Modulation of Amyloidogenic Protein Self-Assembly Using Tethered Small Molecules

Abstract

<p></p><p>Protein-protein interactions (PPIs) are involved in many of life’s essential biological functions yet are also an underlying cause of several human diseases, including amyloidosis. The modulation of PPIs presents opportunities to gain mechanistic insights into amyloid assembly, particularly through the use of methods which can trap specific intermediates for detailed study. Such information can also provide a starting point for drug discovery. Here, we demonstrate that covalently tethered small molecule fragments can be used to stabilize specific oligomers during amyloid fibril formation, facilitating the structural characterization of these assembly intermediates. We exemplify the power of covalent tethering using the naturally occurring truncated variant (ΔN6) of the human protein β2-microglobulin (β2m), which assembles into amyloid fibrils associated with dialysis-related amyloidosis. Using this approach, we have trapped tetramers formed by ΔN6 under conditions which would normally lead to fibril formation and found that the degree of tetramer stabilization depends on the site of the covalent tether and the nature of the protein-fragment interaction. The covalent protein-ligand linkage enabled structural characterization of these trapped oligomeric species using X-ray crystallography and NMR, providing insight into why tetramer stabilization inhibits amyloid assembly. Our findings highlight the power of “post-translational chemical modification" as a tool to study biological molecular mechanisms. </p><br><p></p>

Interfacial dynamics and growth modes ofβ2-microglobulin dimers

“…More recent DMD folding simulations indicate that the wt- β 2 m can also populate a similar conformer provided the disulfide bond is established. 30 While the role of the experimentally detected intermediate state in the aggregation pathway of D76N still lacks consensus, 32 we decided to use the topologically similar I 2 as a model system to explore the dimerization phase of β 2 m. 14 By conducting extensive protein-protein docking simulations, we found a prominent role for the DE- and EF-loops as adhesion regions, with the N-terminal region becoming also relevant at acidic pH. 28 Our analyses also predict that Trp60, located at the apex of the DE-loop, is no longer the leading hot-spot at acidic pH, losing its role for Arg3 located at the N-terminus.…”