Amyloid structure at last !

As a neurologist, I’ve been extremely interested in amyloid  since I started in the late 60s.  The senile plaque of Alzheimers disease is made of amyloid.  The stuff was insoluble gunk. All we had back in the day was Xray diffraction patterns showing two prominent reflections at 4 and 9 Angstroms, so we knew there was some sort of repetitive structure.

My notes on papers on the subject over the past 20 years contain  about 100,000 characters (but relatively little enlightenment until recently).

A while ago I posted some more homework problems — https://luysii.wordpress.com/2021/09/30/another-homework-assignment/

Homework assignment #1:  design a sequence of 10 amino acids which binds to the same sequence in the reverse order forming a plane 4.8 Angstroms thick.

Homework assignment #2 design a sequence of 60 amino acids which forms a similar plane 4.8 Angstroms thick, such that two 60 amino acid monomers bind to each other.

Feel free to use any computational or theoretical devices currently at our disposal, density functional theory, force fields, rosetta etc. etc.

Answers to follow shortly

Hint:  hundreds to thousands of planes can stack on top of each other.

 

If you have a subscription to Cell take a look at a marvelous review full of great pictures and diagrams [ Cell vol. 184 pp. 4857 – 4873 ’21 ].

 

Despite all that reading I never heard anyone predict that a significantly long polypeptide chain could flatten out into a 4.8 Angstrom thick sheet, essentially living in 2 dimensions.  All the structures we had  (alpha helix, beta pleated sheet < they were curved >, beta barrel, solenoid, Greek key) live in 3 dimensions.

 

 

So amyloid is not a particular protein, but a type of conformation a protein can assume (like the structures mentioned above).

 

 

So start with NH – CO – CHR.  NH  CO and C in the structure all lie in the same plane (the H and the side chain of the amino acid < R >  project out of the plane).

 

Here’s a bit of elaboration for those of you whose organic chemistry is a distant memory.  The carbon in the carbonyl bond (CO) has 3 bonding orbitals in one plane 120 degrees apart, with the 4th orbital perpendicular to the plane — this is called sp2 hybridization.  The nitrogen can also be hybridized to sp2.  This lets the pair of electrons above the plane roam around moving toward the carbon.  Why is this good?  Because any time you let electrons roam around you increase their entropy (S) and anything increasing entropy lowers their free energy (F)which is given by the formula F = H – TS where H is enthalpy (a measure of bond strength, and T is the absolute temperature in Kelvin.

 

 

So N and CO are in one plane, and so are the bonds from  N and C to the adacent atoms (C in both cases).

 

You can fit the plane atoms into a  rectangle 4.8 Angstroms high.  Well that’s one 2 dimensional rectangle, but the peptide bond between NH and CO in adjacent rectangles allows you to tack NH – CO – C s together while keeping them in a 3 dimensional parallelopiped 4.8 Angstroms high.

 

 

Notice that in the rectangle the NH and CO bonds are projecting toward the top and bottom of the rectangle, which means that in each plane  NH – CO – CHR s, the NH and CO are pointing out of the 2 dimensional plane (and in opposite directions to boot). This is unlike protein structure in which the backbone NHs and COs hydrogen bond to each other.  There is nothing in this structure for them to bond to.

 

 

What they do is hydrogen bond to another 3 dimensional parallelopiped (call it a sheet, but keep in mind that this is NOT the beta sheet you know about from the 3 dimensional structures of proteins we’ve had for years).

 

 

So thousands of sheets stacked together form the amyloid fibril.

 

Where does the 9 Angstrom reflection of cross beta come from?  Consider the  [ NH – CHR – CO ]  backbone as it lies in the 4.8  thick plane (I never thought such a thing would be even possible ! ).  It curves around like a snake lying flat.  Where are the side chains?  They are in the 4.8 thick plane, separating parts of the meandering backbone from each other — by an average of 9 Angstroms

 

Here is an excellent picture of the Alzheimer culprit — the aBeta42 peptide as it forms the amyloid of the senile plaque

https://pubmed.ncbi.nlm.nih.gov/27355699/#&gid=article-figures&pid=figure-7-uid-6.

 

 

You can see the meandering backbone and the side chains keeping the backbone apart.

 

 

That’s just the beginning of the paper, and I’ll have lots more to say about amyloid as I read further.   Once again, biology instructs chemistry and biochemistry giving it more “things in heaven and earth, Horatio, than are dreamt of in your philosophy.”

Amyloid

Amyloid goes way back, and scientific writing about has had various zigs and zags starting with Virchow (1821 – 1902) who named it because he thought it was made out of sugar.  For a long time it was defined by the way it looks under the microscope being birefringent when stained with Congo red (which came out 100 years ago,  long before we knew much about protein structure (Pauling didn’t propose the alpha helix until 1951).

Birefringence itself is interesting.  Light moves at different speeds as it moves through materials — which is why your legs look funny when you stand in shallow water.  This is called the refractive index.   Birefringent materials have two different refractive indexes depending on the orientation (polarization) of the light looking at it.  So when amyloid present in fixed tissue on a slide, you see beautiful colors — for pictures and much more please see — https://onlinelibrary.wiley.com/doi/full/10.1111/iep.12330

So there has been a lot of confusion about what amyloid is and isn’t and even the exemplary Derek Lowe got it wrong in a recent post of his

“It needs to be noted that tau is not amyloid, and the TauRx’s drug has failed in the clinic in an Alzheimer’s trial.”

But Tau fibrils are amyloid, and prions are amyloid and the Lewy body is made of amyloid too, if you subscribe to the current definition of amyloid as something that shows a cross-beta pattern on Xray diffraction — https://www.researchgate.net/figure/Schematic-representation-of-the-cross-b-X-ray-diffraction-pattern-typically-produced-by_fig3_293484229.

Take about 500 dishes and stack them on top of each other and that’s the rough dimension of an amyloid fibril.  Each dish is made of a beta sheet.  Xray diffraction was used to characterize amyloid because no one could dissolve it, and study it by Xray crystallography.

Now that we have cryoEM, we’re learning much more.  I have , gone on and on about how miraculous it is that proteins have one or a few shapes — https://luysii.wordpress.com/2010/08/04/why-should-a-protein-have-just-one-shape-or-any-shape-for-that-matter/

So prion strains and the fact that alpha-synuclein amyloid aggregates produce different clinical disease despite having the same amino acid sequence was no surprise to me.

But it gets better.  The prion strains etc. etc may not be due to different structure but different decorations of the same structure by protein modifications.

The same is true for the different diseases that tau amyloid fibrils produce — never mind that they’ve been called neurofibrillary tangles and not amyloid, they have the same cross-beta structure.

A great paper [ Cell vol. 180 pp. 633 – 644 ’20 ] shows how different the tau protofilament from one disease (corticobasal degeneration) is from another (Alzheimer’s disease).  Figure three shows the side chain as it meanders around forming one ‘dish’ in the model above.  The meander is quite different in corticobasal degeneration (CBD) and Alzheimers.

It’s all the stuff tacked on. Tau is modified on its lysines (some 15% of all amino acids in the beta sheet forming part) by ubiquitination, acetylation and trimethylation, and by phosphorylation on serine.

Figure 3 is worth more of a look because it shows how different the post-translational modifications are of the same amino acid stretch of the tau protein in the Alzheimer’s and CBD.  Why has this not been seen before — because the amyloid was treated with pronase and other enzymes to get better pictures on cryoEM.  Isn’t that amazing.  Someone is probably looking to see if this explains prion strains.

The question arises — is the chain structure in space different because of the modifications, or are the modifications there because the chain structure in space is different.  This could go either way we have 500+ enzymes (protein kinases) putting phosphate on serine and/or threonine, each looking at a particular protein conformation around the two so they don’t phosphorylate everything — ditto for the enzymes that put ubiquitin on proteins.

Fascinating times.  Imagine something as simple as pronase hiding all this beautiful structure.