The world’s longest allosteric effect

I think there is some very interesting protein physical chemistry to be discovered/worked out based on a recent report [ Nature vol. 537 pp. 107 – 111 ’16 ]. It involves a long (2,200 Angstrom) coiled coil protein called EEA1 (Early Endosome Antigen 1). It contains 1,400 amino acids 1,275 of which form a coiled coil.

If you are conversant with the alpha helix and how two of them form a coiled coil, jump to ****. Otherwise here is some background and links to pictures which should help.

The alpha helix is a type of protein secondary structure in which the protein backbone assumes the shape of a coiled spring. There are 3.64 amino acids per turn. A single turn is 5.4 Angstroms high and 11 Angstroms wide. The alpha helix is right handed. That is to say, that if you orient the chain so that your thumb points from the N terminal to C terminal amino acid, the chain will twist in the direction of the fingers of the right hand as it rises. For some reason I can’t provide a link to a very large number of images for you hit. However, when I go to Google and type images of alpha helices you see them immediately — you’ll have to do the same to get there.

Coiled coils have two alpha helices winding around each other. This means that for secure interactions, the same types of amino acids must repeat again and again. A 7 residue periodicity (abcdefg)n in the distribution of nonpolar and charged amino acid residues is a feature characteristic of proteins which form alpha helices coiled about each other (coiled coil molecules). The 7 amino acids are lettered a – g from amino to carboxy. Positions a and d are usually hydrophobic amino acids (Leu, Ile, Val, Ala), positions e and g are usually polar or charged. The nonpolar a and d side chains associate by means of complementary knobs into holes packing. Each individual alpha helix is right handed, but the two helices wind around each other with a left handed turn. There are 3.64 amino acids per turn of an alpha helix, so for a regular repeating structure an amino acid should appear at the same position in space on the alpha helix (which forms a rigid rod). To see all the pictures you want — go to Google and type “Images of the Alpha Helix”.

To get the number of amino acids down so there are 3.5 per/turn (so the structure can repeat exactly every 7 amino acids –e.g. after 2 alpha helical turns) left handed supercoiling of each helix occurs (it’s a chicken and the egg situation). The helices are at an angle of 18 degrees to each other, and every 3.5 amino acids still form a 5.4 Angstrom (when one helix is viewed in isolation), but due to the tilt, they take up 5.1 Angstroms. This means that the same type of amino acid is found at positions 1, 8, 15, 22 etc. All intermediate filament proteins (keratin, neurofilaments, vimentin, etc.) contain a coiled coil structure. So to see all the pictures you could want — go to Google and type “Images of coiled coil proteins”

****
So the 1,275 amino acids of EEA1 divided by 3.5 and multiplied by 5.1 give you a coiled coil of fairly enormous length for a protein (1,858 Angstroms) — average protein diameter (if there is such a thing) is under 50 Angstroms

Functionally, EEA1 seems to be used as a tether with one end free and the other end hooked to a target membrane which wants to ‘catch’ the early endosome. The target membrane isn’t specified in the paper. Apparently EEA1 when not binding the endosome, is in a fully extended state, at around 2,000 Angstroms.

A protein called Rab5 is found on the early endosome membrane, and when EEA1 contacts it, the long coiled coil helix collapses, dragging the endosome toward the target membrane.  This is entropy in action, there being far more configurations of a collapsed protein than a rigidly extended one. To feel entropy for yourself, just pull on a rubber band, entropic effects just like this one are what you feel pulling back.

The collapse of EEA1  is an allosteric effect and a very long one, although the authors note long range allosteric effects are “not uncommon among coiled coil proteins”.

EEA1 is more complicated than initialy described. It contains amino acids which disrupt the 7 amino acid periodicity of the coiled coil (making it a jointed structure). The authors then made an EEA1 protein without the joints (so it was a perfect very long coiled coil). Binding of this protein to Rab5 on an endosome doesn’t result in collapse. So clearly normal EEA1 collapses at the ‘joints’.

The authors talk about some hypotheses as to how this happens in the Supplementary material (but I was unable to find).

So here’s a good research proejct for an enterprising grad student: either find out why and how a protein with multiple joints should exist in a fully extended configuration, or figure out how binding of Rab5 at one end of EEA1 produces such profound allosteric changes through this long linear protein. Happy hunting and thinking.

I must say it’s a pleasure to get back to chemistry after writing about the neurologic and medical issues of the presidential candidates.

Addendum 29 September — I wrote one the following to one of the authors (Dr. Grill) sending him the post above

Dr. Grill

Greatly enjoyed the paper.  I could never find the discussion of possible mechanism in the supplementary material.  You might enjoy the following post written about the paper

Luysii
He replied as follows:

“Dear Luysii thank you very much for the kind words, and I really like your title!

With the supplementary discussion, besides the method part there is an additional supplement file on the Nature website that is easy to miss…I attach it here for you. We discuss this a bit more, but I must admit that this is not very satisfactory at the moment. We just don’t know how this works, and much of our efforts at the moment are dedicated to understand”
So for other readers of the original paper who also can’t find the supplement with the authors’ speculations as to what is going on– here  is what he sent.

” A key question is how Rab5 can induce such a long-range global molecular transition in flexibility of EEA1. Indeed, long-range allosteric effects have been observed for other coiled-coil proteins. In the case of myosin, the presence of discontinuities in the coiled-coil heptads drive structural changes to flexibility. Other tethering factors may bend through large breaks in coiled-coil structure acting as joints, although it remains to be shown whether and how conformational changes are triggered by Rab binding, as shown for EEA1.

Furthermore, a dynamically flexible coiled-coil is mostly extended, provided its ends are free60. However, when the ends of this coiled coil are tethered, bent, or when torsion is locally applied, compensatory structural changes are propagated and even amplified through the length of the structure. Our results suggest that a change in intrinsic static curvature may contribute but is not the major cause for the reduction in end-to-end distance. However, a more rigorous assessment would require visualizing the thermal fluctuations of the bound and unbound EEA1 very rapidly and in three dimensions.

Force generation due to entropic effects plays a key role in many processes in biology ranging from DNA cytoskeletal filaments to motor proteins. Switching a molecule from stiff to flexible could be an effective and general mechanism of many coiled-coil proteins for generating an attractive force, thereby pulling two objects together or allowing reactions otherwise hindered by polymer rigidity. Future experiments will test to what extent the entropic collapse is a general mechanism used not only by membrane tethers but also in other biological processes.”

 

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