Domain movement in gelsolin: a calcium-activated switch.

The actin-binding protein gelsolin is involved in remodeling the actin cytoskeleton during growth-factor signaling, apoptosis, cytokinesis, and cell movement. Calcium-activated gelsolin severs and caps actin filaments. The 3.4 angstrom x-ray structure of the carboxyl-terminal half of gelsolin (G4-G6) in complex with actin reveals the basis for gelsolin activation. Calcium binding induces a conformational rearrangement in which domain G6 is flipped over and translated by about 40 angstroms relative to G4 and G5. The structural reorganization tears apart the continuous beta sheet core of G4 and G6. This exposes the actin-binding site on G4, enabling severing and capping of actin filaments to proceed.

The structures of the neurotrophin 4 homodimer and the brain-derived neurotrophic factor/neurotrophin 4 heterodimer reveal a common Trk-binding site.

The neurotrophins are growth factors that are involved in the development and survival of neurons. Neurotrophin release by a target tissue results in neuron growth along the neurotrophin concentration gradient, culminating in the eventual innervation of the target tissue. These activities are mediated through trk cell surface receptors. We have determined the structures of the heterodimer formed between brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4), as well as the structure of homodimer of NT4. We also present the structure of the Neurotrophin 3 homodimer, which is refined to higher resolution than previously published. These structures provide the first views of the architecture of the NT4 protomer. Comparison of the surface of a model of the BDNF homodimer with the structures of the neurotrophin homodimers reveals common features that may be important in the binding between the neurotrophins and their receptors. In particular, there exists an analogous region on the surface of each neurotrophin that is likely to be involved in trk receptor binding. Variations in sequence on the periphery of this common region serve to confer trk receptor specificity.

Equus caballus gelsolin--cDNA sequence and protein structural implications.

We have generated and characterized the cDNA from equine smooth muscle that encodes gelsolin, an actin-modulating protein. Overlapping cDNA clones synthesized by the reverse transcriptase/polymerase chain reaction and clones isolated from a horse genomic library provided the complete primary structure for the intracellular isoform of gelsolin, while cDNA complemented with protein sequence data produced the full-length primary transcript of the gelsolin isoform found circulating in equine plasma. The deduced amino acid sequences of the intracellular and secreted versions of equine gelsolin infer polypeptides of 731 and 755 residues with apparent molecular masses of 80.7 kDa and 83.2 kDa, respectively. Multiple sequence alignment analysis of equine, human, porcine, and murine orthologs of gelsolin demonstrates prominent similarities among all of these proteins, with the horse and human molecules exhibiting the largest degree of likeness with respect to polypeptide length and overall sequence composition. Both horse and human plasma gelsolins are comprised of 755 amino acids with 94% of the residues identical, while the degree of sequence identity in the shorter (731 residues) cytoplasmic gelsolins is 95%. Analysis of the sequences and structures of the six related domains that comprise gelsolin emphasizes the strong correlation that exists between primary structural conservation among mammalian gelsolins and maintenance of the three-dimensional domain fold characteristic of members of this protein family.

The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation.

The structure of gelsolin has been determined by crystallography and comprises six structurally related domains that, in a Ca2+-free environment, pack together to form a compact globular structure in which the putative actin-binding sequences are not sufficiently exposed to enable binding to occur. We propose that binding Ca2+ can release the connections that join the N- and C-terminal halves of gelsolin, enabling each half to bind actin relatively independently. Domain shifts are proposed in response to Ca2+ as bases for models of how gelsolin acts to sever, cap, or nucleate F-actin filaments. The structure also invites discussion of polyphosphoinositide binding to segment 2 and suggests how mutation at Asp-187 could initiate a series of events that lead to deposition of amyloid plaques, as observed in victims of familial amyloidosis (Finnish type).

Multiple pathways for denaturation of horse plasma gelsolin.

Gelsolin purified from horse plasma carries a surface charge distribution that greatly influences how the protein unfolds, aggregates, or precipitates as a function of temperature or concentration of chemical denaturant. Modification of gelsolin with fluorescein isothiocyanate replaces positive charges on amine groups with bulky, negatively charged fluorescein moieties. This postpones thermally induced precipitation by about 10 degrees C [Koepf, E.K., and Burtnick, L.D. 1993. Eur. J. Biochem. 212: 713-718]. Interaction with cations such as Ca2+ or guanidinium+ also alters the surface charge on gelsolin. This affects the structure of the protein in solution, modifies the pathway for unfolding, and moderates the onset of precipitation induced by chemical denaturants or heat. Denaturation of gelsolin is not interpretable in terms of a simple two-state cooperative mechanism. The pathway to a denatured state and intermediate structures present along the way depend upon the agent used to unfold the protein.

The cellulose-binding domain of endoglucanase A (CenA) from Cellulomonas fimi: evidence for the involvement of tryptophan residues in binding.

Cellulomonas fimi endo-beta-1,4-glucanase A (CenA) contains a discrete N-terminal cellulose-binding domain (CBDCenA). Related CBDs occur in at least 16 bacterial glycanases and are characterized by four highly conserved Trp residues, two of which correspond to W14 and W68 of CBDCenA. The adsorption of CBDCenA to crystalline cellulose was compared with that of two Trp mutants (W14A and W68A). The affinities of the mutant CBDs for cellulose were reduced by approximately 50- and 30-fold, respectively, relative to the wild type. Physical measurements indicated that the mutant CBDs fold normally. Fluorescence data indicated that W14 and W68 were exposed on the CBD, consistent with their participation in binding to cellobiosyl residues on the cellulose surface.

Fluorescent responses of acrylodan-labeled plasma gelsolin.

Under nondenaturing conditions, 1 mol of horse plasma gelsolin reacts with 1.9 +/- 0.5 mol (mean +/- SD, n = 6) of the sulfhydryl-specific fluorescent reagent 6-acryloyl-2-dimethylaminonaphthalene (acrylodan). The degree of labeling in 6 M guanidine-HCl increases to about 3 mol of acrylodan per mole of gelsolin. Viscosity studies show that the modified gelsolin retains its ability to sever F-actin filaments. Circular dichroism spectra in the peptide bond absorption region are indistinguishable for labeled and unmodified gelsolin at room temperature. The thermal stability of gelsolin, as monitored by circular dichroism, is unaffected by reaction with acrylodan. While circular dichroism spectra of acrylodan-labeled gelsolin recorded at room temperature are not influenced significantly by Ca2+, fluorescence studies reveal a number of Ca(2+)-dependent changes in the protein. Ca2+ causes a decrease and red-shift in fluorescence emission, an increase in sensitivity to quenching by I-, and a decrease in polarization of the fluorescence of acrylodan-labeled gelsolin. Together, these changes in fluorescence properties indicate there to be an increased exposure of the label to the solvent when gelsolin binds Ca2+. Fluorescence polarization experiments in which acrylodan-labeled gelsolin is titrated with actin emphasize that Ca2+ is required for these two proteins to interact.

Horse plasma gelsolin labelled with fluorescein isothiocyanate responds to calcium and actin.

Reaction between horse plasma gelsolin and fluorescein-5-isothiocyanate (FITC) resulted in incorporation of 4.8 +/- 0.6 fluorescein groups/gelsolin molecule. The sites of modification were not clustered in any one portion of the gelsolin polypeptide chain; all major peptides produced by proteolytic digestion with alpha-chymotrypsin exhibited a fluorescence characteristic of fluorescein. FITC-gelsolin has a peptide-backbone circular dichroism spectrum at 20 degrees C that is indistinguishable from that of native gelsolin, but FITC-gelsolin is considerably more resistant than native gelsolin to thermally induced precipitation. FITC-gelsolin is fully able to carry out severing of F-actin filaments, the prime function of gelsolin in plasma. An opening up of the structure of gelsolin on binding Ca2+ is evident from an increased susceptibility of FITC-gelsolin to quenching by I-. Ca2+ dependence of the interaction between gelsolin and actin is evident in titrations both of intensity and polarization of the fluorescence of FITC-gelsolin solutions. A Ca(2+)-sensitive interaction between gelsolin and tropomyosin also is observed.

Interaction of plasma gelsolin with tropomyosin.

Horse plasma gelsolin labelled with benzophenone-4-isothiocyanate can be photochemically cross-linked to rabbit cardiac tropomyosin. The cross-linking proceeds with greater efficiency in calcium-containing buffers. Further evidence for interaction between these proteins is provided by retention of fluorescently labelled gelsolin on tropomyosin-agarose affinity columns and by the ability of tropomyosin to cause an increase in the fluorescence intensity of gelsolin labelled with fluorescein-5-isothiocyanate. Both of these effects require the presence of calcium ions.

Monomer and excimer fluorescence of horse plasma gelsolin labelled with N-(1-pyrenyl)iodoacetamide.

Horse plasma gelsolin was labelled with the sulfhydryl-specific fluorescent reagent N-(1-pyrenyl)iodoacetamide. The level of incorporation of probe was 1.6 +/- 0.3 mol pyrene/mol gelsolin. The circular dichroism spectrum of pyrenyl-gelsolin and its ability to interact with muscle actin were not different from that found for unmodified gelsolin. The emission from pyrenyl-gelsolin was dominated by a broad emission band centred near 483 nm, characteristic of the presence of pyrene excimers. Analysis of excitation spectra for the monomer and excimer-type fluorescence suggested that ground-state interactions may occur between adjacent pyrenes in the gelsolin structure. In the case either of excimer formation or of ground-state pyrene-pyrene interactions in doubly labelled gelsolin molecules, the modified Cys residues must be in close proximity in the folded protein structure. Thermal denaturation of gelsolin could be monitored by observing the decrease in excimer emission that accompanied heating and unfolding of the tertiary structure. While heat treatment alone did not eliminate excimer fluorescence, digestion of gelsolin with chymotrypsin completely abolished such emission. Also, pyrenyl-gelsolin prepared and studied in 6 M guanidine-HCl exhibited fluorescence characteristic of pyrene monomers exclusively.

Stabilization of the structure of horse plasma vitamin D binding protein by disulfide bonds.

Vitamin D binding protein (DBP) was isolated from horse plasma in a four-step procedure that involved Affi-Gel Blue affinity chromatography, gel filtration, hydroxylapatite chromatography, and anion exchange high-pressure liquid chromatography. The yield of DBP from 80 mL of plasma was 6-7 mg. Horse plasma DBP closely resembles other plasma DBPs, being a tryptophan-free protein of Mr 53,000. It is able to bind to and block the polymerization of monomeric actin. The secondary structure of DBP was calculated from circular dichroism measurements to be 39% alpha-helix, 42% beta-sheet, and 19% random coil. Circular dichroism and fluorescence studies revealed that the disulfide bonds of DBP contribute substantial structural stabilization to the molecule with respect to thermal denaturation. The thermal stability of DBP can be used to advantage. Incorporation of a brief treatment at 70 degrees C into the preparative scheme enables omission of one chromatographic step, without detectable alteration of the purified product.

Hybridization of rabbit cardiac tropomyosin with nonpolymerizable tropomyosin species.

Rabbit cardiac tropomyosin was hybridized with its nonpolymerizable form, produced by treatment with carboxypeptidase A, and with a naturally occurring nonpolymerizable tropomyosin from horse platelets. Hybridization was achieved by heating equimolar mixtures to 60 degrees C in the presence of 10 mM dithiothreitol, followed by recooling. Samples of intact and carboxypeptidase-truncated tropomyosins treated in this way show lower viscosities at low ionic strength than predicted assuming random reformation of the coiled coils, suggesting that hybrids formed with one intact COOH-terminus are unable to polymerize normally. Hybridization of cardiac and platelet tropomyosins was detected by observation of the fluorescence of pyrene groups attached to cysteine residues on platelet tropomyosin.

Interaction of horse plasma gelsolin with the hydrophobic fluorescent probe 2-(N-methylanilino)naphthalene-6-sulfonic acid.

Addition of horse plasma gelsolin to solutions of the fluorescent probe 2-(N-methylanilino)naphthalene-6-sulfonic acid (MANS) results in both a considerable enhancement and blue-shift of the MANS emission, indicative of hydrophobic interaction between MANS and gelsolin. Titrations suggest each gelsolin to bind two to three molecules of MANS, with a dissociation constant for each site of 0.24 microM. The peptide bond circular dichroism of gelsolin is unaffected by interaction with MANS, and viscosity data indicate that MANS does not inhibit the effects of gelsolin on actin polymerization. Fluorescence polarization data confirm gelsolin to be a globular protein and thermal denaturation studies suggest a cooperative melting transition for plasma gelsolin near 46 degrees C.

Coupled responses of the regions near cysteine-190 and the carboxy terminus of rabbit cardiac tropomyosin: fluorescence and circular dichroism studies.

Rabbit cardiac tropomyosin was labeled at Cys-190 with either N-(1-pyrenyl)iodoacetamide (Py) or 6-acryloyl-2-(dimethylamino)naphthalene (AD, acrylodan). Half of the labeled sample then was treated with carboxypeptidase A to produce an identically labeled nonpolymerizable form of tropomyosin, NPTM. Investigation of temperature-dependent changes in pyrene excimer emission, acrylodan fluorescence polarization, and tyrosyl circular dichroism in different samples of tropomyosin and NPTM reveals that absence of the COOH-terminal portion of tropomyosin modifies the response of the Cys-190 region to heat. Removal of the COOH terminus releases certain conformational constraints from the coiled-coil back to and including the Cys-190 region without causing a severe drop in the net alpha-helical content of the protein. Observation of changes in pyrene excimer fluorescence and in fluorescence polarization of acrylodan with time after addition of carboxypeptidase A to samples of labeled tropomyosin directly demonstrates this relaxation process. Thermally induced reduction in tyrosyl circular dichroism, together with consideration of the distribution of tyrosyl residues on tropomyosin, also supports the proposal.

Characterization of horse plasma gelsolin.

Gelsolin can be purified from horse blood plasma by treating the plasma sequentially with an anion-exchange medium in the presence and then the absence of free Ca2+. The purified gelsolin migrates as a 90-kilodalton protein on electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate. It has an absorption coefficient of 1.4 mL/(mg.cm) and is similar in amino acid composition to other plasma gelsolins. Horse plasma gelsolin has an intrinsic sedimentation coefficient of 4.8S and a Stokes' radius of 3.8 nm. Hydrodynamic calculations suggest it to be a rather globular protein of 75,000 relative mass, a value similar to those calculated for human and pig plasma gelsolins from their amino acid sequences. Horse plasma gelsolin is able to nucleate actin polymerization, i.e., to abolish the lag observed between the initiation of polymerization of monomeric actin by the addition of salts and the rapid elongation phase of actin filament growth. This nucleation activity also results in lower final viscosities of F-actin solutions, as the existence of a larger number of filaments in samples that contain gelsolin requires that their average length be shorter.

Interaction of muscle and non-muscle tropomyosins with deoxyribonuclease I.

The sulfhydryl-selective fluorescent reagent acrylodan (6-acryloyl-2-dimethylaminonaphthalene) was used to label tropomyosins from rabbit cardiac muscle and from equine platelets. Addition of bovine pancreatic deoxyribonuclease I to solutions of acrylodan-modified tropomyosins significantly altered the emission properties of the samples. Muscle and non-muscle tropomyosin fluorescence were affected in qualitatively similar manners; emission maxima were red-shifted by about 8 nm to 522-525 nm and maximal intensities were reduced by approximately 15%. Addition of KI to each of the fluorescent samples caused a greater degree of fluorescence quenching in the presence of DNase I than in its absence. The slopes of Stern-Volmer plots were 15-25% steeper in the presence of DNase I. Fluorescence polarization values for acrylodan-labelled tropomyosin samples were 25-35% lower in the presence of DNase I. Each of these effects could be saturated by addition of about a two-fold molar excess of DNase I to tropomyosin. Together they suggest that interaction with DNase I causes localized unfolding of tropomyosin, thereby allowing the fluorescent label to become more exposed to the solvent and less restricted in its local motions. Circular dichroism measurements support this idea. Addition of DNase I to solutions of either labelled or unlabelled tropomyosin results in a net 14-18% loss in ellipticity near 220 nm, indicative of unfolding of alpha-helix.

Interaction of iodoacetamidofluorescein-labelled tropomyosin with deoxyribonuclease I.

5-Iodoacetamidofluorescein (IAF) reacted with rabbit cardiac muscle tropomyosin (TM) to yield a highly fluorescent product, IAF-TM. The extent of labelling reached one fluorescein group per TM molecule in solutions at pH 8.5. While fluorescence polarization values for IAF-TM solutions were unaffected by the presence or absence of KCl, addition of pancreatic deoxyribonuclease I (DNase I) resulted in a 10% drop, suggestive of a greater freedom of motion of the fluorescein label in the presence of DNase I. Furthermore, a 15% increase in slopes of Stern-Volmer plots for IAF-TM in the presence of DNase I demonstrated a greater susceptibility of the fluorescein group to dynamic quenching by iodide. These results suggest that interaction between DNase I and TM produces a localized unfolding of the coiled coil near the IAF reactive site on TM.

Fluorescence from pyrene-labeled native and reconstituted chicken gizzard tropomyosins.

Sulfhydryl groups at Cys-36 on the beta chain and at Cys-190 on the gamma chain of chicken gizzard tropomyosin were reacted with the pyrene-containing sulfhydryl-specific reagents N-(1-pyrenyl)iodoacetamide and N-(1-pyrenyl)maleimide. Tropomyosin prepared and labeled under nondenaturing conditions displayed significant pyrene monomer emission but low levels of pyrene excimer fluorescence. In contrast, tropomyosin subjected to denaturation and renaturation prior to labeling, or labeled in the denatured state prior to renaturation, displayed considerable excimer emission. Furthermore, labeling of isolated beta or gamma chains in denaturant, followed by reconstitution, gave separate samples of beta beta- and gamma gamma-tropomyosin that exhibited even greater pyrene excimer to monomer emission ratios. As pyrene excimers can form only when an excited pyrene is immediately adjacent to a ground state pyrene, i.e., when the labeled Cys residues on the two chains in a tropomyosin coiled coil share the same cross section, these results support conclusions based upon chemical crosslinking studies [C. Sanders, L. D. Burtnick, and L. B. Smillie (1986) J. Biol. Chem. 261, 12774-12778] that native gizzard tropomyosin exists predominantly as a beta gamma-heterodimer. In addition, the low degree of labeling of native gizzard tropomyosin and the differences in degrees of labeling of beta beta- and gamma gamma-tropomyosins in the absence of denaturants reflect on the accessibilities of the sulfhydryl groups in these tropomyosin isoforms. Circular dichroism measurements indicate that the labeled proteins form stable coiled coil structures that have thermal stabilities comparable to that of the native protein.