N-terminal stathmin-like peptides bind tubulin and impede microtubule assembly

Microtubules are major cytoskeletal components involved in numerous cellular functions such as mitosis, cell motility, or intracellular traffic. These cylindrical polymers of alphabeta-tubulin assemble in a closely regulated dynamic manner. We have shown that the stathmin family proteins sequester tubulin in a nonpolymerizable ternary complex, through their stathmin-like domains (SLD) and thus contribute to the regulation of microtubule dynamics. We demonstrate here that short peptides derived from the N-terminal part of SLDs impede tubulin polymerization with various efficiencies and that phosphorylation of the most potent of these peptides reduces its efficiency as in full-length stathmin. To understand the mechanism of action of these peptides, we undertook a NMR-based structural analysis of the peptide-tubulin interaction with the most efficient peptide (I19L). Our results show that, while disordered when free in solution, I19L folds into a beta-hairpin upon binding to tubulin. We further identified, by means of saturation transfer difference NMR, hydrophobic residues located on the beta2-strand of I19L that are involved in its tubulin binding. These structural data were used together with tubulin atomic coordinates from the tubulin/RB3-SLD crystal structure to model the I19L/tubulin interaction. The model agrees with I19L acting through an autonomous tubulin capping capability to impede tubulin polymerization and provides information to help understand the variation of efficiency against tubulin polymerization among the peptides tested. Altogether these results enlighten the mechanism of tubulin sequestration by SLDs, while they pave the way for the development of protein-based compounds aimed at interfering with tubulin polymerization.     

Polymerization and colchicine binding. Two independent properties of tubulin

When stored frozen in 1 M sucrose and 1 mM GTP, tubulin loses polymerizing ability exponentially. Since addition of diethiothreitol does not change the decay half-life, this decrease in activity can not be attributed to disulfide bond formation. When tubulin is stored frozen in dithiothreitol and GTP only, the decay half-life increases by a factor of four, indicating that sucrose destabilizes polymerizing ability. Frozen storage in sucrose has the opposite effect on colchicine binding, which remains at 100% for 40 days. This temporal divergence indicates that colchine binding and polymerization are two independent properties of tubulin.     

Direct observation of steady-state microtubule dynamics

Different types of unusual dynamic behavior have been reported for steady-state microtubules. While almost all earlier reports relied on kinetic measurements of bulk polymerization, we have directly visualized the steady-state addition of subunits to individual microtubules through the use of tubulin derivitized with biotin. Biotinylated tubulin was used both as an internal "seed" for polymerization and as a marker for assembly onto the ends of microtubules composed of purified tubulin. Biotinylated segments were distinguished from unmodified tubulin by double-label immunofluorescence. Microtubule lengths, number concentrations, and segment lengths have been monitored with time at steady state under two buffer conditions. The results indicate that the microtubule steady state under these conditions is a balance between a majority of slowly growing microtubules and a minority of rapidly depolymerizing ones as described by the "dynamic instability" model (Mitchison T., and M. Kirschner, 1984, Nature (Lond.)., 312:232-242). Microtubules show no evidence of treadmilling; instead most show progressive growth off both ends at steady state. Although solvent conditions markedly influence the growth rates, qualitatively the behavior is unchanged.     

Beyond Lumry-Eyring: an unexpected pattern of operational reversibility/irreversibility in protein denaturation

We have found that, contrary to naïve intuition, the degree of operational reversibility in the thermal denaturation of lipase from Thermomyces lanuginosa (an important industrial enzyme) in urea solutions is maximum when the protein is heated several degrees above the end of the temperature‐induced denaturation transition. Upon cooling to room temperature, the protein seems to reach a state with enzymatic activity similar to that of the initial native state, but with higher denaturation temperature and radically different behavior in terms of susceptibility to irreversible denaturation. These results show that patterns of operational reversibility/irreversibility in protein denaturation may be more complex than the often‐taken‐for‐granted, two‐situation classification (reversible vs. irreversible). Furthermore, they are consistent with the possibility of existence of different native or native‐like states separated by high kinetic barriers under native conditions and they suggest experimental procedures to reach and study such “alternative” native states. Proteins 2008. © 2007 Wiley‐Liss, Inc.     

Kainate reversibly aggregates brain tubulin in vitro

The effect of kainate, an heterocyclic analogue of glutamate on tubulin polymerization was studied. We demonstrate that kainate induces a dose-dependent aggregation of rat brain tubulin either purified by DEAE-Sephadex A-50 or in the presence of MAPs into a mesh-like structure. Such polymer is cold-, CaCl2- and colchicine-insensitive. Removal of kainate from the incubation medium yields free tubulin competent for polymerizing into normal microtubules in the presence of GTP.     

Hydrophobic surfaces of tubulin probed by time-resolved and steady-state fluorescence of nile red

Binding of Nile Red to tubulin enhances and blue-shifts fluorescence emission to about 623 nm with a "shoulder" around 665 nm. Binding is reversible and saturable with an apparent Kd of approximately 0.6 microM. Nile Red does not alter tubulin polymerization, and polymerization in 2-(N-morpholino)ethanesulfonic acid (Mes) buffer does not alter the spectrum of the Nile Red-tubulin complex. In contrast, polymerization in glutamate buffer results in a red shift, reduction of intensity, and a decrease in lifetime, suggesting an increase in "polarity" of the binding environment. Lifetimes of 4.5 and 0.6 ns fluorescence in Mes buffer are associated with the 623-nm peak and the 665-nm shoulder, respectively. Indirect excitation spectra for these components are distinct and the 4.5-ns component exhibits tryptophan to Nile Red energy transfer. Acrylamide quenching yields linear Stern-Volmer plots with unchanged lifetimes, indicating static quenching. Apparent quenching constants are wavelength-dependent; global analysis reveals a quenchable component corresponding to the 4.5 ns component and an "unquenchable" component superposing the 0.6-ns spectrum. Analysis of anisotropy decay required an "associative" model which yielded rotational correlation times of greater than 50 ns for the 4.5-ns lifetime and 0.3 ns for the 0.6-ns lifetime. Dilution of tubulin in Mes results in an apparent red shift of emission without lifetime changes, due only to loss of the 623-nm component. These data are reconciled in terms of a model with two binding sites on the tubulin dimer. The more "nonpolar" site is located in a region of subunit-subunit contact which accounts for the fluorescence changes upon dilution; this permits estimation of a subunit dissociation constant of 1 microM.     

Evidence that the tightly bound magnesium in tubulin is associated with the N-site GTP

In an attempt to determine whether the tightly bound Mg2+ found in purified tubulin in associated with the N-site GTP or the E-site GDP or GTP, we removed the E-site nucleotide by several means: (i) alkaline phosphatase treatment; (ii) displacement using excess GMPPCP; and (iii) polymerizing tubulin in the presence of alkaline phosphatase and non-hydrolyzable analogues. The Mg2+ content remained equal to about 1 mol/mol tubulin under conditions where denaturation did not occur. Moreover, the Mg/GTP ratio always remained equal to 1. These results indicate that the Mg2+ is associated with the N-site GTP.     

Structural microtubule cap: stability,catastrophe, rescue,and third state

Microtubules polymerize from GTP-liganded tubulin dimers, but are essentially made of GDP-liganded tubulin. We investigate the tug-of-war resulting from the fact that GDP-liganded tubulin favors a curved configuration, but is forced to remain in a straight one when part of a microtubule. We point out that near the end of a microtubule, the proximity of the end shifts the balance in this tug-of-war, with some protofilament bending as result. This somewhat relaxes the microtubule lattice near its end, resulting in a structural cap. This structural cap thus is a simple mechanical consequence of two well-established facts: protofilaments made of GDP-liganded tubulin have intrinsic curvature, and microtubules are elastic, made from material that can yield to forces, in casu its own intrinsic forces. We explore possible properties of this structural cap, and demonstrate 1) how it allows both polymerization from GTP-liganded tubulin and rapid depolymerization in its absence; 2) how rescue can occur; 3) how a third, meta-stable intermediate state is possible and can explain some experimental results; and 4) how the tapered tips observed at polymerizing microtubule ends are stabilized during growth, though unable to accommodate a lateral cap. This scenario thus supports the widely accepted GTP-cap model by suggesting a stabilizing mechanism that explains the many aspects of dynamic instability.     

The 90-kDa Heat Shock Protein Hsp90 Protects Tubulin against Thermal Denaturation

Hsp90 and tubulin are among the most abundant proteins in the cytosol of eukaryotic cells. Although Hsp90 plays key roles in maintaining its client proteins in their active state, tubulin is essential for fundamental processes such as cell morphogenesis and division. Several studies have suggested a possible connection between Hsp90 and the microtubule cytoskeleton. Because tubulin is a labile protein in its soluble form, we investigated whether Hsp90 protects it against thermal denaturation. Both proteins were purified from porcine brain, and their interaction was characterized in vitro by using spectrophotometry, sedimentation assays, video-enhanced differential interference contrast light microscopy, and native polyacrylamide gel electrophoresis. Our results show that Hsp90 protects tubulin against thermal denaturation and keeps it in a state compatible with microtubule polymerization. We demonstrate that Hsp90 cannot resolve tubulin aggregates but that it likely binds early unfolding intermediates, preventing their aggregation. Protection was maximal at a stoichiometry of two molecules of Hsp90 for one of tubulin. This protection does not require ATP binding and hydrolysis by Hsp90, but it is counteracted by geldanamycin, a specific inhibitor of Hsp90.     

Tubulin-colchicine complex (TC) inhibits microtubule depolymerization by a capping reaction exerted preferentially at the minus end

The effects of colchicine and tubulin-colchicine complex (TC) on microtubule depolymerization were studied using the axoneme-subunit system described previously [Bergen LG, Borisy GG; J Cell Biol 84:141-150, 1980]. This system allows the independent analysis of the polymerization kinetics at both the plus and minus ends of a microtubule. Depolymerization was induced by isothermal dilution with 10 volumes of an experimental solution containing colchicine, TC, or buffer alone. Colchicine alone (5-100 microM) blocked depolymerization at the minus end, whereas depolymerization at the plus end occurred at almost control rates. A similar effect was produced by TC (0.4:1-1:1 molar ratio to free tubulin). High molar ratios of TC to tubulin (10:1) blocked depolymerization at both plus and minus ends, and intermediate molar ratios of TC:T allowed depolymerization of the plus ends but at attenuated rates. The blockage was not readily reversible; TC-affected ends neither shortened upon dilution nor grew longer upon incubation with additional tubulin. We conclude that TC at suprastoichiometric ratios to tubulin inhibits microtubule depolymerization by a capping reaction and that this effect is exerted preferentially at the minus end.     

Interaction of tubulin with octyl glucoside and deoxycholate. 1. Binding and hydrodynamic studies

Tubulin purified from calf brain cytoplasm, normally a compact water-soluble dimer, is able to interact with the mild detergents octyl glucoside (a minimum of 60 detergent molecules) and deoxycholate (95 +/- 8 molecules). Binding is cooperative and approaches saturation below the critical micelle concentration of the amphiphiles. Binding is accompanied by a quenching of the intrinsic protein fluorescence, but no spectral shape changes indicating denaturation such as in the case of sodium dodecyl sulfate are observed. Glycerol, which is known to be preferentially excluded from the tubulin domain and to favor the folded and associated forms of this protein, inhibits the binding of the mild detergents. Octyl glucoside induces a rapidly equilibrating tubulin self-association reaction characterized by a bimodal sedimentation velocity profile with boundaries at approximately 5 and 12 S. Full dissociation of this detergent restores the normal sedimentation behavior to 90% of the protein. Binding of deoxycholate slows the sedimentation velocity of tubulin from s(0)20,w = 5.6 +/- 0.2 S to s(0)20,w = 4.8 +/- 0.3 S. Measurements of the molecular weight of the tubulin-deoxycholate complex indicate an increase from 100,000 to 143,000 +/- 5,000. The diffusion rate consistently decreases from (5.3 +/- 0.5) X 10(-7) to (3.8 +/- 0.2) X 10(-7) cm2 S-1. This is most simply interpreted as an expansion of the undissociated tubulin dimer upon detergent binding (a change in the frictional ratio, f/f min, from 1.35 to 1.86). It is concluded that tubulin shows a reversible transition between the water-soluble state and amphipathic detergent-bound forms which constitute a model system of tubulin-membrane interactions.     

Microtubule elongation and guanosine 5'-triphosphate hydrolysis. Role of guanine nucleotides in microtubule dynamics

The tubulin concentration dependence of the rates of microtubule elongation and accompanying GTP hydrolysis has been studied over a large range of tubulin concentration. GTP hydrolysis followed the elongation process closely at low tubulin concentration and became gradually uncoupled at higher concentrations, reaching a limiting rate of 35-40 s-1. The kinetic parameters for microtubule growth were different at low and high tubulin concentrations. Elongation of microtubules has also been studied in solutions containing GDP and GTP in variable proportions. Only traces of GTP present in GDP were necessary to confer a high stability (low critical concentration) to microtubules. Pure GDP-tubulin was found unable to elongate microtubules in the absence of GTP but blocked microtubule ends with an equilibrium dissociation constant of 5-6 microM. These data were accounted for by a model within which, in the presence of GTP-tubulin at high concentration, microtubules grow at a fast rate with a large GTP cap; the GTP cap may be quite short in the region of the critical concentration; microtubule stability is linked to the strong interaction between GTP and GDP subunits at the elongating site; dimeric GDP-tubulin does not have the appropriate conformation to undergo reversible polymerization. These results are discussed with regard to possible role of GDP and GTP and of GTP hydrolysis in microtubule dynamics.     

Advanced analyses of kinetic stabilities of iggs modified by mutations and glycosylation

The stability of Immunoglobulin G (IgG) affects production, storage and usability, especially in the clinic. The complex thermal and isothermal transitions of IgGs, especially their irreversibilities, pose a challenge to the proper determination of parameters describing their thermodynamic and kinetic stability. Here, we present a reliable mathematical model to study the irreversible thermal denaturations of antibody variants. The model was applied to two unrelated IgGs and their variants with stabilizing mutations as well as corresponding non‐glycosylated forms of IgGs and Fab fragments. Thermal denaturations of IgGs were analyzed with three transitions, one reversible transition corresponding to C_H2 domain unfolding followed by two consecutive irreversible transitions corresponding to Fab and C_H3 domains, respectively. The parameters obtained allowed us to examine the effects of these mutations on the stabilities of individual domains within the full‐length IgG. We found that the kinetic stability of the individual Fab fragment is significantly lowered within the IgG context, possibly because of intramolecular aggregation upon heating, while the stabilizing mutations have an especially beneficial effect. Thermal denaturations of non‐glycosylated variants of IgG consist of more than three transitions and could not be analyzed by our model. However, isothermal denaturations demonstrated that the lack of glycosylation affects the stability of all and not just of the C_H2 domain, suggesting that the partially unfolded domains may interact with each other during unfolding. Investigating thermal denaturation of IgGs according to our model provides a valuable tool for detecting subtle changes in thermodynamic and/or kinetic stabilities of individual domains.     

The reversible heat denaturation of chymotrypsinogen

Within a restricted range of pH and protein concentration crystalline chymotrypsinogen undergoes thermal denaturation which is wholly reversed upon cooling. At a given temperature an equilibrium exists between native and reversibly denatured protein. Within the pH range 2 to 3 the amount of denatured protein is a function of the third power of the hydrogen ion activity. The presence of small amounts of electrolyte causes aggregation of the reversibly denatured protein. A specific anion effect has been observed at pH 2 but not at pH 3. Both the reversible denaturation reaction and the reversal reaction have been found to be first order reactions with respect to protein and the kinetic and thermodynamic constants for both reactions have been approximated at pH 2 and at pH 3. Renatured chymotrypsinogen has been found to be identical with native chymotrypsinogen with respect to crystallizability, solubility, activation to δ-chymotrypsin, sedimentation rate, and behavior upon being heated. Irreversible denaturation of chymotrypsinogen has been found to depend on pH, temperature, protein concentration, and time of heating. Irreversible denaturation results in an aggregation of the denatured protein.     

Decrease in stability of human albumin with increase in protein concentration

The stability (reflected in denaturation temperature, Td) of defatted human albumin monomer, monitored by differential scanning calorimetry, decreases with increasing protein concentration. This is shown to be compatible with a simple model in which reversible polymerization of denatured monomer promotes unfolding. This model also predicts an increase in transition cooperativity with decreasing protein concentration whereas experimentally cooperativity decreases because the rate of thermally induced polymerization of unfolded monomer is slow relative to the scan rate of the calorimeter. The denaturation of undefatted human albumin monomer, subsaturated with high affinity endogenous long-chain fatty acid (LCFA), was previously observed by differential scanning calorimetry to be a biphasic process. Td for the first endotherm, associated with the denaturation of LCFA-poor species, decreases with increasing protein concentration similar to that for defatted monomer whereas Td for the second endotherm, associated with denaturation of LCFA-rich species, is independent of concentration. The magnitude of the concentration dependence of Td relates directly to the extent of polymerization of denatured monomer, which decreases with increasing level of bound ligand. The bimodal thermogram observed for undefatted monomer persists upon simultaneous extrapolation of Td values to low concentration and low scan rate thereby demonstrating that this biphasic denaturation arising from ligand redistribution during denaturation is a true thermodynamic phenomenon and not an artifact of specific experimental conditions or the method used to induce denaturation.     

The relationship between polymerization of complement component C9 and membrane channel formation.

C9 was studied with the objective to clarify the relationship between the process of C9 polymerization and membrane channel formation. Conditions that favor C9 polymerization include low ionic strength and calcium ion in the buffer. Moreover, polymerization is dependent on the concentration of C9. Calcium ion evokes about a threefold increase in the affinity constant for C9 self-association, and at 0 degrees C it imparts reversible amphiphilic properties in the molecule. These were discerned by measuring increases in the degree of reversible nonspecific binding of C9 to hydrophobic (tyramine-zymosan) and hydrophilic (arginyl-glutamyl-zymosan) supports as well as to erythrocytes. At 0 degrees C the hydrophilic-to-amphiphilic alteration of C9 is reversible, but upon incubation at 37 degrees C this transition is rendered permanent with the formation of poly(C9). A functional relationship between C9 polymerization and cytolysis was demonstrated by showing that polymerizing C9 can lyse reduced and alkylated erythrocytes. By studying comparative radiolabeling of tyrosine side chains within thrombin-nicked C9 and its polymerized form, it was demonstrated that upon polymerization the membrane-binding site of C9 becomes exposed. It is concluded that the process of circular polymerization of C9 causes a hydrophilic-to-amphiphilic transition that is required for membrane perforation and channel formation.     

Monotonic versus oscillating microtubule assembly: a cryo-electron microscope study

Depending on the free GTP concentration, microtubules can assemble following either a monotonic or an oscillatory mode. We have used cryoelectron microscopy to compare the tubulin assemblies characteristic of each polymerization pathway. We focus on the first assembly peak. At this particular time point, despite their strikingly different subsequent evolution, both systems are similar with regard to the extent of tubulin polymerization and to the microtubule length distribution. The present study shows that whilst the observed microtubule structures are the same in both systems, the oscillatory system shows quantities of closed ring-like tubulin oligomers, far in excess of those observed in the monotonic system. Furthermore, the conversion of the oscillating system to a monotonic one by GTP addition during the first oscillation is accompanied by a marked decrease in the number of rings. Based on these results we propose that the GTP dependent step which governs microtubule oscillations is the opening of inactive tubulin oligomers.     

The denaturation of orosomucoid

Highly purified human orosomucoid exhibits apparent multistate behavior on thermal denaturation, with ΔH(cal) for the transition being 119 kcal/mol at pH 7.4. Asialoorosomucoid denatured similarly. A domain structure could not be demonstrated for orosomucoid with cyanogen bromide fragments, although some reannealing of these did occur. It is suggested that the source of the apparent multistate behavior may lie in the existence of polypeptide variants, known to be present in orosomucoid. Although some polymerization occurs on heating, the unpolymerized material shows reversible thermal denaturation behavior. The presence of low concentrations of ethanol induces a significant endotherm which moves to lower temperatures with increasing ethanol concentration. This endotherm was irreversible in the continued presence of ethanol.     

Microtubules and protein secretion in rat lacrimal glands. Inhibitory effect of the tubulin . colchicine complex isolated from lacrimal glands upon brain tubulin polymerization. Identification of the complex by gel electrophoresis.

The specific inhibitory effect of colchicine upon protein secretion by lacrimal glands could be related to the formation of a complex between colchicine and tubulin from the soluble fraction of the gland. By gel electrophoresis under nondissociating conditions, it is shown that this complex is similar to the colchicine . tubulin complex from brain. The complex isolated from lacrimal glands is highly inhibitory upon brain tubulin assembly since as low as 0.07 microM complex impedes the polymerization of 8 microM tubulin by 50%, compared to 3 microM for free colchicine. Therefore, a small percentage of complexed tubulin (0.9%) is enough for polymerization to be blocked. In lacrimal glands the complex might prevent the polymerization of tubulin, and colchicine shift the tubulin in equilibrium microtubules equilibrium to microtubules disassembly. The disorganization of the labile microtubular system could lead to a modification of the transport of the secretory granules and to a perturbation of secretion.     

The effect of substrate binding on the conformation and structural stability of Herpes simplex virus type 1 thymidine kinase

The structure of Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) is known at high resolution in complex with a series of ligands and exhibits important structural similarities to the nucleoside monophosphate (NMP) kinase family, which are known to show large conformational changes upon binding of substrates. The effect of substrate binding on the conformation and structural stability of TK(HSV1), measured by thermal denaturation experiments, far-UV circular dichroism (CD) and fluorescence is described, and the results indicate that the conformation of the ligand-free TK(HSV1) is less ordered and less stable compared to the ligated enzyme. Furthermore, two crystal structures of TK(HSV1) in complex with two new ligands, HPT and HMTT, refined to 2.2 A are presented. Although TK(HSV1):HPT does not exhibit any significant deviations from the model of TK(HSV1):dT, the TK(HSV1):HMTT complex displays a unique conformationally altered active site resulting in a lowered thermal stability of this complex. Moreover, we show that binding affinity and binding mode of the ligand correlate with thermal stability of the complex. We use this correlation to propose a method to estimate binding constants for new TK(HSV1)substrates using thermal denaturation measurements monitored by CD spectroscopy. The kinetic and structural results of both test substrates HPT and HMTT show that the CD thermal denaturation system is very sensitive to conformational changes caused by unusual binding of a substrate analog.