Tubulin needs a lot of help from its friends

Our neurons (and us) would be the size of amoebas if weren’t for tubulin which forms the superhighways (microtubules) along which cargo is shipped to the end of axons.   Your average NBA player has axons over 3 feet long going from his sacral spinal cord to his calf muscles.   Split the difference and call it a meter.  Diffusion is way too slow to get anything that far. The trucks schlepping things back and forth on the microtubular highway are called Kinesin and dynein. I think in terms of nanoMeters (10^-9 meters).  Each tubulin dimer is 80 nanoMeters long, and K & D essentially jump from one to the other in 80 nanoMeter steps.

How many jumps do Kinesin and Dynein have to make to go a meter? Just 10^9/80 — call it 10,000,000. Kinesin and Dynein also have to jump from one microtubule to another, as the longest microtubule in our division is at most 100 microns (.1 milliMeter).  So even in the best of cases they have to make at least 10,000 transfers between microtubules.  It’s a miracle they get the job done at all.

To put this in perspective, consider a tractor trailer (not a truck — the part with the motor is the tractor, and the part pulled is the trailer — the distinction can be important, just like the difference between rifle and gun as anyone who’s been through basic training knows quite well).  Say the trailer is 48 feet long, and let that be comparable to the 80 nanoMeters Kinesin and Dynein have to jump. That’s 10,000,000 jumps of 48 feet or 90,909 miles.  It’s amazing they get the job done.

Now that you’re sufficiently impressed with tubulin’s importance, it’s time to see why it needs help.  First a bit of history.  Christian Anfinsen was a Swarthmore football player who happened to win the Nobel prize 50 years ago for his work on the protein ribonuclease, an enzyme.  If you heat it, enzymatic activity is lost (the protein is said to be denatured).  This is because the exact 3 dimensional path of the protein backbone forming the catalytic site of ribonuclease was lost. However if you leave the denatured protein alone (under the proper conditions) it folds back up to the correct 3 dimensional shape.  His point was that the amino acid sequence of the protein was all that was needed to determine ‘the’ three dimensional shape of the protein.  This was at a time when we didn’t know that most proteins have a variety of shapes not just one.

Unfortunately tubulin does not fold up to the shape found in microtubules.  It needs significant help from two friends, prefoldin and TRiC.  TRiC is a monster conglomerate of 2 copies each of 8 different proteins with a molecular mass over 1,000,000 Daltons (e.g. a megaDalton).  What is one Dalton — it’s the mass of a hydrogen atom.   TRiC is made of two back to back rings (with built in lids) each ring consisting of 8 different but related proteins).  Each of the proteins has a domain which binds ATP and a domain which binds the protein to be folded.  There is a central cavity 90 x 90 x 50 Angstroms in size.  Since each hydrogen atom is about 1 Angstrom in diameter, it can fit 405,000 hydrogen atoms inside, or about 200,000 carbons, hydrogens, oxygens and nitrogens — enough room for most proteins.

Prefoldin is equally amazing.  It basically looks like a Portuguese man o’ war — https://en.wikipedia.org/wiki/Portuguese_man_o%27_war.  It is made of 2 copes of one protein and 4 of another.  The tentacles are long alpha helices projecting down from the body.

The tentacles interact with tubulin, carrying it in an unstructured form, thrusting one of its tentacles into the central chamber of TRiC carrying unstructured tubulin with it.   ATP addition leads to lid closure and tubulin encapsulation in the chamber.

A magnificent paper [ Cell vol. 185 pp. 4770 – 4787 ’22 ] describes what happens to tubulin in the TRiC chamber at near atomic resolution.  They are literally watching tubulin fold as it passes from one of the 8 different proteins making up the TRiC ring to another.  The disordered carboxy terminal chains of TRiC are postulated to function as a tethered solvent allowing the intially disordered amino acid sequence of tubulin, to slither into their correct positions more easily.

I’m sure it’s behind a paywall, but if you can look at the figures in the paper, you’ll be bound to be impressed.

So Anfinsen turned out to be wrong, and some 10%  of newly translated proteins turn our to need TRiC’s help.  And yet he wasn’t, because AlphaFold uses only the amino acid sequence of proteins to predict their three dimensional structure.

One further point.  The ancestral bacterial protein for tubulin is called FtsZ.  It happily folds to the correct structure by itself.  However tubulin developed new domains, some of which are for the motor proteins Dynein and Kinesin, and others are for microtubule associated proteins such as tau, the major component of the neurofibrillary tangle of Alzheimer’s disease. These domains are on the surface of the protein, making it harder to fold by itself.

All this information would have been impossible to get 10 years ago, and it’s all due to the sharpening of our technological tools.

A few Thanksgiving thankyou’s

The following was published 5 years ago, but with time and ever more research our organization seems even more miraculous (see last paragraph).  It’s amazing that it lasts as long as it does, and for that we should be thankful.   Call this prayer if you wish.

As CEO of a very large organization, it’s time to thank those unsung divisions that make it all possible.  Fellow CEOs should take note and act appropriately regardless of the year it’s been for them.

First: thanks to the guys in shipping and receiving.  Kinesin moves the stuff out and Dynein brings it back home.  Think of how far they have to go.  The head office sits in area 4 of the cerebral cortex and K & D have to travel about 3 feet down to the motorneurons in the first sacral segment of the spinal cord controlling the gastrocnemius and soleus, so the boss can press the pedal on his piano when he wants. Like all good truckers, they travel on the highway.  But instead of rolling they jump.  The highway is pretty lumpy being made of 13 rows of tubulin dimers.

Now chemists are very detail oriented and think in terms of Angstroms (10^-10 meters) about the size of a hydrogen atom. As CEO and typical of cell biologists, I have to think in terms of the big picture, so I think in terms of nanoMeters (10^-9 meters).  Each tubulin dimer is 80 nanoMeters long, and K & D essentially jump from one to the other in 80 nanoMeter steps.  Now the boss is shrinking as he gets older, but my brothers working for players in the NBA have to go more than a meter to contract the gastrocnemius and soleus (among other muscles) to help their bosses jump.  So split the distance and call the distance they have to go one Meter.  How many jumps do Kinesin and Dynein have to make to get there? Just 10^9/80 — call it 10,000,000. The boys also have to jump from one microtubule to another, as the longest microtubule in our division is at most 100 microns (.1 milliMeter).  So even in the best of cases they have to make at least 10,000 transfers between microtubules.  It’s a miracle they get the job done at all.

To put this in perspective, consider a tractor trailer (not a truck — the part with the motor is the tractor, and the part pulled is the trailer — the distinction can be important, just like the difference between rifle and gun as anyone who’s been through basic training knows quite well).  Say the trailer is 48 feet long, and let that be comparable to the 80 nanoMeters K and D have to jump. That’s 10,000,000 jumps of 48 feet or 90,909 miles.  It’s amazing they get the job done.

Second: Thanks to probably the smallest member of the team.  The electron.  Its brain has to be tiny, yet it has mastered quantum mechanics because it knows how to tunnel through a potential barrier.   In order to produce the fuel for K and D it has to tunnel some 20 Angstroms from the di-copper center (CuA) to heme a in cytochrome C oxidase (COX).  Is the electron conscious? Who knows?  I don’t tell it what to do.   Now COX is just a part of one of our larger divisions, the power plant (the mitochondrion).

Third: The power plant.  Amazing to think that it was once (a billion years or more ago) a free living bacterium.  Somehow back in the mists of time one of our predecessors captured it.  The power plant produces gas (ATP) for the motors to work.  It’s really rather remarkable when you think of it.   Instead of carrying a tank of ATP, kinesin and dynein literally swim in the stuff, picking it up from the surroundings as they move down the microtubule.  Amazingly the entire division doesn’t burn up, but just uses the ATP when and where needed.  No spontaneous combustion.

There are some other unsung divisions to talk about (I haven’t forgotten you ladies in the steno pool, and your incredible accuracy — 1 mistake per 100,000,000 letters [ Science vol. 328 pp. 636 – 639 ’10 ]).  But that’s for next time.

To think that our organization arose by chance, working by finding a slightly better solution to problems it face boggles this CEO’s mind (but that’s the current faith — so good to see such faith in an increasingly secular world).

Call the thankfulness of the CEO prayer if you wish.

Addendum 29 November ’22 — from a friend “We also have to thank all the tau molecules that stabilize the microtubules— until the misbehavior of ERK and JNK1 overdecorate them with holiday lighting (phosphates) and they fall apart. So after Thanksgiving, be careful not to overcommercialize the holiday season.”

The cryoEM work of the last 5 years has shown us the structure of large molecular machines made of multiple proteins, DNAs and RNAs which are even more impressive (to me) than single protein structure.   One example [ Nature vol. 609 pp. 630 – 639 ’22 ] shows the Holliday junction which allows strand migration between the strands of two DNA duplexes.   Pictured is the complex from bacteria which is confined in a rectangle with sides 220 and 120 Angstroms (not sure how thick it is).  The complex contains a molecular motor which slides the junction.  You could spend your life just studying this one structure.  It’s hard for me to see how it arose.

What is legionella trying to tell us?

10 years out of Med School, a classmate in the Public Health service had to deal with the first recognized outbreak of Legionnaire’s disease, at the Bellevue Stratford hotel in Philly, about one air mile from Penn Med where we went.   The organism wasn’t known at the time and caused 182 cases with 29 deaths.  We’ve learned a lot more about Legionella Pneumophila since 1976 and the organism continues to instruct us.

The most recent lesson concerns one of the 300 or so proteins Legionella injects into a cell it attacks.  This is remarkable in itself.  The organism uses them to hijack various cellular mechanisms to build a home for itself in the cell (the LCV — Legionella Containing Vacuole).  Contrast this with diphtheria which basically uses one protein (diphtheria toxin) to kill the cell.

One of the 300 proteins is called SidJ and looks like a protein kinase (of which our genome has over 500).  However [ Science vol. 364 pp. 787 – 792 ’19 ] shows that SidJ carries out a different different reaction.SidJ is activated by host-cell calmodulin to polyglutamylate the SidE family of ubiquitin (Ub) ligases inhibiting them. Crystal structures of the SidJ-calmodulin complex reveal a protein kinase fold that catalyzes ATP-dependent isopeptide bond formation between the amino group of free glutamate and the gamma carboxyl group in the catalytic center of SidE a ubiquitin ligase.   This, instead of just esterifying the hydroxyl group of serine or threonine or tyrosine with the terminal phosphate of ATP as a kinase is supposed to do.

Why is this important? The only protein known to have polyglutamic acid added to it is tubulin, the protein from which microtubules (neurotubules to the neurologist).  The work is important because some of the 500+ protein kinases in our genome might be doing something else.  Has the chemistry each and every member of the group been studied?  Probably not..

The authors close with “In summary, our results underscore the diversity and catalytic versatility of the protein kinase superfamily. We propose that ATP-dependent ligation reactions may be a common feature among the vast diversity of eukaryotic protein kinase–like enzymes found in nature (25). There are more than 500 protein kinases in humans and our results suggest that they should be ex- amined for alternative activities.”

I couldn’t agree more.

A few Thanksgiving thank you’s

As CEO of a very large organization, it’s time to thank those unsung divisions that make it all possible.  Fellow CEOs should take note and act appropriately regardless of the year it’s been for them.

First: thanks to the guys in shipping and receiving.  Kinesin moves the stuff out and Dynein brings it back home.  Think of how far they have to go.  The head office sits in area 4 of the cerebral cortex and K & D have to travel about 3 feet down to the motorneurons in the first sacral segment of the spinal cord controlling the gastrocnemius and soleus, so the boss can press the pedal on his piano when he wants. Like all good truckers, they travel on the highway.  But instead of rolling they jump.  The highway is pretty lumpy being made of 13 rows of tubulin dimers.

Now chemists are very detail oriented and think in terms of Angstroms (10^-10 meters) about the size of a hydrogen atom. As CEO and typical of cell biologists, I have to think in terms of the big picture, so I think in terms of nanoMeters (10^-9 meters).  Each tubulin dimer is 80 nanoMeters long, and K & D essentially jump from one to the other in 80 nanoMeter steps.  Now the boss is shrinking as he gets older, but my brothers working for players in the NBA have to go more than a meter to contract the gastrocnemius and soleus (among other muscles) to help their bosses jump.  So split the distance and call the distance they have to go one Meter.  How many jumps do Kinesin and Dynein have to make to get there? Just 10^9/80 — call it 10,000,000. The boys also have to jump from one microtubule to another, as the longest microtubule in our division is at most 100 microns (.1 milliMeter).  So even in the best of cases they have to make at least 10,000 transfers between microtubules.  It’s a miracle they get the job done at all.

To put this in perspective, consider a tractor trailer (not a truck — the part with the motor is the tractor, and the part pulled is the trailer — the distinction can be important, just like the difference between rifle and gun as anyone who’s been through basic training knows quite well).  Say the trailer is 48 feet long, and let that be comparable to the 80 nanoMeters K and D have to jump. That’s 10,000,000 jumps of 48 feet or 90,909 miles.  It’s amazing they get the job done.

Second: Thanks to probably the smallest member of the team.  The electron.  Its brain has to be tiny, yet it has mastered quantum mechanics because it knows how to tunnel through a potential barrier.   In order to produce the fuel for K and D it has to tunnel some 20 Angstroms from the di-copper center (CuA) to heme a in cytochrome C oxidase (COX).  Is the electron conscious? Who knows?  I don’t tell it what to do.   Now COX is just a part of one of our larger divisions, the power plant (the mitochondrion).

Third: The power plant.  Amazing to think that it was once (a billion years or more ago) a free living bacterium.  Somehow back in the mists of time one of our predecessors captured it.  The power plant produces gas (ATP) for the motors to work.  It’s really rather remarkable when you think of it.   Instead of carrying a tank of ATP, kinesin and dynein literally swim in the stuff, picking it up from the surroundings as they move down the microtubule.  Amazingly the entire division doesn’t burn up, but just uses the ATP when and where needed.  No spontaneous combustion.

There are some other unsung divisions to talk about (I haven’t forgotten you ladies in the steno pool, and your incredible accuracy — 1 mistake per 100,000,000 letters [ Science vol. 328 pp. 636 – 639 ’10 ]).  But that’s for next time.

To think that our organization arose by chance, working by finding a slightly better solution to problems it face boggles this CEO’s mind (but that’s the current faith — so good to see such faith in an increasingly secular world).