Structure of a low-population intermediate state in the release of an enzyme product. eLife (2015)
Enzymes can increase the rate of biomolecular reactions by several orders of magnitude. Although the steps of substrate capture and product release are essential in the enzymatic process, complete atomic-level descriptions of these steps are difficult to obtain because of the transient nature of the intermediate conformations, which makes them largely inaccessible to standard structure determination methods. We describe here the determination of the structure of a low-population intermediate in the product release process by human lysozyme through a combination of NMR spectroscopy and molecular dynamics simulations. We validate this structure by rationally designing two mutations, the first engineered to destabilise the intermediate and the second to stabilize it, thus slowing down or speeding up, respectively, product release. These results illustrate how product release by an enzyme can be facilitated by the presence of a metastable intermediate with transient weak interactions between the enzyme and the product.
A molecular chaperone breaks the catalytic cycle that generates toxic Abeta oligomers. Nat. Struct. Mol. Biol. (2015)
Alzheimer's disease is a highly debilitating and increasingly prevalent neurodegenerative disorder whose pathogenesis has been associated with the aggregation of the amyloid-beta peptide (Abeta42). Recent studies have revealed that the formation of highly neurotoxic oligomers during Abeta aggregation is strongly catalysed by the surfaces of larger aggregates. These findings indicate that a particularly effective way to limit Abeta42 toxicity would be through agents that inhibit this catalytic cycle. Here we show that a molecular chaperone, a Brichos domain, has the ability to specifically block the catalytic production of oligomers. We further demonstrate by a series of biophysical techniques that the Brichos domain achieves this inhibition in vitro by binding on the surface of the fibrils and redirecting the reactive flux from the monomeric to the fibrillar states via a pathway that involves a minimal formation of toxic oligomeric intermediates. We then verify by means of cytotoxicity and electrophysiology experiments using brain tissue that this mechanism also occurs in vivo. These results suggest that biological systems have evolved to achieve effective and efficient suppression of the toxic effects of protein misfolding and aggregation by targeting the individual microscopic pathways that create toxic oligomers, rather than perturbing the overall aggregation reaction.
A method of rational design of protein variants with enhanced solubility.
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P. Sormanni, F. A. Aprile and M. Vendruscolo. J. Mol. Biol. 427, 478-490 (2015).
The s2D method: Simultaneous sequence-based prediction of the statistical populations of ordered and disordered regions in proteins
P. Sormanni, C. Camilloni, P. Fariselli and M. Vendruscolo.
J. Mol. Biol. 427, 982-996 (2015).