Gräslund, A. | Search bioRxiv

bioRxiv2021

Amino acid substitutions in human growth hormone affect coiled-coil content and receptor binding

The interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) has great relevance to human diseases such as acromegaly and cancer. HGH has been extensively engineered by other workers to improve binding and other properties. We used a computational screen to select substitutions at single hGH positions within the hGHR-binding site. We find that, while many successfully slow down dissociation of the hGH-hGHR complex once bound, they also slow down the association of hGH to hGHR. We are particularly interested in E174 which belongs to the hGH zinc-binding triad, and which spans coiled-coil helices and obeys the coiled-coil heptad pattern. Surprisingly, substituting E174 with A leads to substantial increase in an experimental measure of coiled-coil content. E174A is known to increase affinity of hGH against hGHR; here we show that this is simply because the off-rate is slowed down more than the on-rate, in line with what has been found for other affinity-improving mutations. For E174Y (and mutations at other sites) the slowdown in on-rate was greater, leading to decreased affinity. The results point to a link between coiled-coiling, zinc binding, and hGHR-binding affinity in hGH, and also suggest rules for choosing affinity-increasing substitutions.

bioengineering↗

bioRxiv2020

Amyloid-β oligomers are captured by the DNAJB6 chaperone: Direct detection of interactions that can prevent primary nucleation

A human molecular chaperone protein, DNAJB6, is an efficient inhibitor of amyloid aggregation owing to a unique motif with conserved S/T-residues with high capacity for hydrogen bonding. Global analysis of kinetics data previously showed that especially the primary nucleation rate is inhibited. It was concluded that DNAJB6 achieves this remarkably effective and sub-stoichiometric inhibition by interacting not with the monomeric unfolded conformations of the amyloid-{beta} (A{beta}) peptide but with aggregated species. The pre-nucleation oligomeric aggregates are transient and difficult to study experimentally. Here we employed an approach to directly detect oligomeric forms of A{beta} formed in solution by subsequent analysis with native mass spectrometry (native MS). Results show that the signals from the various forms of A{beta} (1-40) oligomers were reduced considerably in the presence of DNAJB6, but not with a mutational variant of DNAJB6 in which the S/T-residues were substituted. With focus on DNAJB6 we could also detect signals that appear to represent DNAJB6 dimers and trimers to which varying amounts of A{beta} is bound. These data provide direct experimental evidence that it is the oligomeric forms of A{beta} that are captured by DNAJB6 in a manner which is dependent on the S/T residues. Strong binding of A{beta} oligomers to DNAJB6 should indeed inhibit the formation of amyloid nuclei, in agreement with the previously observed decrease in primary nucleation rate.

biochemistry↗