Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure

Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments.     

Crystal structure of a TOG domain: conserved features of XMAP215/Dis1-family TOG domains and implications for tubulin binding

Members of the XMAP215/Dis1 family of microtubule-associated proteins (MAPs) are essential for microtubule growth. MAPs in this family contain several 250 residue repeats, called TOG domains, which are thought to bind tubulin dimers and promote microtubule polymerization. We have determined the crystal structure of a single TOG domain from the Caenorhabditis elegans homolog, Zyg9, to 1.9 A resolution, and from it we describe a structural blueprint for TOG domains. These domains are flat, paddle-like structures, composed of six HEAT-repeat elements stacked side by side. The two wide faces of the paddle contain the HEAT-repeat helices, and the two narrow faces, the intra- and inter-HEAT repeat turns. Solvent-exposed residues in the intrarepeat turns are conserved, both within a particular protein and across the XMAP215/Dis1 family. Mutation of some of these residues in the TOG1 domain from the budding yeast homolog, Stu2p, shows that this face indeed participates in the tubulin contact.     

What tubulin drugs tell us about microtubule structure and dynamics

A wide range of small molecules, including alkaloids, macrolides and peptides, bind to tubulin and disturb microtubule assembly dynamics. Some agents inhibit assembly, others inhibit disassembly. The binding sites of drugs that stabilize microtubules are discussed in relation to the properties of microtubule associated proteins. The activities of assembly inhibitors are discussed in relation to different nucleotide states of tubulin family protein structures.     

The solution structure of the N-terminal domain of human tubulin binding cofactor C reveals a platform for tubulin interaction

Human Tubulin Binding Cofactor C (TBCC) is a post-chaperonin involved in the folding and assembly of α- and β-tubulin monomers leading to the release of productive tubulin heterodimers ready to polymerize into microtubules. In this process it collaborates with other cofactors (TBC's A, B, D, and E) and forms a supercomplex with TBCD, β-tubulin, TBCE and α-tubulin. Here, we demonstrate that TBCC depletion results in multipolar spindles and mitotic failure. Accordingly, TBCC is found at the centrosome and is implicated in bipolar spindle formation. We also determine by NMR the structure of the N-terminal domain of TBCC. The TBCC N-terminal domain adopts a spectrin-like fold topology composed of a left-handed 3-stranded α-helix bundle. Remarkably, the 30-residue N-terminal segment of the TBCC N-terminal domain is flexible and disordered in solution. This unstructured region is involved in the interaction with tubulin. Our data lead us to propose a testable model for TBCC N-terminal domain/tubulin recognition in which the highly charged N-terminus as well as residues from the three helices and the loops interact with the acidic hypervariable regions of tubulin monomers.     

Crystallographic studies of RNA hairpins in complexes with recombinant MS2 capsids: implications for binding requirements.

The coat protein of bacteriophage MS2 is known to bind specifically to an RNA hairpin formed within the MS2 genome. Structurally this hairpin is built up by an RNA double helix interrupted by one unpaired nucleotide and closed by a four-nucleotide loop. We have performed crystallographic studies of complexes between MS2 coat protein capsids and four RNA hairpin variants in order to evaluate the minimal requirements for tight binding to the coat protein and to obtain more information about the three-dimensional structure of these hairpins. An RNA fragment including the four loop nucleotides and a two-base-pair stem but without the unpaired nucleotide is sufficient for binding to the coat protein shell under the conditions used in this study. In contrast, an RNA fragment containing a stem with the unpaired nucleotide but missing the loop nucleotides does not bind to the protein shell.     

Structure of GABARAP in two conformations: implications for GABA(A) receptor localization and tubulin binding.

GABARAP recognizes and binds the gamma2 subunit of the GABA(A) receptor, interacts with microtubules and the N-ethyl maleimide sensitive factor, and is proposed to function in GABA(A) receptor trafficking and postsynaptic localization. We have determined the crystal structure of human GABARAP at 1.6 A resolution. The structure comprises an N-terminal helical subdomain and a ubiquitin-like C-terminal domain. Structure-based mutational analysis demonstrates that the N-terminal subdomain is responsible for tubulin binding while the C-terminal domain contains the binding site for the GABA(A). A second GABARAP crystal form was determined at 1.9 A resolution and documents that GABARAP can self-associate in a head-to-tail manner. The structural details of this oligomerization reveal how GABARAP can both promote tubulin polymerization and facilitate GABA(A) receptor clustering.     

Transmission intensity and drug resistance in malaria population dynamics: implications for climate change

Although the spread of drug resistance and the influence of climate change on malaria are most often considered separately, these factors have the potential to interact through altered levels of transmission intensity. The influence of transmission intensity on the evolution of drug resistance has been addressed in theoretical studies from a population genetics' perspective; less is known however on how epidemiological dynamics at the population level modulates this influence. We ask from a theoretical perspective, whether population dynamics can explain non-trivial, non-monotonic, patterns of treatment failure with transmission intensity, and, if so, under what conditions. We then address the implications of warmer temperatures in an East African highland, where, as in other similar regions at the altitudinal edge of malaria's distribution, there has been a pronounced increase of cases from the 1970s to the 1990s. Our theoretical analyses, with a transmission model that includes different levels of immunity, demonstrate that an increase in transmission beyond a threshold can lead to a decrease in drug resistance, as previously shown, but that a second threshold may occur and lead to the re-establishment of drug resistance. Estimates of the increase in transmission intensity from the 1970s to the 1990s for the Kenyan time series, obtained by fitting the two-stage version of the model with an explicit representation of vector dynamics, suggest that warmer temperatures are likely to have moved the system towards the first threshold, and in so doing, to have promoted the faster spread of drug resistance. Climate change and drug resistance can interact and need not be considered as alternative explanations for trends in disease incidence in this region. Non-monotonic patterns of treatment failure with transmission intensity similar to those described as the 'valley phenomenon' for Uganda can result from epidemiological dynamics but under poorly understood assumptions.     

Botulinum neurotoxin inhibitor binding dynamics and kinetics relevant for drug design

BackgroundA natural product analog, 3-(4-nitrophenyl)-7H-furo[3,2-g]chromen-7-one, which is a nitrophenyl psoralen (NPP) was found to be an effective inhibitor of botulinum neurotoxin type A (BoNT/A).MethodsIn this work, we performed enzyme inhibition kinetics and employed biochemical techniques such as isothermal calorimetry (ITC) and fluorescence spectroscopy as well as molecular modeling to examine the kinetics and binding mechanism of NPP inhibitor with BoNT/A LC.ResultsStudies of inhibition mechanism and binding dynamics of NPP to BoNT/A light chain (BoNT/A LC) showed that NPP is a mixed type inhibitor for the zinc endopeptidase activity, implying that at least part of the inhibitor-enzyme binding site may be different from the substrate-enzyme binding site. By using biochemical techniques, we demonstrated NPP forms a stable complex with BoNT/A LC. These observations were confirmed by Molecular Dynamics (MD) simulation, which demonstrates that NPP binds to the site near the active site.ConclusionThe NPP binding interferes with BoNT/A LC binding to the SNAP-25, hence, inhibits its cleavage. Based on these results, we propose a modified strategy for designing a molecule to enhance the efficiency of the inhibition against the neurotoxic effect of BoNT.General significanceInsights into the interactions of NPP with BoNT/A LC using biochemical and computational approaches will aid in the future development of effective countermeasures and better pharmacological strategies against botulism.     

Structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design

Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. The coronavirus main protease (M^pro), which plays a pivotal role in viral gene expression and replication through the proteolytic processing of replicase polyproteins, is an attractive target for anti-CoV drug design. In this study, the crystal structures of infectious bronchitis virus (IBV) M^pro and a severe acute respiratory syndrome CoV (SARS-CoV) M^pro mutant (H41A), in complex with an N-terminal autocleavage substrate, were individually determined to elucidate the structural flexibility and substrate binding of M^pro. A monomeric form of IBV M^pro was identified for the first time in CoV M^pro structures. A comparison of these two structures to other available M^pro structures provides new insights for the design of substrate-based inhibitors targeting CoV M^pros. Furthermore, a Michael acceptor inhibitor (named N3) was cocrystallized with IBV M^pro and was found to demonstrate in vitro inactivation of IBV M^pro and potent antiviral activity against IBV in chicken embryos. This provides a feasible animal model for designing wide-spectrum inhibitors against CoV-associated diseases. The structure-based optimization of N3 has yielded two more efficacious lead compounds, N27 and H16, with potent inhibition against SARS-CoV M^pro.     

Calculation of cyclodextrin binding affinities: energy, entropy, and implications for drug design

The second generation Mining Minima method yields binding affinities accurate to within 0.8 kcal/mol for the associations of alpha-, beta-, and gamma-cyclodextrin with benzene, resorcinol, flurbiprofen, naproxen, and nabumetone. These calculations require hours to a day on a commodity computer. The calculations also indicate that the changes in configurational entropy upon binding oppose association by as much as 24 kcal/mol and result primarily from a narrowing of energy wells in the bound versus the free state, rather than from a drop in the number of distinct low-energy conformations on binding. Also, the configurational entropy is found to vary substantially among the bound conformations of a given cyclodextrin-guest complex. This result suggests that the configurational entropy must be accounted for to reliably rank docked conformations in both host-guest and ligand-protein complexes. In close analogy with the common experimental observation of entropy-enthalpy compensation, the computed entropy changes show a near-linear relationship with the changes in mean potential plus solvation energy.     

The crystal structure of nucleoplasmin-core: implications for histone binding and nucleosome assembly.

The efficient assembly of histone complexes and nucleosomes requires the participation of molecular chaperones. Currently, there is a paucity of data on their mechanism of action. We now present the structure of an N-terminal domain of nucleoplasmin (Np-core) at 2.3 A resolution. The Np-core monomer is an eight-stranded beta barrel that fits snugly within a stable pentamer. In the crystal, two pentamers associate to form a decamer. We show that both Np and Np-core are competent to assemble large complexes that contain the four core histones. Further experiments and modeling suggest that these complexes each contain five histone octamers which dock to a central Np decamer. This work has important ramifications for models of histone storage, sperm chromatin decondensation, and nucleosome assembly.     

Structural basis for ligand binding and specificity in adrenergic receptors: implications for GPCR-targeted drug discovery

Crystal structures of engineered human beta 2-adrenergic receptors (ARs) in complex with an inverse agonist ligand, carazolol, provide three-dimensional snapshots of the disposition of seven transmembrane helices and the ligand-binding site of an important G protein-coupled receptor (GPCR). As expected, beta 2-AR shares substantial structural similarities with rhodopsin, the dim-light photoreceptor of the rod cell. However, although carazolol and the 11- cis-retinylidene moiety of rhodopsin are situated in the same general binding pocket, the second extracellular (E2) loop structures are quite distinct. E2 in rhodopsin shows beta-sheet structure and forms part of the chromophore-binding site. In the beta 2-AR, E2 is alpha-helical and seems to be distinct from the receptor's active site, allowing a potential entry pathway for diffusible ligands. The structures, together with extensive structure-activity relationship (SAR) data from earlier studies, provide insight about possible structural determinants of ligand specificity and how the binding of agonist ligands might alter receptor conformation. We review key features of the new beta 2-AR structures in the context of recent complementary work on the conformational dynamics of GPCRs. We also report 600 ns molecular dynamics simulations that quantified beta 2-AR receptor mobility in a membrane bilayer environment and show how the binding of an agonist ligand, adrenaline (epinephrine), causes conformational changes to the ligand-binding pocket and neighboring helices.     

Refined solution structure of omega-conotoxin GVIA: implications for calcium channel binding.

The polypeptide omega-conotoxin GVIA (GVIA) is an N-type calcium channel blocker from the venom of Conus geographus, a fish-hunting cone shell. Here we describe a high-resolution solution structure of this member of the 'inhibitor cystine knot' protein family. The structure, based on NMR data acquired at 600 MHz, has mean pairwise RMS differences of 0.25 +/- 0.06 and 1.07 +/- 0.14 A over the backbone heavy atoms and all heavy atoms, respectively. The solvent-accessible side chains are better defined than in previously published structures and provide an improved basis for docking GVIA with models of the calcium channel. Moreover, some side chain interactions important in GVIA folding in vitro and in stabilizing the native structure are defined clearly in the refined structure. Two qualitatively different backbone conformations in the segment from Thr11 to Asn14 persisted in the restrained simulated annealing calculations until a small number of lower bound constraints was included to prevent close contacts from occurring that did not correspond with peaks in the NOESY spectrum. It is possible that GVIA is genuinely flexible at this segment, spending a finite time in the alternative conformation, and this may influence its interaction with the calcium channel.     

A new colchicine binding assay for tubulin

A simple and sensitive procedure is described for assaying tubulin, the subunit protein of microtubules. Tubulin is equilibrated with [³H]colchicine after which the remaining free colchicine is removed by adsorption to activated charcoal. Values for the amount of tubulin in various rat tissues have been determined by both the charcoal method and by gel filtration on Sephadex G-100, and in all cases the values determined by the two methods correspond closely.     

Covalent binding of acetaldehyde to tubulin: evidence for preferential binding to the alpha-chain

The covalent binding of [14C]acetaldehyde to purified beef brain tubulin was characterized. As we have found for several other proteins, tubulin bound acetaldehyde to form both stable and unstable adducts. Unstable adducts (Schiff bases) were stabilized, and rendered detectable, by treating incubated reaction mixtures with the reducing agent sodium borohydride. In short-term incubations, the majority of the adducts formed were unstable, but the percentage of total adducts that were stable gradually increased with time. Stable adduct formation was greatly increased by the inclusion of sodium cyanoborohydride in reaction mixtures (reductive ethylation). When reaction mixtures were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the alpha- and beta-chains of the heterodimeric tubulin molecule, the alpha-chain of free tubulin, but not intact microtubules, was the preferential site of stable adduct formation under both reductive and nonreductive conditions. Denaturation studies showed that the native tubulin conformation was necessary for the alpha-chain to show enhanced reactivity toward acetaldehyde. Competition binding studies showed that alpha-tubulin could effectively compete with beta-tubulin and bovine serum albumin for a limited amount of acetaldehyde. Unstable acetaldehyde adducts with free tubulin or microtubules did not exhibit alpha-chain selectivity. Analysis of reaction mixtures indicates that lysine residues are the major group of the protein participating in adduct formation. These data indicate that the alpha-chain of free tubulin is the preferential site of stable acetaldehyde-tubulin adduct formation. Further, these data raise the possibility that alpha-tubulin may be a selective target for acetaldehyde adduct formation in cellular systems.     

The binding energetics of first- and second-generation HIV-1 protease inhibitors: implications for drug design

KNI-764 is a powerful HIV-1 protease inhibitor with a reported low susceptibility to the effects of protease mutations commonly associated with drug resistance. In this paper the binding thermodynamics of KNI-764 to the wild-type and drug-resistant mutant V82F/I84V are presented and the results compared to those obtained with existing clinical inhibitors. KNI-764 binds to the wild-type HIV-1 protease with very high affinity (3.1 x 10(10) M(-1) or 32 pM) in a process strongly favored by both enthalpic and entropic contributions to the Gibbs energy of binding (Delta G = -RTlnK(a)). When compared to existing clinical inhibitors, the binding affinity of KNI-764 is about 100 fold higher than that of indinavir, saquinavir, and nelfinavir, but comparable to that of ritonavir. Unlike the existing clinical inhibitors, which bind to the protease with unfavorable or only slightly favorable enthalpy changes, the binding of KNI-764 is strongly exothermic (-7.6 kcal/mol). The resistant mutation V82F/I84V lowers the binding affinity of KNI-764 26-fold, which can be accounted almost entirely by a less favorable binding enthalpy to the mutant. Since KNI-764 binds to the wild type with extremely high affinity, even after a 26-fold decrease, it still binds to the resistant mutant with an affinity comparable to that of other inhibitors against the wild type. These results indicate that the effectiveness of this inhibitor against the resistant mutant is related to two factors: extremely high affinity against the wild type achieved by combining favorable enthalpic and entropic interactions, and a mild effect of the protease mutation due to the presence of flexible structural elements at critical locations in the inhibitor molecule. The conclusions derived from the HIV-1 protease provide important thermodynamic guidelines that can be implemented in general drug design strategies.