Understanding ß-strand mediated protein-protein interactions: tuning binding behaviour of intrinsically disordered sequences by backbone modification.

A significant challenge in chemical biology is to understand and modulate protein-protein interactions (PPIs). Given that many PPIs involve a folded protein domain and a peptide sequence that is intrinsically disordered in isolation, peptides represent powerful tools to understand PPIs. Using the interaction between small ubiquitin-like modifier (SUMO) and SUMO-interacting motifs (SIMs), here we show that N-methylation of the peptide backbone can effectively restrict accessible peptide conformations, predisposing them for protein recognition. Backbone N-methylation in appropriate locations results in faster target binding, and thus higher affinity, as shown by relaxation-based NMR experiments and computational analysis. We show that such higher affinities occur as a consequence of an increase in the energy of the unbound state, and a reduction in the entropic contribution to the binding and activation energies. Thus, backbone N-methylation may represent a useful modification within the peptidomimetic toolbox to probe ß-strand mediated interactions.

Bioorthogonal, Bifunctional Linker for Engineering Synthetic Glycoproteins.

Post-translational glycosylation of proteins results in complex mixtures of heterogeneous protein glycoforms. Glycoproteins have many potential applications from fundamental studies of glycobiology to potential therapeutics, but generating homogeneous recombinant glycoproteins using chemical or chemoenzymatic reactions to mimic natural glycoproteins or creating homogeneous synthetic neoglycoproteins is a challenging synthetic task. In this work, we use a site-specific bioorthogonal approach to produce synthetic homogeneous glycoproteins. We develop a bifunctional, bioorthogonal linker that combines oxime ligation and strain-promoted azide-alkyne cycloaddition chemistry to functionalize reducing sugars and glycan derivatives for attachment to proteins. We demonstrate the utility of this minimal length linker by producing neoglycoprotein inhibitors of cholera toxin in which derivatives of the disaccharide lactose and GM1os pentasaccharide are attached to a nonbinding variant of the cholera toxin B-subunit that acts as a size- and valency-matched multivalent scaffold. The resulting neoglycoproteins decorated with GM1 ligands inhibit cholera toxin B-subunit adhesion with a picomolar IC50.

Microsecond Backbone Motions Modulate the Oligomerization of the DNAJB6 Chaperone.

DNAJB6 is a prime example of an anti-aggregation chaperone that functions as an oligomer. DNAJB6 oligomers are dynamic and subunit exchange is critical for inhibiting client protein aggregation. The T193A mutation in the C-terminal domain (CTD) of DNAJB6 reduces both chaperone self-oligomerization and anti-aggregation of client proteins, and has recently been linked to Parkinson's disease. Here, we show by NMR, including relaxation-based methods, that the T193A mutation has minimal effects on the structure of the ß-stranded CTD but increases the population and rate of formation of a partially folded state. The results can be rationalized in terms of ß-strand peptide plane flips that occur on a timescale of ≈100 µs and lead to global changes in the overall pleat/flatness of the CTD, thereby altering its ability to oligomerize. These findings help forge a link between chaperone dynamics, oligomerization and anti-aggregation activity which may possibly lead to new therapeutic avenues tuned to target specific substrates.

Microsecond Backbone Motions Modulate the Oligomerization of the DNAJB6 Chaperone.

DNAJB6 is a prime example of an anti-aggregation chaperone that functions as an oligomer. DNAJB6 oligomers are dynamic and subunit exchange is critical for inhibiting client protein aggregation. The T193A mutation in the C-terminal domain (CTD) of DNAJB6 reduces both chaperone self-oligomerization and anti-aggregation of client proteins, and has recently been linked to Parkinson's disease. Here, we show by NMR, including relaxation-based methods, that the T193A mutation has minimal effects on the structure of the ß-stranded CTD but increases the population and rate of formation of a partially folded state. The results can be rationalized in terms of ß-strand peptide plane flips that occur on a timescale of ≈100 µs and lead to global changes in the overall pleat/flatness of the CTD, thereby altering its ability to oligomerize. These findings help forge a link between chaperone dynamics, oligomerization and anti-aggregation activity which may possibly lead to new therapeutic avenues tuned to target specific substrates.

Towards optimizing peptide-based inhibitors of protein-protein interactions: predictive saturation variation scanning (PreSaVS).

A simple-to-implement and experimentally validated computational workflow for sequence modification of peptide inhibitors of protein-protein interactions (PPIs) is described.

Visualizing and trapping transient oligomers in amyloid assembly pathways.

Oligomers which form during amyloid fibril assembly are considered to be key contributors towards amyloid disease. However, understanding how such intermediates form, their structure, and mechanisms of toxicity presents significant challenges due to their transient and heterogeneous nature. Here, we discuss two different strategies for addressing these challenges: use of (1) methods capable of detecting lowly-populated species within complex mixtures, such as NMR, single particle methods (including fluorescence and force spectroscopy), and mass spectrometry; and (2) chemical and biological tools to bias the amyloid energy landscape towards specific oligomeric states. While the former methods are well suited to following the kinetics of amyloid assembly and obtaining low-resolution structural information, the latter are capable of producing oligomer samples for high-resolution structural studies and inferring structure-toxicity relationships. Together, these different approaches should enable a clearer picture to be gained of the nature and role of oligomeric intermediates in amyloid formation and disease.

Modulation of Amyloidogenic Protein Self-Assembly Using Tethered Small Molecules.

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 ß2-microglobulin (ß2m), 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, off-pathway oligomers 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.

The role of the IT-state in D76N ß2-microglobulin amyloid assembly: A crucial intermediate or an innocuous bystander?

The D76N variant of human ß2-microglobulin (ß2m) is the causative agent of a hereditary amyloid disease. Interestingly, D76N-associated amyloidosis has a distinctive pathology compared with aggregation of WT-ß2m, which occurs in dialysis-related amyloidosis. A folding intermediate of WT-ß2m, known as the IT-state, which contains a nonnative trans Pro-32, has been shown to be a key precursor of WT-ß2m aggregation in vitro However, how a single amino acid substitution enhances the rate of aggregation of D76N-ß2m and gives rise to a different amyloid disease remained unclear. Using real-time refolding experiments monitored by CD and NMR, we show that the folding mechanisms of WT- and D76N-ß2m are conserved in that both proteins fold slowly via an IT-state that has similar structural properties. Surprisingly, however, direct measurement of the equilibrium population of IT using NMR showed no evidence for an increased population of the IT-state for D76N-ß2m, ruling out previous models suggesting that this could explain its enhanced aggregation propensity. Producing a kinetically trapped analog of IT by deleting the N-terminal six amino acids increases the aggregation rate of WT-ß2m but slows aggregation of D76N-ß2m, supporting the view that although the folding mechanisms of the two proteins are conserved, their aggregation mechanisms differ. The results exclude the IT-state as the origin of the rapid aggregation of D76N-ß2m, suggesting that other nonnative states must cause its high aggregation rate. The results highlight how a single substitution at a solvent-exposed site can affect the mechanism of aggregation and the resulting disease.

Structural mapping of oligomeric intermediates in an amyloid assembly pathway.

Transient oligomers are commonly formed in the early stages of amyloid assembly. Determining the structure(s) of these species and defining their role(s) in assembly is key to devising new routes to control disease. Here, using a combination of chemical kinetics, NMR spectroscopy and other biophysical methods, we identify and structurally characterize the oligomers required for amyloid assembly of the protein ΔN6, a truncation variant of human ß2-microglobulin (ß2m) found in amyloid deposits in the joints of patients with dialysis-related amyloidosis. The results reveal an assembly pathway which is initiated by the formation of head-to-head non-toxic dimers and hexamers en route to amyloid fibrils. Comparison with inhibitory dimers shows that precise subunit organization determines amyloid assembly, while dynamics in the C-terminal strand hint to the initiation of cross-ß structure formation. The results provide a detailed structural view of early amyloid assembly involving structured species that are not cytotoxic.