Microtubule-destabilizing agents: structural and mechanistic insights from the interaction of colchicine and vinblastine with tubulin.

Microtubules (MTs) are dynamic structures of the eukaryotic cytoskeleton that, during cell division, form the mitotic spindle. Perturbing them leads to mitotic arrest and ultimately to cell death. Consistently, MTs and their building block, αß tubulin, are one of the best characterized targets in anti-cancer chemotherapy. Drugs that interfere with MTs either stabilize or destabilize them. The latter class is the subject of this review. These ligands bind to the colchicine site or to the vinca domain, two distinct sites located at a distance from each other on tubulin. Nevertheless the effects of both classes of ligands share a common theme, they prevent the formation of MT specific contacts, therefore triggering their disassembly.

[The stathmin-tubulin interaction and the regulation of the microtubule assembly]. / L'interaction stathmine-tubuline et la régulation de la dynamique des microtubules.

Stathmin family proteins interact with tubulin and negatively regulate its assembly in microtubules. One stathmin molecule forms a complex with two alphabeta tubulin heterodimers in an interaction that is weakened upon stathmin phosphorylation. The X-ray structure of crystals of the complex reveals a head-to-tail arrangement of the two tubulins which are connected by a long stathmin alpha helix. By holding tubulins in a curved complex that is not incorporated in microtubules, stathmin lowers the pool of "assembly competent" tubulin. An alternate mechanism has been also proposed to account for the stathmin action in vivo; it involves a direct interaction of stathmin with microtubule (+) ends. More experiments are needed to evaluate the relative contribution of this alternative mechanism to the regulation of tubulin assembly by stathmin.

Mechanism of neutralization of influenza virus infectivity by antibodies.

We have determined the mechanism of neutralization of influenza virus infectivity by three antihemagglutinin monoclonal antibodies, the structures of which we have analyzed before as complexes with hemagglutinin. The antibodies differ in their sites of interaction with hemagglutinin and in their abilities to interfere in vitro with its two functions of receptor binding and membrane fusion. We demonstrate that despite these differences all three antibodies neutralize infectivity by preventing virus from binding to cells. Neutralization occurs at an average of one antibody bound per four hemagglutinins, a ratio sufficient to prevent the simultaneous receptor binding of hemagglutinins that is necessary to attach virus to cells.

An antibody that prevents the hemagglutinin low pH fusogenic transition.

We have determined the structure of a complex of influenza hemagglutinin (HA) with an antibody that binds simultaneously to the membrane-distal domains of two HA monomers, effectively cross-linking them. The antibody prevents the low pH structural transition of HA that is required for its membrane fusion activity, providing evidence that a rearrangement of HA membrane-distal domains is an essential component of the transition.

Cleavage of vesicular stomatitis virus matrix protein prevents self-association and leads to crystallization.

The matrix protein (M) of vesicular stomatitis virus is responsible for the budding of newly formed virions out of host cells. In vitro, it has been shown to self-associate, a property that may be related to the role of M in virus assembly but also prevents crystallization. Using limited proteolysis by thermolysin, we have isolated and characterized two soluble fragments of the protein that remain noncovalently associated. The digestion product does not self-associate nor is it recruited in aggregates formed by intact M molecules. These results identify a peptide, located at the surface of the protein and disorganized by thermolysin cleavage, responsible for M self-association. The thermolysin-resistant core of M has been crystallized and the crystals diffract to 2-A resolution.

The 4 A X-ray structure of a tubulin:stathmin-like domain complex.

Phosphoproteins of the stathmin family interact with the alphabeta tubulin heterodimer (tubulin) and hence interfere with microtubule dynamics. The structure of the complex of GDP-tubulin with the stathmin-like domain of the neural protein RB3 reveals a head-to-tail assembly of two tubulins with a 91-residue RB3 alpha helix in which each copy of an internal duplicated sequence interacts with a different tubulin. As a result of the relative orientations adopted by tubulins and by their alpha and beta subunits, the tubulin:RB3 complex forms a curved structure. The RB3 helix thus most likely prevents incorporation of tubulin into microtubules by holding it in an assembly with a curvature very similar to that of the depolymerization products of microtubules.

Structural evidence for a programmed general base in the active site of a catalytic antibody.

The crystal structure of the complex of a catalytic antibody with its cationic hapten at 1.9-A resolution demonstrates that the hapten amidinium group is stabilized through an ionic pair interaction with the carboxylate of a combining-site residue. The location of this carboxylate allows it to act as a general base in an allylic rearrangement. When compared with structures of other antibody complexes in which the positive moiety of the hapten is stabilized mostly by cation-pi interactions, this structure shows that the amidinium moiety is a useful candidate to elicit a carboxylate in an antibody combining site at a predetermined location with respect to the hapten. More generally, this structure highlights the advantage of a bidentate hapten for the programmed positioning of a chemically reactive residue in an antibody through charge complementarity to the hapten.

A neutralizing antibody Fab-influenza haemagglutinin complex with an unprecedented 2:1 stoichiometry: characterization and crystallization.

The haemagglutinin HA is a trimer of identical subunits and is the more abundant viral surface glycoprotein of the influenza virus. It is the target of antibodies that neutralize viral infectivity. Antibodies that bind to HA with 3:1 and 1:1 stoichiometries have been identified. Here, an antibody whose Fab binds to HA with an unprecedented 2:1 Fab:HA stoichiometry is characterized. The complex has been crystallized and synchrotron data to 3.5 A resolution have been collected. Molecular replacement confirms the stoichiometry of the complex.

Structural evidence for recognition of a single epitope by two distinct antibodies.

The structure of a complex between the hemagglutinin of influenza virus and the Fab of a neutralizing antibody was determined by X-ray crystallography at 2.8 A resolution. This antibody and another which has only 56% sequence identity bind to the same epitope with very similar affinities and in the same orientation. One third of the interactions is conserved in the two complexes; a significant proportion of the interactions that differ are established by residues of the H3 complementarity-determining regions (CDR) which adopt distinct conformations in the two antibodies. This demonstrates that there is a definite flexibility in the selection of antibodies that bind to a given epitope, despite the high affinity of their complexes. This flexibility allows the humoral immune response to be redundant, a feature that may be useful in achieving longer lasting protection against evolving viral pathogens.

Neither phosphorylation nor the amino-terminal part of rabies virus phosphoprotein is required for its oligomerization.

Rabies virus (PV strain) phosphoprotein (P) was expressed in bacteria. This recombinant protein binds specifically to the nucleoprotein-RNA complex purified from infected cells. Chemical cross-linking and gel-filtration studies indicated that the P protein forms oligomers. Analytical centrifugation data demonstrated the co-existence of monomeric and oligomeric forms of rabies virus P protein and suggested that there is an equilibrium between these species. As P expressed in bacteria is not phosphorylated, this result indicates that P phosphorylation is not required for its oligomerization. Although an alignment of several rhabdovirus P sequences revealed that the amino-terminal domain of P has a conserved predicted propensity to form helical coiled coils, an amino-terminally truncated form of P protein, lacking the first 52 residues, was also shown to be oligomeric. Therefore, the amino-terminal domain of rabies virus P is not necessary for its oligomerization.

Diverse structural solutions to catalysis in a family of antibodies.

BACKGROUND: Small organic molecules coupled to a carrier protein elicit an antibody response on immunisation. The diversity of this response has been found to be very narrow in several cases. Some antibodies also catalyse chemical reactions. Such catalytic antibodies are usually identified among those that bind tightly to an analogue of the transition state (TSA) of the relevant reaction; therefore, catalytic antibodies are also thought to have restricted diversity. To further characterise this diversity, we investigated the structure and biochemistry of the catalytic antibody 7C8, one of the most efficient of those which enhance the hydrolysis of chloramphenicol esters, and compared it to the other catalytic antibodies elicited in the same immunisation. RESULTS: The structure of a complex of the 7C8 antibody Fab fragment with the hapten TSA used to elicit it was determined at 2.2 A resolution. Structural comparison with another catalytic antibody (6D9) raised against the same hapten revealed that the two antibodies use different binding modes. Furthermore, whereas 6D9 catalyses hydrolysis solely by transition-state stabilisation, data on 7C8 show that the two antibodies use mechanisms where the catalytic residue, substrate specificity and rate-limiting step differ. CONCLUSIONS: Our results demonstrate that substantial diversity may be present among antibodies catalysing the same reaction. Therefore, some of these antibodies represent different starting points for mutagenesis aimed at boosting their activity. This increases the chance of obtaining more proficient catalysts and provides opportunities for tailoring catalysts with different specificities.

A complex of influenza hemagglutinin with a neutralizing antibody that binds outside the virus receptor binding site.

The structure of a complex of influenza hemagglutinin (HA) with a neutralizing antibody shows that the antibody binds to HA at a distance from the virus receptor binding site. Comparison of the properties of this antibody and its Fab with those of an antibody that recognizes an epitope overlapping the receptor binding site leads to two main conclusions. First, inhibition of receptor binding is an important component of neutralization. Second, the efficiency of neutralization by the antibodies ranks in the same order as their avidities for HA, and their large size makes these antibodies highly efficient at neutralization, regardless of the location of their epitope in relation to the virus receptor binding site. These observations provide rationales for the range of antibody specificities that are detected in immune sera and for the distribution of sequence changes on the membrane-distal surface of influenza HAs that occur during 'antigenic drift.'

Crossreactivity, efficiency and catalytic specificity of an esterase-like antibody.

The antibody D2.3 catalyzes the hydrolysis of several p-nitrobenzyl and p-nitrophenyl esters with significant rate enhancement; product inhibition is observed with the former compounds but not with the latter. Whereas enzyme specificity has been extensively studied by X-ray crystallography, structural data on catalytic antibodies have thus far related only to one of the reactions they catalyze. To investigate the substrate specificity and the substrate relative to product selectivity of D2.3, we have determined the structures of its complexes with two p-nitrophenyl phosphonate transition state analogs (TSAs) and with the reaction product, p-nitrophenol. The complexes with these TSAs, determined at 1.9 A resolution, and that with p-nitrobenzyl phosphonate determined previously, differ mainly by the locations and conformations of the ligands. Taken together with kinetic data, the structures suggest that a hydrogen bond to an atom of the substrate distant by eight covalent bonds from the carbonyl group of the hydrolyzed ester bond contributes to catalytic efficiency and substrate specificity. The structure of Fab D2.3 complexed with p-nitrophenol was determined at 2.1 A resolution. Release of p-nitrophenol is facilitated due to the unfavourable interaction of the partial charge of the nitro group of p-nitrophenolate with the hydrophobic cavity where it is located, and to the absence of a direct hydrogen bond between the product and the Fab. Catalytic specificity and the manner of product release are both affected by interactions with substrate atoms remote from the reaction center that were not programmed in the design of the TSA used to elicit this antibody. Selection of a catalytic antibody that makes use of TSA unprogrammed features has been made practical because of the screening for catalytic efficiency incorporated in the procedure used to obtain it.

Antigen distortion allows influenza virus to escape neutralization.

The structure of the hemagglutinin (HA) of a mutant influenza virus that escapes neutralization by a monoclonal antibody shows that the mutation causes changes in HA structure which avoid an energetically less favorable conformation. However, the structure of the mutant HA.Fab complex indicates that the antibody binds selectively to mutant HA in a wild type-like distorted conformation. The association of an antibody with a less favored HA conformation represents an alternative to previously described mechanisms of escape from neutralization by antibodies.

The stathmin/tubulin interaction in vitro.

Stathmin is a highly conserved ubiquitous cytoplasmic protein, phosphorylated in response to extracellular signals and during the cell cycle. Stathmin has recently been shown to destabilize microtubules, but the molecular mechanisms of this function remained unclear. We show here that stathmin directly interacts with tubulin. We assessed the conditions of this interaction and determined some its quantitative parameters using plasmon resonance, gel filtration chromatography, and analytical ultracentrifugation. The stathmin/tubulin interaction leads to the formation of a 7.7 S complex with a 60-A Stokes radius, associating one stathmin with two tubulin heterodimer molecules as determined by direct quantification by Western blotting. This interaction is sensitive to pH and ionic environment. Its equilibrium dissociation constant, determined by plasmon resonance measurement of kinetic constants, has an optimum value of 0.5 microM at pH 6.5. The affinity was lowered with a fully "pseudophosphorylated" 4-Glu mutant form of stathmin, suggesting that it is modulated in vivo by stathmin phosphorylation. Finally, analysis of microtubule dynamics by video microscopy shows that, in our conditions, stathmin reduces the growth rate of microtubules with no effect on the catastrophe frequency. Overall, our results suggest that the stathmin destabilizing activity on microtubules is related to tubulin sequestration by stathmin.

X-ray structures of a hydrolytic antibody and of complexes elucidate catalytic pathway from substrate binding and transition state stabilization through water attack and product release.

The x-ray structures of the unliganded esterase-like catalytic antibody D2.3 and its complexes with a substrate analogue and with one of the reaction products are analyzed. Together with the structure of the phosphonate transition state analogue hapten complex, these crystal structures provide a complete description of the reaction pathway. At alkaline pH, D2.3 acts by preferential stabilization of the negatively charged oxyanion intermediate of the reaction that results from hydroxide attack on the substrate. A tyrosine residue plays a crucial role in catalysis: it activates the ester substrate and, together with an asparagine, it stabilizes the oxyanion intermediate. A canal allows facile diffusion of water molecules to the reaction center that is deeply buried in the structure. Residues bordering this canal provide targets for mutagenesis to introduce a general base in the vicinity of the reaction center.

Mechanism of inactivation of a catalytic antibody by p-nitrophenyl esters.

Antibody CNJ206 catalyses the hydrolysis of p-nitrophenyl esters with significant rate enhancement; however, after a few cycles, 90% of the catalytic activity of CNJ206 is irreversibly lost. This report investigates the properties of the inactivated Fab (fragment antigen binding). After inactivation, the residual esterase activity of CNJ206 is similar to that of the catalytic antibody inhibited by the transition-state analogue (TSA) used to elicit it; the affinity of CNJ206 for the TSA is also dramatically lowered. Here we propose a simple scheme that accounts for the steady-state kinetics of inactivation. The following lines of evidence, when taken together, suggest that stable acylated tyrosine side chains within or close to the Fab combining site are involved in the inactivation process: isoelectric focusing and matrix-assisted-laser-desorption-ionisation-time-of-flight (MALDI-TOF) mass spectrometry show that incubation with substrate results in several acylated Fab species; inactivation is stable at pH 8, is reversed by mild hydroxylamine treatment and follows the same kinetics as inhibition of binding, which is slowed down by the presence of the TSA hapten. Analysis of the Fab-TSA X-ray structure shows that three tyrosine residues are potential candidates for the inactivation of CNJ206 by its substrates, Tyr L96 being the most likely one; this also suggests that site-directed mutation of one or more of these residues might prevent substrate inactivation and significantly improve catalysis.

Structural convergence in the active sites of a family of catalytic antibodies.

The x-ray structures of three esterase-like catalytic antibodies identified by screening for catalytic activity the entire hybridoma repertoire, elicited in response to a phosphonate transition state analog (TSA) hapten, were analyzed. The high resolution structures account for catalysis by transition state stabilization, and in all three antibodies a tyrosine residue participates in the oxyanion hole. Despite significant conformational differences in their combining sites, the three antibodies, which are the most efficient among those elicited, achieve catalysis in essentially the same mode, suggesting that evolution for binding to a single TSA followed by screening for catalysis lead to antibodies with structural convergence.

Similarities of hydrolytic antibodies revealed by their X-ray structures: a review.

Numerous antibodies have been programmed to catalyse the hydrolysis of esters as well as other acyl transfer reactions. They were raised against stable analogues that model the structure of the tetrahedral transition states of these reactions. The three-dimensional structures of four hydrolytic antibodies complexed to their respective phosphonate transition state analogues (TSAs) reveal a similar orientation of hapten relative to the antibody. Analysis of the four combining sites suggests that residues binding the phosphonate TSA stabilise the oxyanion intermediate of the reaction and play a preponderant role in catalysis. Comparison of catalytic antibodies selected from the same hybridoma fusion indicates a high similarity of the motifs that catalyse the hydrolysis of a given substrate.