Now is the winter of our discontent

One of the problems with being over 80 is that you watch your friends get sick.  In the past month, one classmate developed ALS and another has cardiac amyloidosis complete with implantable defibrillator.  The 40 year old daughter of a friend who we watched since infancy has serious breast cancer and is undergoing surgery radiation and chemo.  While I don’t have survivor’s guilt (yet), it isn’t fun.

Reading and thinking about molecular biology has been a form of psychotherapy for me (for why, see the reprint of an old post on this point at the end).

Consider ALS (Amyotrophic Lateral Sclerosis, Lou Gehrig disease).  What needs explaining is not why my classmate got it, but why we all don’t have it.  As you know human neurons don’t replace themselves (forget the work in animals — it doesn’t apply to us).  Just think what the neurons  which die in ALS have to do.  They have to send a single axon several feet (not nanoMeters, microMeters, milliMeters — but the better part of a meter) from their cell bodies in the spinal cord to the muscle the innervate (which could be in your foot).

Supplying the end of the axon with proteins and other molecules by simple diffusion would never work.  So molecular highways (called microtubules) inside the axon are constructed, along which trucks (molecular motors such as kinesin and dynein) drag cargos of proteins, and mRNAs to make more proteins.

We know a lot about microtubules, and Cell vol. 179 pp. 909 – 922 ’19 gives incredible detail about them (even better with lots of great pictures).  Start with the basic building block — the tubulin heterodimer — about 40 Angstroms wide and 80 Angstroms high.  The repeating unit of the microtubule is 960 Angstroms long, so 12 heterodimers are lined up end to end in each repeating unit — this is the protofilament of the microtubule, and our microtubules have 13 of them, so that’s 156 heterodimers per microtubule repeat length which is 960 Angstroms or 96 nanoMeters (96 billionths of a meter).  So a microtubule (or a bunch of microtubules extending a meter has 10^7 such repeats or about 1 billion heterodimers.  But the axon of a motor neuron has a bunch of microtubules in it (between 10 and 100), so the motor neuron firing to  the muscle moving my finger has probably made billions and billions of heterodimers.  Moreover it’s been doing this for 80 plus years.

This is why, what needs explaining is not ALS, but why we don’t all have it.

Here’s the old post

The Solace of Molecular Biology

Neurology is fascinating because it deals with illnesses affecting what makes us human. Unfortunately for nearly all of my medical career in neurology ’62 – ’00 neurologic therapy was lousy and death was no stranger. In a coverage group with 4 other neurologists taking weekend call (we covered our own practices during the week) about 1/4 of the patients seen on call weekend #1 had died by on call weekend #2 five weeks later.

Most of the deaths were in the elderly with strokes, tumors, cancer etc, but not all. I also ran a muscular dystrophy clinic and one of the hardest cases I saw was an infant with Werdnig Hoffman disease — similar to what Steven Hawking has, but much, much faster — she died at 1 year. Initially, I found the suffering of such patients and their families impossible to accept or understand, particularly when they affected the young, or even young adults in the graduate student age.

As noted earlier, I started med school in ’62, a time when the genetic code was first being cracked, and with the background then that many of you have presently understanding molecular biology as it was being unravelled wasn’t difficult. Usually when you know something you tend to regard it as simple or unimpressive. Not so the cell and life. The more you know, the more impressive it becomes.

Think of the 3.2 gigaBases of DNA in each cell. At 3 or so Angstroms aromatic ring thickness — this comes out to a meter or so stretched out — but it isn’t, rather compressed so it fits into a nucleus 5 – 10 millionths of a meter in diameter. Then since DNA is a helix with one complete turn every 10 bases, the genome in each cell contains 320,000,000 twists which must be unwound to copy it into RNA. The machinery which copies it into messenger RNA (RNA polymerase II) is huge — but the fun doesn’t stop there — in the eukaryotic cell to turn on a gene at the right time something called the mediator complex must bind to another site in the DNA and the RNA polymerase — the whole mess contains over 100 proteins and has a molecular mass of over 2 megaDaltons (with our friend carbon containing only 12 Daltons). This monster must somehow find and unwind just the right stretch of DNA in the extremely cramped confines of the nucleus. That’s just transcription of DNA into RNA. Translation of the messenger RNA (mRNA) into protein involves another monster — the ribosome. Most of our mRNA must be processed lopping out irrelevant pieces before it gets out to the cytoplasm — this calls for the spliceosome — a complex of over 100 proteins plus some RNAs — a completely different molecular machine with a mass in the megaDaltons. There’s tons more that we know now, equally complex.

So what.

Gradually I came to realize that what needs explaining is not the poor child dying of Werdnig Hoffman disease but that we exist at all and for fairly prolonged periods of time and in relatively good shape (like my father who was actively engaged in the law and a mortgage operation until 6 months before his death at age100). Such is the solace of molecular biology. It ain’t much, but it’s all I’ve got (the religious have a lot more). You guys have the chemical background and the intellectual horsepower to understand molecular biology — and even perhaps to extend it.

 

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.

The uses of disorder

There was a lot of shock and awe about a report showing how seemingly minor changes in an aliphatic group on benzene led to markedly different conformations in its protein target (lysozyme from bacteriophage T4) http://pipeline.corante.com/archives/2015/06/18/tiny_and_not_so_tiny_changes.php.

Our noses are being rubbed in just how floppy proteins are, in contrast to the first glimpses of protein structure obtained by Xray crystallography. Back then we knew so little about proteins, that seeing all the atoms laid out in alpha helices and beta sheets was incredibly compelling. We talked about the structure of a protein rather than a structure. Even back then, with hemoglobin (one of the first solved proteins) it was obvious that proteins had to have more than one structure. The porphyrin ring in heme that oxygen binds to is buried deep in hemoglobin, and the initial structure had to move in some way to allow oxygen to find its way in (because the initial structure showed no obvious channel for oxygen). So hemoglobin had to breathe.

We now know that many proteins have intrinsically disordered segments. Amazingly, the most recent estimate I could find in my notes (or in Wikipedia) is this — It is estimated that over 30% of eukaryotic proteins have stretches of over 30 amino acids that are intrinsically disordered [ J. Mol. Biol. vol. 337 pp. 635 – 645 ’04 ]. Does anyone out there know of more recent data?

We’re a lot smarter now — here’s a comment on Derek’s post — “I have always thought crystal structures of proteins/enzymes are more a guide than actually useful. You are crystallizing a protein first-proteins don’t pack like that in vivo. Then you are settling on the conformation that freezes out- is this the lowest energy form? Then you are ignoring hte fact that these are highly dynamic structures that are constantly moving, sliding, shaking, adjusting. Then if you put a ligand in there you get the lowest energy form-which is what it would look like after reaction and before ligand dissociation- this is quite different from what it can look like at other stages of the reaction.”

Here is an interesting example of the uses of protein disorder going on right now in just about every neuron in your body. Most neurons have long processes, far too long for diffusion to move a needed protein to their ends. For that purpose we have microtubules (aka neurotubules in neurons) stretching the length of the processes, onto which two types of motors attach (dyneins which moves things to negative end of the microtubule and kinesins which move things to the positive end).

The microtubule is built from a heterodimer of two proteins (alpha and beta tubulin). Each contains about 450 amino acids and forms a globule 40 Angstroms (4 nanoMeters) in diameter. The heterodimers pack end to end to form a protofilament. 13 protofilaments line up side by side to form the microtubule, a hollow structure about 250 Angstroms in diameter. In cells microtubules are 1 to 10 microns long, but in nerve process they can be ‘up to’ 100 microns in length. Even at 1 micron (1,000 nanoMeters) that’s 13 * 250 heterodimers in a microtubule.

Any protein structure this important has a lot of modifications imposed on it to alter structure and function. Examples include phosphorylation and the addition of glutamic acid chains (polyglutamylation). The carboxy terminal tails of alpha and beta tubulin are flexible and stick out from the tubulin rod (which is why they aren’t seen on Xray crystallography). The carboxy terminal tail is the site of post-translational glutamylation. The enzyme polyglutamylating the carboxy terminal tail of beta tubular is TTLL7 (you don’t want to know what the acronym stands for). It binds to the alpha/beta tubular heterodimer by an intrinsically disordered region of its own (becoming structured in the process), then it binds to the intrinsically disordered carboxyl terminal tails, structuring them and modifying them. It’s basically a mating dance. There is a precedent for this — see https://luysii.wordpress.com/2013/12/29/the-mating-dance-of-a-promiscuous-protein/

So disordered regions of proteins although structureless are far from functionless