An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated β-sheet structure for amyloid
Melinda Balbirnie, Robert Grothe, and David S. Eisenberg
PNAS, February 2001
27; 98(5): 2375–2380
This paper is a really landmark study on amylogenic peptides and their relationship to prions – a weird class of inheritable proteins.
High level summary
Most proteins sit in your cells and have one or more jobs to do, which they do in a single conformation – a single 3D structure – or set of similar conformations. An analogy here would be that most tools we use in life have a single shape and behavior, which makes them good for doing what they’re built for (wrenches and wrenched shaped, fishing rods and fishing-rod shaped, etc).
Prion proteins (prions) are different. They represent a class of proteins and can exist in two states: a “normal” state which has a variety of different functions, and a prion state, which we’ll call the amylogenic state. Prion proteins can interconvert from the normal state to the amylogenic state. The exact detail of this conversion are poorly understood, but suffice to say that most of the time the protein sits in its normal state, but should it convert into this amylogenic state it becomes infectious, and can cause other normal state proteins to flip into this amylogenic state. As an analogy, imagine a village where everyone is healthy, but one day one of the villagers gets sick. That villager can infect all the other villagers simply by getting close to them, and soon the whole village is sick. In the village analogy, everyone gets better, but in cells, there is no way for these amylogenic prions to convert back to the healthy form. In this way, one amylogenic prion can (theoretically) infect (seed) all the prion proteins in a cell or even an organism. Once a prion protein turns amylogenic, it converts into a shape which makes it sticky to other prion proteins, causing them to clump together into aggregates of some type.
This sounds bad – and it can be! In many organisms, this event is associated with incurable brain diseases. Examples of this are mad cow disease in cattle, Creutzfeldt–Jakob disease (CJD) in humans and scrapie in sheep. Thankfully, these diseases are pretty rare, so even though you’ve probably heard about CJD, very few people have died from it. However, prions are also observed in single cell organisms, especially yeast, where they are not lethal and are hypothesized to be important for evolution and survival.
This paper focuses on one of those yeast prion proteins – a very well studied protein called Sup35. As a prion, Sup35 can exist in two conformations (normal and amylogenic), and when Sup35 is amylogenic, it aggregates together to form long fibrils. In this paper, the authors discovered that by chopping a tiny bit out of the protein, that tiny bit can form fibrils which show very similar properties to the fibrils made from the full length Sup35. By a tiny bit I really mean a tiny bit – around 1% of the protein!
Sup35 is known to be split into three main regions, and one of those regions was already known to be crucial for this amylogenic prion structure. However, the fact that you could re-create the Sup35 fibrils with such a tiny fragment (a peptide) is really surprising. A lot of the paper is focused on showing how this peptide fibril and the full length sup35 fibril are similar, and they don’t show that the peptide fibril can trigger the infectious behaviour I mentioned before, but even so this has provided extensive insight into the mechanism of prion aggregation and fibril formation.
The authors also provide a theory to explain some of the fibers’ properties, which would be general to many different types of prions, by examining a range of data to construct a possible structural model.
Building on the general gist above, this paper is really focused on the prion amyloid state, as opposed to the many other properties of prions which make them interesting. The whole amyloid field-of-study is slightly confusing as a result of overlapping nomenclature, so before I go on I’m going to quickly summarize some of the vocabulary;
Amyloid fold – is a specific, super-stable beta-sheet rich protein fold. The current working hypothesis is that this fold represents an energetic minima, which various different proteins sequences can fold or misfold into. Once this fold forms, it’s really hard to unfold from it.
Amyloids (AKA amyloid deposits) – are protein deposits which can be formed from many different proteins, where the proteins which make up the deposit are in the amyloid fold. Generally, amyloids deposists are made up of a single protein type, although to be totally honest I’m not sure if anyone’s looked at heterogeneity in amyloids. Amyloids also, generally, form fibrils.
Amyloids are often associated with diseases – frequently neurodegeneration (Alzheimer’s disease, CJD) but also other diseases like diabetes or atherosclerosis. However, they also often have non-disease associated roles.
OK – so back to the paper. Sup35 is a 685 amino acid protein and has three domains – N, M, C. The N domain is known to be the prion forming domain, so the authors tested a bunch of different constructs from this N-domain to try and find a region which in isolation could form fibrils – focusing on a region in the N domain known to be crucial for prion aggregation. They found many peptides which formed fibrils and have the characteristic X-ray cross section behavior, and found one specific peptide GNNQQNY (which runs from residue 7 to residue 13 in Sup35) which formed fibrils with the same amyloid properties as the full length Sup35. These properties include
- Structural transition from a soluble coiled-coil to beta-sheet
- Fibril formation
- Consistent X-ray diffraction pattern
- Congo red binding
- Cooperative aggregation kinetics
Additionally, they show how by adding Sup35 to a solution of GNNQQNY you can increase the rate of aggregation – i.e. Sup35 can seed aggregate formation.
Beyond this, they go on to show how the GNNQQNY crystal structure indicates that the peptide forms a dehydrated beta-sheet structure where internal hydrogen bonds are satisfied through self-interaction, as opposed to via water, as is commonly seen in normal beta sheets. Additionally they were able to interrogate beta-sheet direction and unit cell size. The authors hypothesize that this “dry beta-sheet” may provide an explanation for why amyloid deposits are highly stable.