1-231)), or complete C-terminal truncation (apoE4-(1-191)) maintained greater receptor binding abilities than their apoE3 counterparts. The cholesterol-lowering abilities of apoE3-(72-299) and apoE3-(1-231) in apoE-deficient mice were decreased significantly. The structural preference of apoE4 to remain functional in solution may explain the enhanced opportunity of apoE4 isoform to display its pathophysiologic functions in atherosclerosis and Alzheimer disease.

Structural and Functional Organization of the ESCRT-I Trafficking Complex

The endosomal sorting complex required for transport (ESCRT) complexes are central to receptor downregulation, lysosome biogenesis, and budding of HIV. The yeast ESCRT-I complex contains the Vps23, Vps28, and Vps37 proteins, and its assembly is directed by the C-terminal steadiness box of Vps23, the N-terminal half of Vps28, and the C-terminal half of Vps37. The crystal structures of a Vps23:Vps28 core subcomplex and the Vps23:Vps28:Vps37 core were solved at 2.1 and 2.8 A resolution. Each subunit contains a structurally similar pair of helices that form the core. The N-terminal domain of Vps28 has a hydrophobic binding site on its surface that is conformationally dynamic. The C-terminal domain of Vps28 binds the ESCRT-II complex. The structure shows how ESCRT-I is assembled by a compact core from which the Vps23 UEV domain, the Vps28 C domain, and other domains project to bind their partners.

Refolding Hydrogels Self-Assembled from N-(2-Hydroxypropyl)methacrylamide Graft Copolymers by Antiparallel Coiled-Coil Formation

A novel hybrid hydrogel system based on N-(2-hydroxypropyl)methacrylamide copolymers was proposed. It consisted of the hydrophilic polymer backbone and a pair of oppositely charged peptide grafts. Two distinct pentaheptad peptides (CCE and CCK) were anticipated to create a dimerization motif and serve as physical cross-linkers. Consequently, the graft copolymers CCE-P and CCK-P self-assembled into hybrid hydrogels in situ; the process was modulated by the formation of antiparallel heterodimeric coiled-coils. This approach possesses an advantage to decrease the steric hindrance of the polymer backbone on the "in-register" alignment of peptide grafts. Indeed, equimolar mixtures of the graft copolymers, CCE-P/CCK-P, have been observed to self-assemble into hydrogels in PBS solution at neutral pH at concentrations as low as 0.1 wt %. Circular dichroism spectroscopy, sedimentation equilibrium experiments, and microrheology revealed that the self-assembly process corresponded to the two-stranded alpha-helical coiled-coil formation between CCE and CCK. Moreover, the formation of hybrid hydrogels was reversible. Denaturation of the coiled-coil domains with guanidine hydrochloride (GdnHCl) solutions resulted in disassembly of the hydrogels. Removal of GdnHCl by dialysis caused coiled-coil refolding and hydrogel reassembly. Scanning electron microscopy results demonstrated that the concentration of the graft copolymers had a significant impact on the structure and morphology of self-assembled hydrogels.

Identification and Characterization of an 8-kDa Light Chain Associated with Dictyostelium discoideum MyoB, a Class I Myosin

Dictyostelium discoideum MyoB is a single-headed class I myosin. Analysis of purified MyoB by SDS-PAGE indicated the presence of an approximately 9-kDa light chain. A tryptic digest of MyoB yielded a partial sequence for the light chain that exactly matched a sequence in a 73-amino acid, 8,296-Da protein (dictyBase number DDB0188713). This protein, termed MlcB, contains two EF-hand motifs and shares approximately 30% sequence identity with the N- and C-terminal lobes of calmodulin. FLAG-MlcB expressed in Dictyostelium co-immunoprecipitated with MyoB but not with the related class myosins and MyoD. Recombinant MlcB bound Ca2+ with a Kd value of 0.2 microm and underwent a Ca2+-induced change in conformation that increased alpha-helical content and surface hydrophobicity. Mutational analysis showed that the first EF-hand was responsible for Ca2+ binding. In the presence and absence of Ca2+ MlcB was a monomer in solution and bound to a MyoB IQ motif peptide with a Kd value of approximately 0.5 microm. A MyoB head-neck construct with a Ser to Glu mutation at the TEDS site bound MlcB and displayed an actin-activated Mg2+ ATPase activity that was insensitive to Ca2+. We conclude that MlcB represents a novel type of small myosin light chain that binds to IQ motifs in a manner comparable with a single lobe of a typical four-EF-hand protein.

A Point Mutation in NEMO Associated with Anhidrotic Ectodermal Dysplasia with Immunodeficiency Pathology Results in Destabilization of the Oligomer and Reduces Lipopolysaccharide- and Tumor Necrosis Factor-mediated NF-κB Activation

The NEMO (NF-kappaB essential modulator) protein plays a crucial role in the canonical NF-kappaB pathway as the regulatory component of the IKK (IkappaB kinase) complex. The human disease anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has been recently linked to mutations in NEMO. We investigated the effect of an alanine to glycine substitution found in the NEMO polypeptide of an EDA-ID patient. This pathogenic mutation is located within the minimal oligomerization domain of the protein, which is required for the IKK activation in response to diverse stimuli. The mutation does not dramatically change the native-like state of the trimer, but temperature-induced unfolding studied by circular dichroism showed that it leads to an important loss in the oligomer stability. Furthermore, fluorescence studies showed that the tyrosine located in the adjacent zinc finger domain, which is possibly required for NEMO ubiquitination, exhibits an alteration in its spectral properties. This is probably due to a conformational change of this domain, providing evidence for a close interaction between the oligomerization domain and the zinc finger. In addition, functional complementation assays using NEMO-deficient pre-B and T lymphocytes showed that the pathogenic mutation reduced TNF-alpha and LPS-induced NF-kappaB activation by altering the assembly of the IKK complex. Altogether, our findings provide understanding as to how a single point mutation in NEMO leads to the observed EDA-ID phenotype in relation to the NEMO-dependent mechanism of IKK activation.

Effects of Efavirenz Binding on the Subunit Equilibria of HIV-1 Reverse Transcriptase

Recent studies showed that nonnucleoside reverse transcriptase inhibitors (NNRTIs) have variable effects on dimerization of p66 and p51 subunits of HIV-1 reverse transcriptase (RT). Efavirenz, one of three NNRTIs currently used in highly active anti-retroviral therapy, enhances subunit dimerization. Sedimentation equilibrium experiments on each subunit and equimolar mixtures of both subunits were used to measure dissociation constants for the three coupled dimerization reactions of RT in the absence and presence of saturating concentrations of the drug. The dimerization constants of the p51/p51 homodimer, the p66/p66 homodimer, and the p66/p51 heterodimer increased 600-, 50-, and 25-fold, respectively, upon binding of efavirenz. The effects of NNRTIs on RT dimerization are consistent with a thermodynamic linkage between subunit association/dissociation and inhibitor binding. Analysis of crystal structures of the p66/p51 heterodimer reveals that efavirenz binding induces small structural changes at the dimer interface.

Crystal Structure of the Cytoplasmic Domain of the Chloride Channel ClC-0

Ion channels are frequently organized in a modular fashion and consist of a membrane-embedded pore domain and a soluble regulatory domain. A similar organization is found for the ClC family of Cl- channels and transporters. Here, we describe the crystal structure of the cytoplasmic domain of ClC-0, the voltage-dependent Cl- channel from T. marmorata. The structure contains a folded core of two tightly interacting cystathionine beta-synthetase (CBS) subdomains. The two subdomains are connected by a 96 residue mobile linker that is disordered in the crystals. As revealed by analytical ultracentrifugation, the domains form dimers, thereby most likely extending the 2-fold symmetry of the transmembrane pore. The structure provides insight into the organization of the cytoplasmic domains within the ClC family and establishes a framework for guiding future investigations on regulatory mechanisms.

The crystal structure of Aq_328 from the hyperthermophilic bacteria Aquifex aeolicus shows an ancestral histone fold.

The structure of Aq_328, an uncharacterized protein from hyperthermophilic bacteria Aquifex aeolicus, has been determined to 1.9 A by using multi-wavelength anomalous diffraction (MAD) phasing. Although the amino acid sequence analysis shows that Aq_328 has no significant similarity to proteins with a known structure and function, the structure comparison by using the Dali server reveals that it: (1) assumes a histone-like fold, and (2) is similar to an ancestral nuclear histone protein (PDB code 1F1E) with z-score 8.1 and RMSD 3.6 A over 124 residues. A sedimentation equilibrium experiment indicates that Aq_328 is a monomer in solution, with an average sedimentation coefficient of 2.4 and an apparent molecular weight of about 20 kDa. The overall architecture of Aq_328 consists of two noncanonical histone domains in tandem repeat within a single chain, and is similar to eukaryotic heterodimer (H2A/H2B and H3/H4) and an archaeal histone heterodimer (HMfA/HMfB). The sequence comparisons between the two histone domains of Aq_328 and six eukaryotic/archaeal histones demonstrate that most of the conserved residues that underlie the Aq_328 architecture are used to build and stabilize the two cross-shaped antiparallel histone domains. The high percentage of salt bridges in the structure could be a factor in the protein's thermostability. The structural similarities to other histone-like proteins, molecular properties, and potential function of Aq_328 are discussed in this paper.

Human Mitochondrial ClpP Is a Stable Heptamer That Assembles into a Tetradecamer in the Presence of ClpX

The functional form of ClpP, the proteolytic component of ATP-dependent Clp proteases, is a hollow-cored particle composed of two heptameric rings joined face-to-face forming an aqueous chamber containing the proteolytic active sites. We have found that isolated human mitochondrial ClpP (hClpP) is stable as a heptamer and remains a monodisperse species (s(20,w) 7.0 S; M(app) 169, 200) at concentrations > or = 3 mg/ml. Heptameric hClpP has no proteolytic activity and very low peptidase activity. In the presence of ATP, hClpX interacts with hClpP forming a complex, which by equilibrium sedimentation measurements has a M(app) of 1 x 10(6). Electron microscopy confirmed that the complex consisted of a double ring of hClpP with an hClpX ring axially aligned on each end. The hClpXP complex has protease activity and greatly increased peptidase activity, indicating that interaction with hClpX affects the conformation of the hClpP catalytic active site. A mutant of hClpP, in which a cysteine residue was introduced into the handle region at the interface between the two rings formed stable tetradecamers under oxidizing conditions but spontaneously dissociated into two heptamers upon reduction. Thus, hClpP rings interact transiently but very weakly in solution, and hClpX must exert an allosteric effect on hClpP to promote a conformation that stabilizes the tetradecamer. These data suggest that hClpX can regulate the appearance of hClpP peptidase activity in mitochondria and might affect the nature of the degradation products released during ATP-dependent proteolytic cycles.

The structure of human apolipoprotein E2, E3 and E4 in solution: 1. Tertiary and quaternary structure

Three recombinant apoE isoforms fused with an amino-terminal extension of 43 amino acids were produced in a heterologous expression system in E. coli. Their state of association in aqueous phase was analyzed by size-exclusion liquid chromatography, sedimentation velocity and sedimentation equilibrium experiments. By liquid chromatography, all three isoforms consisted of three major species with Stokes radii of 4.0, 5.0 and 6.6 nm. Sedimentation velocity confirmed the presence of monomers, dimers and tetramers as major species of each isoform. The association schemes established by sedimentation equilibrium experiments corresponded to monomer–dimer–tetramer–octamer for apoE2, monomer–dimer–tetramer for apoE3 and monomer–dimer–tetramer–octamer for apoE4. Each of the three isoforms exhibits a distinct self-association pattern. The apolipoprotein multi-domain structure was mapped by limited proteolysis with trypsin, chymotrypsin, elastase, subtilisin and Staphylococcus aureus V8 protease. All five enzymes produced stable intermediates during the degradation of the three apoE isoforms, as described for plasma apoE3. The recombinant apoE isoforms, thus, consist of N- and C-terminal domains. The presence of the fusion peptide did not appear to alter the apolipoprotein tertiary organization. However, a 30 kDa amino-terminal fragment appeared during the degradation of the recombinant apoE isoforms resulting from cleavage in the 273–278 region. This region, not accessible in plasma apoE3, results from a different conformation of the C-terminal domain in the recombinant isoforms. A specific pattern for the apoE4 C-terminal domain was observed during the proteolysis. The region 230–260 in apoE4, in contrast to that of apoE3 and apoE2, was not accessible to proteases, probably due to the existence of a longer helix in this region of apoE4 stabilized by an interdomain interaction.

Design of Amphiphilic Protein Maquettes: Enhancing Maquette Functionality through Binding of Extremely Hydrophobic Cofactors to Lipophilic Domains

We demonstrate coordination of the extremely hydrophobic 13(2)-OH-Ni-bacteriochlorophyll (Ni-BChl) to the lipophilic domain of a novel, designed amphiphilic protein maquette (AP3) dispersed in detergent micelles [Discher et al. (2005) Biochemistry 44, 12329-12343]. Sedimentation velocity and equilibrium experiments and steady-state absorption spectra indicate that Ni-BChl-AP3 is a four-helix bundle containing one Ni-BChl axially ligated by one or two histidines. The nature of the ligation was pursued with ultrafast visible spectroscopy. While it is well established that light excitation of axially ligated mono- and bisimidazole Ni-BChl in solution leads to rapid imidazole dissociation and nanosecond recombination, there is no evidence of axial ligand dissociation in the light-excited Ni-BChl-AP3. This indicates that Ni-BChl is confined within the AP3 protein, ligated to histidines with severely restricted mobility. Dissociation constants show that Ni-BChl binding to AP3 is considerably weaker than the nanomolar range usual for heme and hydrophilic (HP) maquettes; moreover, there is a tendency for the Ni-BChl-AP3 four-helix bundles to dimerize into eight-helix bundles. Nevertheless, the preparation of the Ni-BChl-AP3 four-alpha-helix maquettes, supported by time-resolved spectroscopic analysis of the nature of the ligation, provides a viable new approach to AP maquette designs that address the challenges involved in binding extremely hydrophobic cofactors.

Monomer/dimer equilibrium of the AB-type lectin from mistletoe enables combination of toxin/agglutinin activities in one protein: analysis of native and citraconylated proteins by ultracentrifugation/gel filtration and cell biological consequences of dimer destabilization

The biological activity of a lectin is influenced by its quaternary structure. Viscumin is special among the family members of toxic AB-type plant lectins, because it triggers mitogenicity, toxicity, and agglutination. Its activity profile is dependent on the concentration, motivating a thorough inspection of the status of quaternary structure. Over a broad range of protein concentrations (0.01-25 mg/mL), viscumin occurs as a dimer. At high concentrations, the solutions exhibited nonideality, self-association, and polydispersity in sedimentation equilibrium and velocity experiments caused by irreversible aggregation. Calculation of viscumin's overall shape based on sedimentation velocity data resulted in an elongated dimer form resembling that of crystallized agglutinin. Appearance of monomers was restricted to concentrations in the submicrogram/mL level, as demonstrated by fast protein liquid chromatography gel-filtration analysis. To shift the equilibrium to the monomer for comparative cell biological assays, we performed chemical modification under conditions protecting the lectin activity. Citraconylation was effective to destabilize the dimer. Binding studies by fluorescence-activated cell scan analysis revealed a reduction in cell association upon modification and a tendency for increased sensitivity towards haptenic inhibitors at microg/mL concentrations. Nonetheless, growth inhibition continued to be potent for the ricin-like monomer despite reduced extent of binding. Occurrence of a concentration-dependent monomer/dimer equilibrium appears to achieve the same objectives as the development of two separate protein entities in Ricinus communis, an alternative strategy to emergence of a monomeric toxin, and cell cross-linking dimeric agglutinin.

Ligand-Receptor and Receptor-Receptor Interactions Act in Concert to Activate Signaling in the Drosophila Toll Pathway

In Drosophila, the signaling pathway mediated by the Toll receptor is critical for the establishment of embryonic dorso-ventral pattern and for innate immune responses to bacterial and fungal pathogens. Toll is activated by high affinity binding of the cytokine Spätzle, a dimeric ligand of the cystine knot family. In vertebrates, a related family of Toll-like receptors play a critical role in innate immune responses. Despite the importance of this family of receptors, little is known about the biochemical events that lead to receptor activation and signaling. Here, we show that Spätzle binds to the N-terminal region of Toll and, using biophysical methods, that the binding is complex. The two binding events that cause formation of the cross-linked complex are non-equivalent: the first Toll ectodomain binds Spätzle with an affinity 3-fold higher than the second molecule suggesting that pathway activation involves negative cooperativity. We further show that the Toll ectodomains are able to form low affinity dimers in solution and that juxtamembrane sequences of Toll are critical for the activation or derepression of the pathway. These results, taken together, suggest a mechanism of signal transduction that requires both ligand-receptor and receptor-receptor interactions.