Inspired by Eoin’s interesting talks on prions and prion diseases, and Nick’s discussion of how Cyro-Electron microscopy is going to be the end of an era for Crystallography. I thought I’d look at a paper that discusses aggregation of protein complexes, with some cryo-electron microscopy thrown in for good measure.
Supramolecular assemblies are folded protein complexes forming into much larger units. This formation can be triggered by a mutation on a copy of the constituent homomers of the complex, acting as a self-interacting patch. If this patch were to form in a non-symmetric complex, it would likely form a finite assemble with a limited number of copies of the complex. However, if the complex has dihedral symmetry such that a patch is accessible at multiple separated locations, then complex can potentially form near infinite supramolecular assemblies.
The authors demonstrate that these self-interacting patches can be triggered by clusters of mutations to tyrosine or lysine. Initially three residue mutations show an associated fibril formation. However, the authors show that is some cases a single point mutation is sufficient to form either fibrils or other aggregates known as punctate foci.
To confirm that the protein is folded the authors use transmission electron microscopy to directly show that the units forming the fibril are the size of the complex, and thus remain folded. They also use circular dichroism to show that there is little change to the secondary structure elements of the protein when formed into supramolecular assemblies, thus the complex remains folded.
Single particle cryo-electron microscopy relies on collecting many images of differing orientations of the molecule or complex (in this case: the fibrils). These images are classified, manually and then automatically into 2D classes. These 2D classes are then used to form a three-dimensional reconstruction of the molecule. As the authors have access to atomic resolution crystallographic structures of the complex forming the fibrils, they use a fitting routine to compare the repeated fibril to that in the crystal structure. They use this to show that the interaction can be localised to the small number of nucleotides that are mutated, and propose potential interactions which could lead to the fibril formation. The specificity of the information gained by the cryo-EM seems quite low, and brings into question alternate less intensive techniques may have been more valuable to confirm the interactions undertaken in the fibril formation
The authors suggest an interesting metric, the normal distance to the closest bounding plane of the homomer (nDp), this effectively describes how far from the apex of the quaternary structure of the complex any residue is. Lower nDp areas are at higher risk of becoming self-interacting patches, through mutation of a low number of residues. The compare this to the intrinsic stickiness of the protein, where the stickiness is defined as the ratio of amino acids found at protein surfaces compared to protein- protein interfaces. Implying that residues/motifs forming PPIs are more likely to be interacting surfaces. Areas with a low nDp, and thus higher chance of mutation to form self-interacting patches have a lower propensity of residues typically found in PPIs. There is no significant enrichment in cyclic complexes, implying the potential propensity for dihedral complexes to form supramolecular assemblies, has led to an evolutionary pressure to reduce the stickiness of low nDp regions.
Whether nDp or other correlated metric could be used to create a predictive model of folded protein aggregation is not discussed heavily in the paper, but should be considered one of the potential follow ups from this study. Also, the authors do not fully discuss the significance of the punctate foci: are these forms of aggregation not as potentially damaging as fibrils and thus less selected against.
Overall I found the paper to be an interesting combination of techniques, exploring the relationship between aggregation, residue propensity and complex symmetry.