Category Archives: Chemistry (relatively pure)

Frameshifting

hed oga tet hec atw hoa tet her atw hob ith erp aw

Say what?  It’s a simple sentence made of 3 letter words frameshifted by one

he dog ate the cat who ate the rat who bit her paw

Codons are read as groups of three nucleotides, and frameshifting has always been thought to totally destroy the meaning of a protein, as an entirely different protein is made.

Not so says PNAS vol. 117 pp. 5907 – 5912 ’20. Normally a frameshifted protein has only 7% sequence identity with the original.  This is about what one would expect given that there are 20 amino acids, and chance coincidence would argue for 5%.  But there are more ways for proteins to be similar rather than identical.  One can classify our amino acids in several ways, charged vs. uncharged, aromatic vs. nonaromatic, hydrophilic vs. hydrophobic etc. etc.

The authors looked at 2,900 human proteins, then they frameshifted the original by +1 and compared the hydrophobicity profiles of the two.  Amazingly there was a correlation of .7 between the two, despite sequence identity of 7%.  Similarly frameshifting didn’t disturb the chance of intrinsic disorder.  So frameshifting is embedded in the structure of the universal genetic code, and may have actually contributed to its shaping.  Frameshifting could be an evolutionary mechanism of generating proteins with similar attributes (hydrophobicity, intrinsic order vs. disorder, etc.) but with vastly different sequences.  The evolution, aka natural selection aka deus ex machine aka God could muck about the ready made protein and find something new for it to do.   A remarkable concept.

The gag-pol precursor p180 of the AIDS virus is derived from the gag-pol mRNA by translation involving ribosomal frameshifting within the gag-pol overlap region.  The overlap is 241 nucleotides with pol in the -1 phase with respect to gag (that’s an amazing 80 amino acids).  I was amazed at the efficiency of coding of two different proteins (one and enzyme and one structural), but perhaps they aren’t that different in terms of hydrophobicity (or something else).

I’d love to see the hydropathy profile of the overlap of the two proteins, but I don’t know how to get it.

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.

 

 

The ubiquitin wars

Ubiquitin used to be simple.  All it had to do was form an amide between its carboxy terminal glycine and the epsilon amino group of lysine of a target protein, and bingo — the protein was targeted for degradation by the proteasome.

Before proceeding, it’s worth thinking why this sort of thing doesn’t happen more often, by which I mean amide formation between carboxyl groups on aspartic and glutamic acid on one protein and lysines on the surface of another.  That’s where the 3 amino acids are likely to be found, because they are charged at physiological pH, meaning they cost energy (and probably entropy) to put into the relatively hydrophobic interior of a protein where there isn’t a lot of water around to hide their charges.   Also, every noncyclic protein (which is just about all of them) has a carboxy terminal amino acid — why don’t they link up spontaneously to the lysines on the surface of other proteins?

Well, ubiquitin does NOT link up spontaneously.  It has a suite of enzymes to do so.  Like a double play in baseball, 3 enzymes are involved, which move ubiquitin to E1 (the shortstop) to E2 (the second baseman) to E3 (the first baseman).  We have over 600 E3 enzymes, 40 E2s and 9 E1s.  650/20,000 protein coding genes is a significant number — and the 600 E3s are likely there to provide specificity to just what protein gets linked to.

Addendum 21 Feb — Silly me, I should have added in the nearly 100 genes coding for proteins that remove attached ubiquitins (e.g. the deubiquitinases).

A few more fun facts and then down to business.  First ubiquitin is so stable that boiling water doesn’t denature it [ Science vol. 365 pp. 502 – 505 ’19 ].  Second ubiquitin can link to itself, as it contains 7 lysines at amino acids 6, 11, 27, 28, 33, 48 and 63 of the 72 amino acids contained in the protein.

Polyubiquitin chains are often made up of multiple ubiquitin monomers with lengths up to 10 [ Nature. vol. 462 pp. 615 – 619 ’09  2009 ] meaning that there could be a lot of different ones ( 7^10 = 282,475,249.  However chains found in nature seem to use just one type of link, e.g. linking the carboxyl group of one ubiquitin to just one of the 7 lysines over and over, forming a rather monotonous polymer.

On to the interesting paper, namely the ubiquitin wars inside a macrophage invaded by TB [ Nature vol. 577 pp. 682 – 688 ’20 ]  Ubiquitin initially was thought to be a tag marking a protein for destruction.  It’s much more complicated than that.  A host E3 ubiquitin ligase (ANAPC2, a core subunit of the anaphase promoting complex/cyclosome) promotes the attachment of lysine #11 linked ubiquitin chains to lysine #76 of the TB protein Rv0222.  In some way this helps Rv022 to suppress the expression of proinflammatory cytokines.

We do know that the ubiquitination of Rv022 facilitates in some way the recruitment of the protein tyrosine phosphatase SHP1 to the adaptor protein TRAF6 (Tumor necrosis factor Receptor Associated Family member 6) preventing the its ubiquitination and activation.  Of interest is the fact that TRAF6 itself is an E3 ubiquitin ligase which acts on many proteins.

Now to continue and show the further complexity of what’s going on inside our cells.  Autophosphorylated IRAK leaves the TLR (Toll Like Receptor) signaling complex forming a complex with TRAF6 resulting in the oligomerization of TRAF6.  Somehow this activates TAK1, a member of the MAP3 kinase family and this leads to the activation of the family of IKappaB kinases which phosphorylate IKappaB leading to its proteolysis.  Once IKappaB is removed from NFKappaB, translation of NFKappaB to the nucleus occurs where it turns on transcription of cytokines and other proinflammatory genes.

It is really amazing when you think of all the checks and balances going on down there.  How crude our weapons against inflammation are now, compared to what we might have when we know all the mechanisms behind it.

What would Woodward do — take II

“It’s no wonder that truth is stranger than fiction. Fiction has to make sense.”  Mark Twain.

The Harvard Chemistry Department chair arrested?  And for what?  For lying and hiding research work he was doing for China.

“The arrangement between Lieber and the Chinese institution spanned “significant” periods of time between at least 2012 and 2017, according to the affidavit. It says the deal called for Lieber to be paid up to $50,000 a month, in addition to $150,000 per year “for living and personal expenses.”

Who knew betraying your country could be so lucrative?  Of course these are allegations, and have to be proved in court.

What would the great Robert Burns Woodward (https://en.wikipedia.org/wiki/Robert_Burns_Woodward) say to this?  He’s already spinning in his grave over the slings and arrows heaped on pure synthetic organic chemistry.  For details see part of an old post at the end.

Interesting how the department has changed.  No Chinese there at all ’60 – ’62 (even postdocs).  There were several Japanese and Sikh postdocs along with a fair number of happy go lucky Australians.

Chemistry applications can be lucrative.  The new Princeton Chemistry Building was built thanks to professor Ted Taylor, whose royalties on Alimta (Pemetrexed), an interesting molecule with what looks like guanine, glutamic acid, benzoic acid and ethane all nicely stitched together to form an antifolate, to the tune of over 1/4 of a billion dollars built it.

It’s interesting to note that the Princeton undergraduate catalog for ’57 – ’58 has Dr. Taylor basically in academic slobbovia — he’s only teaching Chem 304a, a one semester course “Elementary Organic Chemistry for Basic Engineers” (not even advanced engineers)

For details please see  — https://luysii.wordpress.com/2011/05/16/princeton-chemistry-department-the-new-oberlin/

What would Woodward do ?

Sleeper is one of the great Woody Allen movies from the 70s.  Woody plays Miles Monroe, the owner of (what else?) a health food store who through some medical mishap is frozen in nitrogen and is awakened 200 years later.  He finds that scientific research has shown that cigarettes and fats are good for you.  A McDonald’s restaurant is shown with a sign “Over 795 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 Served”

I returned from my father’s 100 year birthday blowout and band camp and began attacking a giant pile of accumulated unread journals.  In the 9 August Nature (p. 630 – 631)  2007 he was amazed to read criticism of a 64 step 22 year synthesis of an exquisitely complex molecule (azadirachtin) — a molecule in which it is easier to count the number of optically INactive carbons than the optically active ones.  Back in the 60s we were all impressed with how Woodward got the 5 asymmetric centers in a 6 membered ring of reserpine (which was in use as an antihypertensive at the time, and whose fairly common side effect of depression was one of the clues leading to the amine theory of affect).  Rip was surprised to find that the criticism was not that the synthesis was incorrect, but that the project shouldn’t have been done at all.  Apparently a significant body of organic chemists think this way.

Political correctness has left few groups which it is safe to disparage.  With apologies to one of them (Christians) I’ve got to ask “What would Woodward do?”

Have you had your molybdenum today?

Chemists don’t usually think of the products of a chemical reaction barreling off and penetrating another structure.   Because of the equipartition of energy, the energy of a given exothermic chemical reaction quickly gets redistributed into electronic, vibration and rotational energy and some translational energy.  It’s exactly why blasting a particular bond with exactly the right energy to break it, isn’t widely used in synthetic organic chemistry — the energy redistributes over the whole molecule too rapidly.  But that’s exactly what is thought to happen in the molybdenum storage protein according to Proc. Natl. Acad. Sci. vol. 116 pp. 26497 – 26504 ’19.

Back off a bit.  Without molybdenum we’d all be dead, as it is a critical component of the plant enzyme breaking the triple nitrogen to nitrogen (aka nitrogenase), so it can be fixed into biologic material of the plant (and ultimately us).  It takes 225 kiloCalories/mole to break N2 apart (compared to 90 kiloCalories/mole for the carbon carbon bond in ethane).

The paper concerned discusses the molybdenum storage protein of a bacterium  (Azotobacter vinelandii).  The protein is a heterohexamer of 3 alpha and 3 beta subunits with a total molecular mass of 180 kiloDaltons.

The mechanism if cleverness itself — here’s a direct quote from the abstract of the paper. “First, we show that molybdate, ATP, and Mg2+ consecutively bind into the open ATP-binding groove of the β-subunit, which thereafter becomes tightly locked by fixing the previously disordered N-terminal arm of the α-subunit over the β-ATP. Next, we propose a nucleophilic attack of molybdate onto the γ-phosphate of β-ATP, analogous to the similar reaction of the structurally related UMP kinase. The formed instable phosphoric-molybdic anhydride becomes immediately hydrolyzed and, according to the current data, the released and accelerated molybdate is pressed through the cage wall, presumably by turning aside the Metβ149 side chain. A structural comparison between MoSto and UMP kinase provides valuable insight into how an enzyme is converted into a molecular machine during evolution. The postulated direct conversion of chemical energy into kinetic energy via an activating molybdate kinase and an exothermic pyrophosphatase reaction to overcome a proteinous barrier represents a novelty in ATP-fueled biochemistry, because normally, ATP hydrolysis initiates large-scale conformational changes to drive a distant process.”

What drives the MO4 away from the ADP ? Probably electrostatic repulsion between two negative charges in the very low dielectric constant environment of the storage protein (said to be around 7 with water at 80) which does relatively little to shield the charges from each other.

Of course the SN2 reaction is like two billiard balls hitting each other with the leaving group barreling off at about the same velocity as the attacking group. How fast is that?

Pretty fast.  To figure out how fast any chemical entity is moving at 300 K (80 F) just divide 2735 by the square root of the molecular mass.  So when Iodine barrels in to methyl bromide at 243 meters second, the bromine leaves at 307 meters second.

Well the C – Br bond length  is 1.9 Angstroms, the atomic radii of Br and C are 1.8 and .7 Angstroms — So methyl bromide is 4.5 Angstroms long or 4.5 x 10^-10 meters.  So 307 meters/ second means that the bromine ion takes  roughly 10^-3 seconds to go a meter, and 10^-3 * ( 1/4.5) * 10^-10 ) seconds to go the diameter of the methyl bromide molecule.  (Of course this ignores the solvent that’s in the way impeding the Bromine anion’s progress — but that’s another story).  I put this numerology in because chemists (including me) usually don’t think about reactions this way and it’s rather humbling to do so.

How a chemical measuring stick actually works

The immune system knows something is up when a foreign peptide fragment is presented to it.  Here’s the hand holding the peptide — https://www.researchgate.net/figure/Overall-structure-of-HLA-peptide-complex_fig1_26490512.

There it sits, lying on top of a bed of beta sheets, with two side rails of alpha helices.  Proteins are big, way too big to fit into the hand, so the fragments must be chopped up into peptides no longer than 9 amino acids long (see the picture of it lying in state).

So the class assignment for today is to figure out how to design a protein which takes peptides from 10 – 16 amino acids long, and shortens them to 9 amino acids.

Obviously a trick question, because the actual amino acids making up the peptide don’t really matter much.  So somehow the protein is reacting to length rather than chemistry.

Tricky no?

ERAP1 (Endoplasmic Reticulum aminopeptidase associated with Antigen Processing has figured it out [ Proc. Natl. Acad. Sci. vol. 116 pp. 22709 – 22715 ’19 ].  It is a huge protein (948 amino acids) with four domains forming a large cavity (which it must have to accomodate a 19 amino acid paptide).  The peptide is chopped up from the amino terminal, stopping when the length reaches 9 amino acids.  The active site is at one end of the cavity, and at the other end there is a site which looks like it should cleave the carboxyterminal amino acid, but it doesn’t because the site is inactive.  However, even catalytically inactive enzymatic sites have enough structure left so they bind the substrate.

So binding of the carboxy terminal amino acid to the back site causes conformational changes transmitted through various alpha helices to the active enzyme at the other end.  It munches away removing amino acid after amino acid until the peptide gets short enough (translation 9 amino acids) so that it doesn’t push on the back site.

Incredibly clever, even though it hurts me as a chemist to see the enzyme essentially ignoring the chemistry of its substrate.

I far prefer this to politics where data is ignored.  Two examples

l. From a review of a book by Paul Krugman in the Jan/Feb 2020 Atlantic

“Krugman is substantively correct on just about every topic he addresses.” Yes except Peak Oil in 2010, Stock Market collapse in Nov 2016 and the coming recession in an article April 2019

2. Former Secretary of Labor Robert Reich in the Guardian 22 Dec ’19 — “How Trump has betrayed the working class” — by employing them and raising their wages no doubt.

How little we really understand about proteins

How little we really understand about proteins.  We ‘know’ that the 7 transmembrane alpha helices of G Protein Coupled Receptors (GPCRs) all contain hydrophobic amino acids, so they dissolve in the (hydrophobic) lipids of the membrane.  GPCRs have been intensively by chemists, molecular biologists, pharmacologists and drug chemists with the net result that as of last year “128 GPCRs are targets for drugs listed in the Food and Drug Administration Orange Book. We estimate that ∼700 approved drugs target GPCRs, implying that approximately 35% of approved drugs target GPCRs.” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820538/

So if you changed the hydrophobic amino acids found in the 7 transmembrane segments of GPCRs to hydrophilic ones — all hell should break loose.

Wrong says Proc. Natl. Acad. Sci. vol. 116 pp. 25668 – 25667 ’19 ].  The trick was to replace hydrophobic amino acids with hydrophilic ones with the same shape.

Thus leucine (L — single amino acid letter code) is replaced by glutamine (Q), Isoleucine (I) and Valine (V) is replaced by Threonine (T) and finally phenylalanine (F) is replaced by Tyrosine (Y).  They call this the QTY code.

Instead of destroying the structure of the GPCRs (CCR5 and CXCR4) they became water soluble, and bound their ligands CCL5 for CCR5  and CXCL12 for CXCR4 to the same extent.

Even more amazing, the QTYdesigned receptors exhibit remarkable thermostability in the presence of arginine and retained ligand-binding activity after heat treatment at 60 °C for 4 h and 24 h, and at 100 °C for 10 min.

I would never have expected this.  Would you?

Why did they even do it?  Because GPCR structures are hard to study. You either have to remove them en bloc from the membrane or dissolve them in other lipids so they don’t denature.  Why these two GPCR’s?    Because their ligands are proteins and can’t snuggle deep down inside the 7 alpha helices embedded in the membrane (they’re just too big), but bind to the outside surface.  CCL5 is an 8 kiloDalton protein (probably 80 amino acids, while CXCL12 has 93.  So just solublizing the GPCR without changing any of the amino acids external to the membrane, produces an object for study.

It would be amusing to do the same thing for a GPCR binding one of the monamines.  I doubt that they would bind, but I never would have believed this possible in the first place.

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.

 

The neuropharmacological brilliance of the meningococcus

The meningococcus can kill you within 12 hours after the spots appear — https://en.wikipedia.org/wiki/Waterhouse–Friderichsen_syndrome.  Who would have thought that it would be teaching us neuropharmacology.   But it is —  showing us how to make a new class of drugs, that no one has ever thought of.

One of the most important ways that the outside of a cell tells the inside what’s going on and what to do is the GPCR (acronym for G Protein Coupled Receptor).  Our 20,000 protein coding genome contains 826 of them. 108 G-protein-coupled receptors (GPCRs) are the targets of 475 Food and Drug Administration (FDA)-approved drugs (slightly over 1/3).   GPCRs are embedded in the outer membrane of the cell, with the protein going back and forth through the membrane 7 times (transmembrane segment 1 to 7 (TM1 – TM7). As the GPCR sits there usually the 7 TMs cluster together, and signaling molecules such as norepinephrine, dopamine, serotonin etc. etc. bind to the center of the cluster.   This is where the 475 drugs try to modify things.

Not so the meningococcus. It binds to the beta2 adrenergic receptor on the surface of brain endothelial cells lining cerebral blood vessels, turning on a signaling cascade which eventually promotes opening junctions of the brain endothelial cells with each other, so the bug can get into the brain.  All sorts of drugs are used to affect beta2 adrenergic receptors, in particular drugs for asthma which activate the receptor causing lung smooth muscle to relax.  All of them are small molecules which bind within the 7 TM cluster.

According to Nature Commun. vol 10 pp. 4752 –> ’19, the little hairs (pili) on the outside of the organism bind to sugars attached to the extracellular surface of the receptor, pulling on it activating the receptor.

This a completely new mechanism to alter GPCR function (which, after all,  is what our drugs are trying to do).  This means that we potentially have a whole new class of drugs, and 826 juicy targets to explore them with.

Here is one clinical experience I had with the meningococcus.  A middle aged man presented with headache, stiff neck and fever.  Normally spinal fluid is as clear as water.  This man’s was cloudy, a very bad sign as it usually means pus (lots of white blood cells).  I started the standard antibiotic (at the time)  for bacterial meningitis — because you don’t wait for the culture to come back which back then took two days.  The lab report showed no white cells, which I thought was screwy, so I went down to the lab to look for myself — there weren’t any.  The cloudiness was due to a huge number of meningococcal bacteria.  I though he was a goner, but amazingly he survived and went home. Unfortunately his immune system was quite abnormal, and the meningitis was the initial presentation of multiple myeloma.

Is the microtubule alive ??

When does inanimate matter become animate?  How about cilia — they beat and move around.  No one would call  the alpha/beta tubulin dimer from which they are formed alive.  The tubulin proteins contain 450 amino acids or so and form a globule 40 Angstroms (4 nanoMeters) in diameter.  The dimer is then 40 x 80 Angstroms and looks like an oil drum.  Then they form protofilaments stacked end to end — e.g. alpha beta alpha beta.  Then 13 protofilaments then align side by side to form the microtubule (which is 250 Angstroms in diameter, with a central hole about half that size.  Do you think you could design a protein to do this?

Lets make it a bit more complicated, and add another 10 protofilaments forming a second incomplete ring.  This is the microtubule doublet, and each cilium has 9 of them all arranged in a circle.

Hopefully you have access to the 31 October cell where the repeating unit of the microtubule doublet is shown in exquisite detail — https://www.cell.com/action/showPdf?pii=S0092-8674%2819%2931081-5. — Cell 179, 909–922 ’19

The structure is from the primitive eukaryote Chlamydomonas, the structure repeats every 960 Angstroms (e.g every 12 alpha/beta tubulin dimers).  So just for one repeating unit which is just under 1/10 of a micron (10,000 Angstroms) there are (13 + 10) * 12 = 276 dimers.  The cilium is 12 microns long so that’s 12 * 276 * 100 = 298,080 alpha tubulin dimers/microtubule doublet. The cilium has 9 of these + another doublet in the center, so thats 2,980,800 alpha tubulin dimers/cilium.

The cell article is far better than this, because it shows how the motor proteins which climb along the outside of the doublet (such as dynein) attach.The article also describes the molecular ruler (basically a 960 Angstrom coil coil which spans the repeat. They found some 38 different proteins associated with the microtubule repeat.  They repeat as well at 80, 160, 240, 480 and 960 Angstrom periodicity.  The proteins in the hole in the center of the microtubule (e.g. the lumen) are rich in a protein module called the EF hand which binds calcium, and which likely causes movement of the microtubule, at which point the damn thing (whose structure we now know) appears alive.

Because of the attachment of the partial ring (B ring) to the complete ring of protofilaments, each of the 23 protofilaments has a unique position in the doublet, and each of the proteins in the lumen is bound to a specific mitotubule profilament. There are 6 different coiled coil proteins inside the A ring, occupying  specific furrows between the protofilaments.

Staggering complexity built from a simple subunit, but then Monticello is only made of bricks.