Accurate calculation of mutational effects on the thermodynamics of inhibitor binding to p38α MAP kinase: a combined computational and experimental study.

A major current challenge for drug design efforts focused on protein kinases is the development of drug resistance caused by spontaneous mutations in the kinase catalytic domain. The ubiquity of this problem means that it would be advantageous to develop fast, effective computational methods that could be used to determine the effects of potential resistance-causing mutations before they arise in a clinical setting. With this long-term goal in mind, we have conducted a combined experimental and computational study of the thermodynamic effects of active-site mutations on a well-characterized and high-affinity interaction between a protein kinase and a small-molecule inhibitor. Specifically, we developed a fluorescence-based assay to measure the binding free energy of the small-molecule inhibitor, SB203580, to the p38α MAP kinase and used it measure the inhibitor's affinity for five different kinase mutants involving two residues (Val38 and Ala51) that contact the inhibitor in the crystal structure of the inhibitor-kinase complex. We then conducted long, explicit-solvent thermodynamic integration (TI) simulations in an attempt to reproduce the experimental relative binding affinities of the inhibitor for the five mutants; in total, a combined simulation time of 18.5 µs was obtained. Two widely used force fields - OPLS-AA/L and Amber ff99SB-ILDN - were tested in the TI simulations. Both force fields produced excellent agreement with experiment for three of the five mutants; simulations performed with the OPLS-AA/L force field, however, produced qualitatively incorrect results for the constructs that contained an A51V mutation. Interestingly, the discrepancies with the OPLS-AA/L force field could be rectified by the imposition of position restraints on the atoms of the protein backbone and the inhibitor without destroying the agreement for other mutations; the ability to reproduce experiment depended, however, upon the strength of the restraints' force constant. Imposition of position restraints in corresponding simulations that used the Amber ff99SB-ILDN force field had little effect on their ability to match experiment. Overall, the study shows that both force fields can work well for predicting the effects of active-site mutations on small molecule binding affinities and demonstrates how a direct combination of experiment and computation can be a powerful strategy for developing an understanding of protein-inhibitor interactions.

Valosin-containing protein (VCP/p97) is an activator of wild-type ataxin-3.

Alterations in the ubiquitin-proteasome system (UPS) have been reported in several neurodegenerative disorders characterized by protein misfolding and aggregation, including the polylgutamine diseases. Machado-Joseph disease (MJD) or Spinocerebellar Ataxia type 3 is caused by a polyglutamine-encoding CAG expansion in the ATXN3 gene, which encodes a 42 kDa deubiquitinating enzyme (DUB), ataxin-3. We investigated ataxin-3 deubiquitinating activity and the functional relevance of ataxin-3 interactions with two proteins previously described to interact with ataxin-3, hHR23A and valosin-containing protein (VCP/p97). We confirmed ataxin-3 affinity for both hHR23A and VCP/p97. hHR23A and ataxin-3 were shown to co-localize in discrete nuclear foci, while VCP/p97 was primarily cytoplasmic. hHR23A and VCP/p97 recombinant proteins were added, separately or together, to normal and expanded ataxin-3 in in vitro deubiquitination assays to evaluate their influence on ataxin-3 activity. VCP/p97 was shown to be an activator specifically of wild-type ataxin-3, exhibiting no effect on expanded ataxin-3, In contrast, we observed no significant alterations in ataxin-3 enzyme kinetics or substrate preference in the presence of hHR23A alone or in combination with VCP. Based on our results we propose a model where ataxin-3 normally functions with its interactors to specify the cellular fate of ubiquitinated proteins.

Ubiquitination directly enhances activity of the deubiquitinating enzyme ataxin-3.

Deubiquitinating enzymes (DUBs) control the ubiquitination status of proteins in various cellular pathways. Regulation of the activity of DUBs, which is critically important to cellular homoeostasis, can be achieved at the level of gene expression, protein complex formation, or degradation. Here, we report that ubiquitination also directly regulates the activity of a DUB, ataxin-3, a polyglutamine disease protein implicated in protein quality control pathways. Ubiquitination enhances ubiquitin (Ub) chain cleavage by ataxin-3, but does not alter its preference for K63-linked Ub chains. In cells, ubiquitination of endogenous ataxin-3 increases when the proteasome is inhibited, when excess Ub is present, or when the unfolded protein response is induced, suggesting that the cellular functions of ataxin-3 in protein quality control are modulated through ubiquitination. Ataxin-3 is the first reported DUB in which ubiquitination directly regulates catalytic activity. We propose a new function for protein ubiquitination in regulating the activity of certain DUBs and perhaps other enzymes.

The deubiquitinating enzyme ataxin-3, a polyglutamine disease protein, edits Lys63 linkages in mixed linkage ubiquitin chains.

Ubiquitin chain complexity in cells is likely regulated by a diverse set of deubiquitinating enzymes (DUBs) with distinct ubiquitin chain preferences. Here we show that the polyglutamine disease protein, ataxin-3, binds and cleaves ubiquitin chains in a manner suggesting that it functions as a mixed linkage, chain-editing enzyme. Ataxin-3 cleaves ubiquitin chains through its amino-terminal Josephin domain and binds ubiquitin chains through a carboxyl-terminal cluster of ubiquitin interaction motifs neighboring the pathogenic polyglutamine tract. Ataxin-3 binds both Lys(48)- or Lys(63)-linked chains yet preferentially cleaves Lys(63) linkages. Ataxin-3 shows even greater activity toward mixed linkage polyubiquitin, cleaving Lys(63) linkages in chains that contain both Lys(48) and Lys(63) linkages. The ubiquitin interaction motifs regulate the specificity of this activity by restricting what can be cleaved by the protease domain, demonstrating that linkage specificity can be determined by elements outside the catalytic domain of a DUB. These findings establish ataxin-3 as a novel DUB that edits topologically complex chains.

Cellular turnover of the polyglutamine disease protein ataxin-3 is regulated by its catalytic activity.

Ataxin-3, a deubiquitinating enzyme, is the disease protein in spinocerebellar ataxia type 3, one of many neurodegenerative disorders caused by polyglutamine expansion. Little is known about the cellular regulation of ataxin-3. This is an important issue, since growing evidence links disease protein context to pathogenesis in polyglutamine disorders. Expanded ataxin-3, for example, is more neurotoxic in fruit fly models when its active site cysteine is mutated (1). We therefore sought to determine the influence of ataxin-3 enzymatic activity on various cellular properties. Here we present evidence that the catalytic activity of ataxin-3 regulates its cellular turnover, ubiquitination, and subcellular distribution. Cellular protein levels of catalytically inactive ataxin-3 were much higher than those of active ataxin-3, in part reflecting slower degradation. In vitro studies revealed that inactive ataxin-3 was more slowly degraded by the proteasome and that this degradation occurred independent of ubiquitination. Slower degradation of inactive ataxin-3 correlated with reduced interaction with the proteasome shuttle protein, VCP/p97. Enzymatically active ataxin-3 also showed a greater tendency to concentrate in the nucleus, where it colocalized with the proteasome in subnuclear foci. Taken together, these and other findings suggest that the catalytic activity of this disease-linked deubiquitinating enzyme regulates several of its cellular properties, which in turn may influence disease pathogenesis.

Lysozyme secretion by submucosal glands protects the airway from bacterial infection.

Submucosal glands are abundant (approximately 1 gland/mm2) secretory structures in the tracheobronchial airways of the human lung. Because submucosal glands express antibacterial proteins, it has been proposed that they contribute to lung defense. However, this concept is challenged by the fact that mice do not have submucosal glands in their bronchial airways, yet are quite resistant to bacterial lung infection. The contribution of airway submucosal glands to host defense is also debated as a pathophysiologic component of cystic fibrosis lung disease. Here, we asked whether submucosal glands protect airways against bacterial infection. By comparing tracheal xenograft airways with and without glands, we found that the presence of glands enhanced bacterial killing in vivo and by airway secretions in vitro. Moreover, immunodepletion studies suggested that lysozyme is a major antibacterial component secreted by submucosal glands. These studies provide evidence that submucosal glands are a major source of antibacterials critical for maintaining sterile airways.