Solution structure and dynamics of ADF from Toxoplasma gondii

Toxoplasma gondii ADF (TgADF) belongs to a functional subtype characterized by strong G-actin sequestering activity and low F-actin severing activity. Among the characterized ADF/cofilin proteins, TgADF has the shortest length and is missing a C-terminal helix implicated in F-actin binding. In order to understand its characteristic properties, we have determined the solution structure of TgADF and studied its backbone dynamics from ¹⁵N-relaxation measurements. TgADF has conserved ADF/cofilin fold consisting of a central mixed β-sheet comprised of six β-strands that are partially surrounded by three α-helices and a C-terminal helical turn. The high G-actin sequestering activity of TgADF relies on highly structurally and dynamically optimized interactions between G-actin and G-actin binding surface of TgADF. The equilibrium dissociation constant for TgADF and rabbit muscle G-actin was 23.81 nM, as measured by ITC, which reflects very strong affinity of TgADF and G-actin interactions. The F-actin binding site of TgADF is partially formed, with a shortened F-loop that does not project out of the ellipsoid structure and a C-terminal helical turn in place of the C-terminal helix α4. Yet, it is more rigid than the F-actin binding site of Leishmania donovani cofilin. Experimental observations and structural features do not support the interaction of PIP2 with TgADF, and PIP2 does not affect the interaction of TgADF with G-actin. Overall, this study suggests that conformational flexibility of G-actin binding sites enhances the affinity of TgADF for G-actin, while conformational rigidity of F-actin binding sites of conventional ADF/cofilins is necessary for stable binding to F-actin.     

Group-specific component (vitamin D binding protein) prevents the interaction between G-actin and profilin

Profilin purified from human platelets formed a 1:1 molar ratio complex with rabbit skeletal muscle G-actin but was displaced by purified serum Gc (vitamin D binding protein) in a dose-dependent fashion as assessed by chromatography and ultrafiltration. This suggested that Gc and profilin competed for the same binding area on G-actin, with Gc-G-actin complexes being more stable than profilin-G-actin complexes in vitro. The binding domain for Gc on G-actin was localized to a 16,000-Da C-terminal fragment of G-actin generated by Staphylococcus aureus V8 protease, as judged by comigration on two-dimensional electrophoresis and also by overlaying electrophoresis gels with 125I-Gc. Previous studies have reported that residues 374 and 375 of G-actin are essential for binding of profilin. In this study, experiments involving tryptic removal of Cys-374 labeled with the fluorescent probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)-ethylenediamine showed that these C-terminal amino acids were not necessary for interaction with Gc.     

The C-terminal fragment of thymopoietin forms F-actin bundles: electron microscopic data

It was shown by electron microscopy that PEP33 a synthetic C-terminal peptide of the thymus hormone thymopoietin, formed bundles of actin 'filaments in the presence of 0.1 M KCl. The structure of PEP33 aggregates localizated in the bundles between actin filaments is very similar to that of aggregates observed in samples of pure PEP33. No changes were revealed in the structure of G-actin in the presence of PEP33. A similar, but a weaker bundling effect of thymopentin (PEP5) was also found. It forms bundles of actin filaments of small size. Further studies can shed light on the physiological importance of actin filament aggregation with the peptides of thymopoietin, the systematic release of which from the thymus produces the phenomena characteristic for the serious neuromuscular disease myasthenia gravis.     

Coupling of folding and binding of thymosin beta4 upon interaction with monomeric actin monitored by nuclear magnetic resonance

Thymosin beta4 is a major actin-sequestering protein, yet the structural basis for its biological function is still unknown. This study provides insight regarding the way this 43-amino acid peptide, mostly unstructured in solution, binds to monomeric actin and prevents its assembly in filaments. We show here that the whole backbone of thymosin beta4 is highly affected upon binding to G-actin. The assignment of all amide protons and nitrogens of thymosin in the bound state, obtained using a combination of NMR experiments and selective labelings, shows that thymosin folds completely upon binding and displays a central extended region flanked by two N- and C-terminal helices. The cleavage of actin by subtilisin in the DNase I binding loop does not modify the structure of thymosin beta4 in the complex, showing that the backbone of the peptide is not in close proximity to segment 42-47 of actin. The combination of our NMR results and previously published mutation and cross-link data allows a better characterization of the binding mode of thymosins on G-actin.     

Structure of thermolysin cleaved microcin J25: extreme stability of a two-chain antimicrobial peptide devoid of covalent links

The structure of a two-chain peptide formed by the treatment of the potent antimicrobial peptide microcin J25 (MccJ25) with thermolysin has been characterized by NMR spectroscopy and mass spectrometry. The native peptide is 21 amino acids in size and has the remarkable structural feature of a ring formed by linkage of the side chain of Glu8 to the N-terminus that is threaded by the C-terminal tail of the peptide. Thermolysin cleaves the peptide at the Phe10-Val11 amide bond, but the threading of the C-terminus through the N-terminal ring is so tight that the resultant two chains remain associated both in the solution and in the gas phases. The three-dimensional structure of the thermolysin-cleaved peptide derived using NMR spectroscopy and simulated annealing calculations has a well-defined core that comprises the N-terminal ring and the threading C-terminal tail. In contrast to the well-defined core, the newly formed termini at residues Phe10 and Val11 are disordered in solution. The C-terminal tail is associated to the ring both by hydrogen bonds stabilizing a short beta-sheet and by hydrophobic interactions. Moreover, unthreading of the tail through the ring is prevented by the bulky side chains of Phe19 and Tyr20, which flank the octapeptide ring. This noncovalent two-peptide complex that has a remarkable stability in solution and in highly denaturing conditions and that survives in the gas phase is the first example of such a two-chain peptide lacking disulfide or interchain covalent bonds.     

The development of ketogenesis at birth in the rat.

The manner in which human liver cathepsin B (EC 3.4.22.1) digests glucagon was determined. After reaction of the proteinase with the substrate for 24h, more than 15 products were formed. During the first 7 h of reaction, eight products were formed; seven of these were dipeptides that originated from the C-terminal portion of the glucagon molecule, whereas the eighth peptide was the remaining large fragment of the hormone, consisting of residues 1-19. Measurement of the rate of formation of the products showed that cathepsin B degraded glucagon by a sequential cleavage of dipeptides from the C-terminal end of the molecule. Cathepsin B from both rat liver and bovine spleen was shown to hydrolyse glucagon by the same mechanism.     

Gc (vitamin D-binding protein) binds the 33.5 K tryptic fragment of actin

Limited proteolysis of G-actin was performed with trypsin and chymotrypsin to compare the binding sites for Gc and DNase. DNase I bound to the N-terminal area corresponding to the major cleavage site on G-actin (residues 62-68) and inhibited proteolysis, but did not bind the 33.5K C-terminal fragment (G-actin33.5) generated. In contrast, Gc did not exert any inhibitory effect upon proteolysis of the intact native G-actin42.0 molecule, although its presence protected G-actin33.5 from further proteolysis. This was shown by gel filtration to be due to the formation of complexes between Gc and G-actin33.5.     

Thymosin beta 4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes

Thymosin beta 4 (T beta 4), a 5-kD peptide which binds G-actin and inhibits its polymerization (Safer, D., M. Elzinga, and V. T. Nachmias. 1991. J. Biol. Chem. 266:4029-4032), appears to be the major G-actin sequestering protein in human PMNs. In support of a previous study by Hannappel, E., and M. Van Kampen (1987. J. Chromatography. 397:279-285), we find that T beta 4 is an abundant peptide in these cells. By reverse phase HPLC of perchloric acid supernatants, human PMNs contain approximately 169 fg/cell +/- 90 fg/cell (SD), corresponding to a cytoplasmic concentration of approximately 149 +/- 80.5 microM. On non-denaturing polyacrylamide gels, a large fraction of G-actin in supernatants prepared from resting PMNs has a mobility similar to the G-actin/T beta 4 complex. Chemoattractant stimulation of PMNs results in a decrease in this G-actin/T beta 4 complex. To determine whether chemoattractant induced actin polymerization results from an inactivation of T beta 4, the G-actin sequestering activity of supernatants prepared from resting and chemoattractant stimulated cells was measured by comparing the rates of pyrenyl-actin polymerization from filament pointed ends. Pyrenyl actin polymerization was inhibited to a greater extent in supernatants from stimulated cells and these results are qualitatively consistent with T beta 4 being released as G-actin polymerizes, with no chemoattractant-induced change in its affinity for G-actin. The kinetics of bovine spleen T beta 4 binding to muscle pyrenyl G-actin are sufficiently rapid to accommodate the rapid changes in actin polymerization and depolymerization observed in vivo in response to chemoattractant addition and removal.     

C-terminal fragments of thymopoietin favor formation of F-actin bundles: Electron microscopic data

Electron microscopy showed that PEP₃₃, a synthetic peptide corresponding to the C-terminal part of the thymic hormone thymopoietin, favors bundling of F-actin filaments in the presence of 0.1 M KCl. The structure of PEP₃₃ aggregates located within bundles between actin filaments is very similar to the structure of aggregates visible in preparations of pure PEP₃₃. No changes were observed in the structure of G-actin in the presence of PEP₃₃. A similar though weaker bundling effect was also detected for PEP₅, or thymopentin, a fragment of PEP₃₃. This peptide favors the formation of bundles of actin filaments of small size. The possible role of aggregation of actin filaments under the action of thymopoietin peptides is discussed in the light of the fact that the systematic release of thymopoietin from thymus leads to a phenomenon characteristic of the serious neuromuscular disease myasthenia gravis.     

Conformation and lipid binding of a C-terminal (198-243) peptide of human apolipoprotein A-I

Human apolipoprotein A-I (apoA-I) is the principle apolipoprotein of high-density lipoproteins that are critically involved in reverse cholesterol transport. The intrinsically flexibility of apoA-I has hindered studies of the structural and functional details of the protein. Our strategy is to study peptide models representing different regions of apoA-I. Our previous report on [1-44]apoA-I demonstrated that this N-terminal region is unstructured and folds into approximately 60% alpha-helix with a moderate lipid binding affinity. We now present details of the conformation and lipid interaction of a C-terminal 46-residue peptide, [198-243]apoA-I, encompassing putative helix repeats 10 and 9 and the second half of repeat 8 from the C-terminus of apoA-I. Far-ultraviolet circular dichroism spectra show that [198-243]apoA-I is also unfolded in aqueous solution. However, self-association induces approximately 50% alpha-helix in the peptide. The self-associated peptide exists mainly as a tetramer, as determined by native electrophoresis, cross-linking with glutaraldehyde, and unfolding data from circular dichroism (CD) and differential scanning calorimetry (DSC). In the presence of a number of lipid-mimicking detergents, above their CMC, approximately 60% alpha-helix was induced in the peptide. In contrast, SDS, an anionic lipid-mimicking detergent, induced helical folding in the peptide at a concentration of approximately 0.003% (approximately 100 microM), approximately 70-fold below its typical CMC (0.17-0.23% or 6-8 mM). Both monomeric and tetrameric peptide can solubilize dimyristoylphosphatidylcholine (DMPC) liposomes and fold into approximately 60% alpha-helix. Fractionation by density gradient ultracentrifugation and visualization by negative staining electromicroscopy demonstrated that the peptide binds to DMPC with a high affinity to form at least two sizes of relatively homogeneous discoidal HDL-like particles depending on the initial lipid:peptide ratio. The characteristics (lipid:peptide weight ratio, diameter, and density) of both complexes are similar to those of plasma A-I/DMPC complexes formed under similar conditions: small discoidal complexes (approximately 3:1 weight ratio, approximately 110 A, and approximately 1.10 g/cm3) formed at an initial 1:1 weight ratio and larger discoidal complexes (approximately 4.6:1 weight ratio, approximately 165 A, and approximately 1.085 g/cm3) formed at initial 4:1 weight ratio. The cross-linking data for the peptide on the complexes of two sizes is consistent with the calculated peptide numbers per particle. Compared to the approximately 100 A disk-like complex formed by the N-terminal peptide in which helical structure was insufficient to cover the disk edge by a single belt, the compositions of these two types of complexes formed by the C-terminal peptide are more consistent with a "double belt" model, similar to that proposed for full-length apoA-I. Thus, our data provide direct evidence that this C-terminal region of apoA-I is responsible for the self-association of apoA-I, and this C-terminal peptide model can mimic the interaction with the phospholipid of plasma apoA-I to form two sizes of homogeneous discoidal complexes and thus may be responsible for apoA-I function in the formation and maintenance of HDL subspecies in plasma.     

Localization of sites for ionic interaction with lipid in the C-terminal third of the bovine myelin basic protein.

The myelin basic protein from bovine brain tissue was purified and the two peptides obtained by cleavage of the polypeptide chain at the single tryptophan residue were isolated. The interaction of these peptides and the intact basic protein with complex lipids was investigated by following the solubilization of lipid-protein complexes into chloroform in a biphasic solvent system. The C-terminal peptide fragment (residues 117-170) and the intact basic protein both formed chloroform-soluble complexes with acidic lipids, but not with neutral complex lipids. The N-terminal fragment (residues 1-115) did not form chloroform-soluble complexes with either acidic or neutral complex lipids. The molar ratio of lipid to protein that caused a 50% loss of protein from the upper phase to the lower chloroform phase was the same for the intact basic protein as for the smaller C-terminal peptide fragment. Phosphatidylserine and phosphatidylinositol were approximately twice as efficient as sulphatide at causing protein redistribution to the chloroform phase. The results are interpreted as indicating that the sites for ionic interactions between lipid and charged groups on the basic protein of myelin are located in the C-terminal region of the protein molecule.     

Activation of heavy meromyosin adenosine triphosphatase by various states of actin.

F-actin monomer (F-monomer) is formed upon the addition of neutral salt to G-actin. Since F-monomer has a digestibility similar to that of F-actin and much lower than that of G-actin, it has been proposed that F-monomer has a conformation different from that of G-actin and similar to the conformation of the subunits in F-actin. To examine whether F-monomer will enhance the magnesium-activated myosin adenosine triphosphatase (Mg2+-ATPase) as much as F-actin, the ability of partially polymerized actin populations at equilibrium to activate the Mg2+-ATPase of heavy meromyosin was investigated. Correlations were made between ATPase activities and the polymerization state of actin as determined by measurements of viscosity and digestibility. No significant activation of the heavy meromyosin ATPase was observed under conditions where G-actin or mixtures of G-actin and F-monomer were present. As polymer formation occurred at higher actin concentrations, or with increased KCl concentrations, substantial activation characteristic of F-actin was observed. The data suggest that F-monomer may undergo a further conformational change as it forms nuclei or joins onto polymers. Alternatively, the site of actin which activates the myosin ATPase may involve the crevice between two adjacent actin subunits.     

Interactions between G-actin and myosin subfragment 1: immunochemical probing of the NH2-terminal segment on actin

The role of the N-terminal segment of actin in myosin-induced polymerization of G-actin was studied by using peptide antibodies directed against the first seven N-terminal residues of alpha-skeletal actin. Light scattering, fluorescence, and analytical ultracentrifugation experiments showed that the Fab fragments of these antibodies inhibited the polymerization of G-actin by myosin subfragment 1 (S-1) by inhibiting the binding of these proteins to each other. Fluorescence measurements using actin labeled with pyrenyliodoacetamide revealed that Fab inhibited the initial step in the binding of S-1 to G-actin. It is deduced from these results and from other literature data that the initial contact between G-actin and S-1 involves residues 1-7 on actin and residues 633-642 on the S-1 heavy chain. This interaction appears to be of major importance for the binding of S-1 and G-actin. The presence of additional myosin contact sites on G-actin was indicated by concentration-dependent recovery of S-1 binding to G-actin without displacement of Fab. The reduced Fab inhibition of S-1 binding to polymerizing and polymerized actin is consistent with the tightening of acto-S-1 binding at these sites or the creation of new sites upon formation of F-actin.     

How a single residue in individual β-thymosin/WH2 domains controls their functions in actin assembly

β-Thymosin (βT) and WH2 domains are widespread, intrinsically disordered actin-binding peptides that display significant sequence variability and different regulations of actin self-assembly in motile and morphogenetic processes. Here, we reveal the structural mechanisms by which, in their 1:1 stoichiometric complexes with actin, they either inhibit assembly by sequestering actin monomers like Thymosin-β4, or enhance motility by directing polarized filament assembly like Ciboulot βT. We combined mutational, functional or structural analysis by X-ray crystallography, SAXS (small angle X-ray scattering) and NMR on Thymosin-β4, Ciboulot, TetraThymosinβ and the long WH2 domain of WASP-interacting protein. The latter sequesters G-actin with the same molecular mechanisms as Thymosin-β4. Functionally different βT/WH2 domains differ by distinct dynamics of their C-terminal half interactions with G-actin pointed face. These C-terminal interaction dynamics are controlled by the strength of electrostatic interactions with G-actin. At physiological ionic strength, a single salt bridge with actin located next to their central LKKT/V motif induces G-actin sequestration in both isolated long βT and WH2 domains. The results open perspectives for elucidating the functions of βT/WH2 domains in other modular proteins.     

Interaction of actin with carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) receptor in liposomes is Ca2+- and phospholipid-dependent

The regulation of binding of G-actin to cytoplasmic domains of cell surface receptors is a common mechanism to control diverse biological processes. To model the regulation of G-actin binding to a cell surface receptor we used the cell-cell adhesion molecule carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1-S) in which G-actin binds to its short cytoplasmic domain (12 amino acids; Chen, C. J., Kirshner, J., Sherman, M. A., Hu, W., Nguyen, T., and Shively, J. E. (2007) J. Biol. Chem. 282, 5749-5760). A liposome model system demonstrates that G-actin binds to the cytosolic domain peptide of CEACAM1-S in the presence of negatively charged palmitoyl-oleoyl phosphatidylserine (POPS) liposomes and Ca(2+). In contrast, no binding of G-actin was observed in palmitoyl-oleoyl phosphatidylcholine (POPC) liposomes or when a key residue in the peptide, Phe-454, is replaced with Ala. Molecular Dynamics simulations on CEACAM1-S in an asymmetric phospholipid bilayer show migration of Ca(2+) ions to the lipid leaflet containing POPS and reveal two conformations for Phe-454 explaining the reversible availability of this residue for G-actin binding. NMR transverse relaxation optimized spectroscopic analysis of (13)C-labeled Phe-454 CEACAM1-S peptide in liposomes plus actin further confirmed the existence of two peptide conformers and the Ca(2+) dependence of actin binding. These findings explain how a receptor with a short cytoplasmic domain can recruit a cytosolic protein in a phospholipid and Ca(2+)-specific manner. In addition, this model system provides a powerful approach that can be applied to study other membrane protein interactions with their cytosolic targets.     

Reactivation of mutant p53 through interaction of a C-terminal peptide with the core domain

A synthetic 22-mer peptide (peptide 46) derived from the p53 C-terminal domain can restore the growth suppressor function of mutant p53 proteins in human tumor cells (G. Selivanova et al., Nat. Med. 3:632-638, 1997). Here we demonstrate that peptide 46 binds mutant p53. Peptide 46 binding sites were found within both the core and C-terminal domains of p53. Lys residues within the peptide were critical for both p53 activation and core domain binding. The sequence-specific DNA binding of isolated tumor-derived mutant p53 core domains was restored by a C-terminal polypeptide. Our results indicate that C-terminal peptide binding to the core domain activates p53 through displacement of the negative regulatory C-terminal domain. Furthermore, stabilization of the core domain structure and/or establishment of novel DNA contacts may contribute to the reactivation of mutant p53. These findings should facilitate the design of p53-reactivating drugs for cancer therapy.     

Actin—membrane interactions: Association of G-actin with the red cell membrane

Chemically tritiated actin from rabbit skeletal muscle was used to investigate the association of G-actin with the red cell membrane. The tritiated actin was shown to be identical to unmodified actin in its ability to polymerize and to activate heavy meromyosin ATPase. Using sealed and unsealed red cell ghosts we have shown that G-actin binds to the cytoplasmic but not the extracellular membrane surface of ghosts. Inside-out vesicles which have been stripped of endogenous actin and spectrin by low-ionic-strength incubation bind little G-actin. However, when a crude spectrin extract containing primarily spectrin, actin, and band 4.1 is added back to stripped vesicles, subsequent binding of G-actin can be increased up to 40-fold. Further, this crude spectrin extract can compete for and abolish G-actin binding to unsealed ghosts. Actin binding to ghosts increases linearly with added G-actin and requires the presence of magnesium. In addition, actin binding is inhibited by cytochalasin B and DNAase I. Negative staining reveals an abundance of actin filaments formed when G-actin is added to reconstituted inside-out vesicles but none when it is added to unreconstituted vesicles. These observations indicate that added G-actin binds to the red cell membrane via filament formation nucleated by some membrane component at the cytoplasmic surface.     

Design, expression and characterization of collagen-like proteins based on the cell adhesive and crosslinking sequences derived from native collagens

Two recombinant collagen-like proteins consisting of cell adhesion domains derived from native type I collagen were designed and synthesized by a genetic engineering method. The cross-linking sequence, GPPGPCCGGG, derived from collagen III was used to promote triple helix formation through the disulfide bonds formed among three chains by flanking the peptide at the C-terminal of the collagen-like proteins. SDS-PAGE and western-blotting data suggested possibility of the formation of a triple helix structure for both recombinant proteins. CD spectra and thermal stability analyses indicated that the triple-helix structure in the collagen-like proteins was pH-dependent and stabilized under acidic environmental condition. Moreover, the collagen-like protein flanked with the cross-linking sequence at the C-terminal showed the most stable triple-helical conformation under acidic conditions.     

Synthesis of the C-terminal domain of the tissue inhibitor of metalloproteinases-1 (TIMP-1).

According to recent investigations, the C-terminal domain of the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) is responsible for some biological effects that are independent of the enzyme-inhibiting effect of the N-terminal domain of the molecule. The C-terminal domain has been prepared for structure-biological activity investigations. After the chemical synthesis and the folding of the linear peptide. LC-MS and MALDI-MS analysis revealed that two isomers with different disulphide bond arrangements were formed. Since more than 30 folding experiments resulted in products with a very similar HPLC-profile, it was concluded that in the absence of the TIMP-1 N-terminal domain no entirely correct folding of the C-terminal domain occurred. Furthermore, it was observed that, in spite of several purification steps, mercury(II) ions were bound to the 6SH-linear peptide; it was demonstrated--using disulphide bonded TIMP-1(Cys145-Cys166) as a model--that mercury(II) ions can cause peptide degradation at pH 7.8 as well as in 0.1% trifluoroacetic acid.     

Crystal Structure of the VgrG1 Actin Cross-linking Domain of the Vibrio cholerae Type VI Secretion System

Vibrio cholerae is the cause of the diarrheal disease cholera. V. cholerae produces RtxA, a large toxin of the MARTX family, which is targeted to the host cell cytosol, where its actin cross-linking domain (ACD) cross-links G-actin, leading to F-actin depolymerization, cytoskeleton rearrangements, and cell rounding. These effects on the cytoskeleton prevent phagocytosis and bacterial engulfment by macrophages, thus preventing V. cholerae clearance from the gut. The V. cholerae Type VI secretion-associated VgrG1 protein also contains a C-terminal ACD, which shares 61% identity with MARTX ACD and has been shown to covalently cross-link G-actin. Here, we purified the VgrG1 C-terminal domain and determined its crystal structure. The VgrG1 ACD exhibits a V-shaped three-dimensional structure, formed of 12 β-strands and nine α-helices. Its active site comprises five residues that are conserved in MARTX ACD toxin, within a conserved area of ∼10 Å radius. We showed that less than 100 ACD molecules are sufficient to depolymerize the actin filaments of a fibroblast cell in vivo. Mutagenesis studies confirmed that Glu-16 is critical for the F-actin depolymerization function. Co-crystals with divalent cations and ATP reveal the molecular mechanism of the MARTX/VgrG toxins and offer perspectives for their possible inhibition.