Have a lotta sugar you need cells to take in? Hope you’ve got enough insulin! Today’s post might get heavy – but it was heavy atoms that let Dorothy Crowfoot Hodgkin figure out what insulin looks like – which let pharma develop optimized versions for treating diabetes.
Insulin is a hormone (chemical messenger) your body uses to help control blood sugar levels. I’m grateful that I don’t have diabetes, but, for people who do, their body has a hard time making and/or “hearing” insulin’s call to “let glucose in!” As a result, they have trouble controlling blood sugar levels, so they often use devices called glucometers to measure blood glucose levels & then, if the levels are too high, they can take insulin.
Different versions of insulin have been created to be fast-acting or slow-acting – and their design was made possible by figuring out its “natural design” – the 3D structure it adopts. Solving the structure of insulin was the crowning achievement for the pioneering crystallographer Dorothy Crowfoot Hodgkin – it took her 34 years, but she did it – and countless lives have been saved because of it.
Diabetes is a disease where the body either doesn’t make (type 1 diabetes (T1D)) or can’t use effectively (type 2 diabetes (T2D)) a hormone called insulin. Hormones are chemical messengers that can relay messages throughout & between cells in your body. Different hormones are made of different building blocks & relay different messages.
Insulin is a peptide hormone, so it’s made up of amino acids (these are the same “letters” as proteins us but proteins are more like novels while peptide hormones are like “text messages”). The text sender is an organ called the pancreas, & the message it relays is – there’s a lot of sugar in the blood – let’s take some into the cells, use some, & store some, why don’t we?
The pancreas basically sends out a “mass text” to cells throughout the body in the form of insulin. The cells respond by opening their doors to glucose (the main monosaccharide (single sugar unit)), importing it from the bloodstream into the cells thus lowering the amount of glucose in the blood. And it tells cells in the liver to “turn on” an enzyme called glycogen synthase in which strings excess glucose into chains (polysaccharides) called glycogen for storage. more here: http://bit.ly/2JwIuRV
Too much sugar is hypERglycemia (remember ovER). Too little sugar is hyPOglycemia (remember below). Both are bad. Which is why having a properly-functioning “early alert” system through insulin signaling is so important. It gives your cells time to prepare & avert a crisis.
But T1D is like the text message never being sent and T2D is like the text gets sent but the phones have the caller on their block list so they never hear the message. If cells are phones, it’s not that they’re completely turned off – they can still get other messages fine, but their bodies have become “desensitized” to it.
Especially for patients with T1D, who can respond normally to insulin, they just can’t make enough of it on their own, insulin has been a cornerstone of treatment. But it’s really important that the administration of insulin is tightly controlled so your body’s not just “taking hits” of the stuff. Sometimes you want something that acts really fast, but other times you want something that gives a more gradual, prolonged effect. So pharmaceutical companies have created “designer insulins” that are fast-acting or slow-acting.
One of the main differences between these forms is their tendency to “oligerimize” – individual insulin units (monomers) can pair up to form dimers and then 3 of those dimers can stick together to form hexamers. It’s the monomer form that’s biologically active, so encouraging this form makes for faster-acting insulin, whereas encouraging the hexameter form slows things down.
So, how do you encourage insulin molecules to hang out or not hang out? It helps if you know how the hanging out comes about! To grasp this there are a few key concepts to keep in mind.
First, and at the core of basically everything in biochemistry, is the deal that opposite charges attract & like charges repel. And these charges don’t even have to be “full” charges. The charge of molecules comes from from uneven number and/or distribution of electrons. Electrons are teeny weeny negatively-charged “subatomic particles” that whizz around dense atomic nuclei where an atom’s positively-charged protons & negatively-charged neutrons hang out. The outermost electrons (called valence electrons), furthest from the pull of the positive protons, are the most energetic & reactive and they can pair up with valence electrons from other atoms to form strong covalent bonds.
Sometimes they can get pulled all the way away from their parent atom, leaving the parent with one fewer electron than protons, so a full charge imbalance (and similarly, but oppositely with the one that gains an extra electron). And we call such fully-charged particles “ions” – “cation” refers to a positively-charged one and “anion” to one that’s negatively-charged. Metals often lose electrons and hang out as cations – so, for example, zinc (Zn) likes to hang out as a divalent (2 charged) cation (+ charged thing), which we can write as Zn²⁺. Keep this in mind because it’s going to come into play in the story I’m telling today!
So ions have a full, “formal” charge. But sometimes, even when electrons are shared and not stolen (so they’re still “owned” by their original atom) they’re shared unevenly, so even though a molecule is neutral overall, parts of it are partly charged. And partly charged regions can get attracted to & repelled by other partly or fully charged regions of things.You get this uneven sharing (a polar covalent bond) when one of the sharing partners is much more electron-hogging (electronegative) than the other.
Oxygen & nitrogen, for example, have much more pulling power than hydrogen – so when they form covalent bonds with H, they convince the H’s lone electron to hang out near them more. So this makes the H partly positive and the O or N partly negative. The partly positive H then frequently gets attracted to a lone pair of electrons on a different O or N, leading to a type of attraction called a hydrogen bond (H-bond).
H-bonding is really important in biochemistry and it’s responsible for everything from water being “sticky” (surface tension, etc.) to DNA strands sticking together. They’re so important because they’re weak enough for individual bonds to easily come apart and together but strong in numbers. So, for example, to “unzip” the strands of double-stranded DNA you have to heat them up or use molecular “helpers” – protein enzymes called helicases spend energy money to break them apart. But the individual strands stay “strand-y” because the the DNA letters (nucleotides) are connected by strong covalent bonds that involve actual electron sharing instead of just charge-based attractions.
H-bonding also plays crucial roles in proteins. Proteins are made up of letters called amino acids, which have generic backbones for linking any of them to any other & unique side chains (R groups) that stick off like charms in a charm bracelet. Amino acids are linked into chains through peptide bonds. These bonds are covalent bonds that form when the carboxyl (-(C=O)-O-) of one amino acid joins with the amino (-NH2/3) group of another amino acid. This leaves you with alternating potential H-bond donors (the H on the NH) and acceptors (the O on the C=O) along the peptide backbone. H-bond forming between these parts of the backbone can lead to common “secondary structure” motifs like alpha helices & beta strands.
And further H-bonding opportunities are provided by some of the unique side chains (R-groups) of the different letters. These letters can interact with other letters (either their side chains or backbones) within their chain to form “tertiary structure.” And they can interact with atoms from other protein chains to form “quaternary structure” (and if you’re wondering about primary structure, that just refers to the sequence of amino acids in a chain.
As a result, peptide chains can interact with one another and you can get multiple chains grouping together to form “oligomers” – oligomers can be multiple copies of the same chain (in which case we call them homooligomers) or different chains (heterooligomers). Sometimes these can be the functional “active” form of a protein, but other time, oligomers can be inactive forms.
This latter situation is the case with insulin – it can form dimers that can form hexamers, but those hexamers are *Not* the active form. Instead the active form is the monomer.
The reason the hexamers form starts with the reason dimers form, which involves H-bonding between the B chains. And here’s where the nomenclature starts to get a bit tricky. I’ve been using the term “chain” to refer to the polypeptide chains encoded for in a protein’s genetic instructors, which the ribosome faithfully links together. Each protein is synthesized as a chain like this. And usually this chain just folds up into a functional protein. Sometimes grouping up with other chains, but that chain stays that chain.
But in the case of insulin it gets made as a longer chain called preproinsulin that gets cleaved in 2 places to give you mature insulin which is made up of 2 stuck together smaller chains in each “monomer” of insulin. The first 24 amino acids form a “signal peptide” – As the protein gets made, these come out of the ribosomal tunnel first and signal to the cell that this protein is destined for secretion (getting shipped out). For such secreted proteins, processing usually takes place in a special compartment in the cell called the endoplasmic reticulum (ER). So the ribosome sends the finished chain in there. And then, since it’s no longer needed, the signaling peptide gets cleaved off, leaving you with proinsulin. This pro-insulin then folds up, and gets cleaved again to give you 2 chains, alpha (21 amino acids) & beta (30 amino acids) (both from that original chain).
These chains stay stuck to one another because they have 2 key disulfide crosslinks. Unlike most side chain interactions, disulfide bonds, which can form between cysteine residues (eg. protein-SH + HS-protein -> protein-S-S-protein) are covalent bonds. So they’re strong. And keep the strands stuck together even though their backbone’s broken. So each mature insulin “monomer” is 51 amino acids in 2 chains from 1 original chain. This is *not* the dimer.
Your pancreas has to stock up on a lot of insulin so it has it ready to ship out when needed. But you don’t want it taking up a bunch of space and you don’t want it “getting loose” so it makes biological sense to store it in a compact, inactive form. The hexametric form is great for this purpose. When insulin gets secreted from the pancreas into the bloodstream, the hexamers fall apart into the active monomers because the zinc concentration is way lower
But turns out that the hexamer-izing can be a problem if you want to use it as a drug to treat diabetes. Because, while the monomers and dimers can easily diffuse into the bloodstream, those big ole hexamers have a harder time getting in there if you just inject them under the skin (subcutaneously). So scientists make “designer insulin” thanks to recombinant protein expression technology (being able to stick protein instructions into cells to have them make it for you). Pharma companies can change insulin’s spelling in a way that didn’t affect its receptor binding but does prevent dimerization and hexamerization, so that it absorbs better and acts faster. Alternatively, they can change the spelling to promote linking up to make it act slower and last longer so you don’t have to inject it as often.
But in order to make those changes, you need to know where to make them, and this is where Dorothy Crowfoot Hodgkin becomes the superhero of our story. Hodgkin was a true pioneer in the technique of x-ray crystallography, which allows you to harness the power of x-rays to figure out what molecules look like at the “atomic scale” (how do all the carbons, etc. link up).
You can learn more about Hodgkin here: http://bit.ly/2TscWlv
And more about crystallography here: http://bit.ly/2QASc8h
But here’s the basic gist of the technique: you get molecules to crystallize (arrange themselves into an orderly 3D lattice) -> beam x-rays at them -> x-rays get scattered by the molecules -> scattered x-rays interfere with one another, some “cancelling out” while others strengthen one another depending on their relative phases (where in their peak-trough-peak-trough… cycle they are) -> these “diffracted” x-rays hit a detector, leaving a pattern of spots called a diffraction pattern -> you work backwards from those spots to figure out where they scattered from.
Doesn’t sound so bad, right? Wrong. Firstly, getting a protein to crystallize into nicely-diffracting crystals is often no easy feat (think trays and trays of screening for the optimal crystallization conditions, which varies from protein to protein). Dorothy was so excited to get her first diffracting crystals, in 1935, that she panicked herself into thinking the diffraction pattern was coming from a salt or some contaminant or something instead of insulin that she rushed in the following morning to check! And these weren’t even very useful crystals in terms of data – but they showed that it could be done! There was hope – and by optimizing and optimizing she was able to grow larger, better-diffracting crystals.
But, even once you have a diffraction pattern, you still have to do the hard math of figuring out what those spots correspond to. Even with powerful computers, which Dorothy didn’t have to help, you’re left with the “phase problem”
when x-rays hit crystals, they get scattered from the electrons of the atoms because they kinda “ring” the electron clouds, getting the electrons to send off their own waves of the same wavelength (kinda like billiard balls getting tossed in a pool and generating rippling waves rather than billiard balls bouncing off pool table walls). The waves add together through wave interference, sometimes canceling out, other times strengthening each other, leading to kinda “megawaves.” You can capture the signals from these megawaves on the detector and deconstruct the megawave to the miniwaves using a math thing called a Fourier transform. But it requires you to know the “phases” – was a wave peaking or troughing when it hit? And you lose this information in crystallography – you only measure the amplitudes (how strong was the wave when it hit). And this leads to the “phase problem”
Carbon and oxygen and hydrogen and all those “usual players” in biochemical molecules don’t vary very much in terms of their electron stock (hydrogen has 1, carbon 4, and your “big guy” oxygen has 8).
Lead? In its neutral form it has 82. So it’s kinda like having hydrogens in one spot – the lead atoms will scatter more strongly than the other atoms in the structure. And this kinda “puts a pin in the map” – if you know what to look for, it “sticks out” in the diffraction pattern, allowing scientists to orient themselves in the signal.
So, to solve the structure, Dorothy used heavy atom soaking – she used EDTA, a metal chelator (basically it’s a molecule that can bite metal in multiple places so it can steal it from the insulin), to remove the zinc. And then she added lead to replace the zinc. In addition to replacing the zinc, the lead bound a few other places too. So this gave her multiple pins in the map. (note: while useful here, this same zinc-replacing ability is one of the reasons lead is dangerous in your body).
And she also made other heavy atom derivatives to try to “map pin” other sites – so she made derivatives with zinc and lead. As well as derivatives with a couple other heavy atoms – uranium (from uranyl fluoride & uranyl acetate) and mercury (from mercuribenzaldehyde). After a lot of hard work, In 1969, 34 years after taking those initial photos, she reported the structure of 2 Zn insulin at a resolution of 2.8A.
It showed that the hexameter assembles around 2 zinc atoms in the 2 Zn form, with the Zn held there through “coordination” to histidines from each monomer, His10. One Zn coordinates with 3 of them and the other Zn the other 3. Glutamate 13 in the B chain is at the center of the hexamer and, unlike what you’d expect, they’re clustered together – and glutamate is negatively-charged normally. And as we talked about, like charges repel. So you’d think they’d push the insulins apart. But here’s part of where the Zn comes in. With its positive charge, it’s able to help neutralize the situation and stabilize the hexamer. So when there’s a lot of Zn you get the hexamer form, good for storage (and diffraction) but when Zn levels are lower, like in the bloodstream, the glutamates can repel. Also helping things out, The pH in the blood is higher than in the storage vesicles in the beta cells, which means there are fewer H+ around to kinda buffer things, so the glutamates really want to get away from each other – so this also helps promote monomerization
The structure showed that the dimers were forming because of hydrogen-bonding between the C-termini of the B chains of the monomers. Knowing this, once recombinant protein expression was possible, pharma companies could change insulin’s spelling in a way that didn’t affect its receptor binding but did prevent dimerization & hexamerization. For example, insulin lispro (Humalog) swaps 2 C-terminal residues in the B chain, Pro28 & Lys29 to Lys28 & Pro29 (check out PDB entries 1lph or 2kjj). Another one, NovoRapid, mutates a proline to an aspartate. As you might remember from #20DaysOfAminoAcids, Proline (Pro, P) is the least flexible amino acid because its side chain loops in on itself, rebinding to the backbone. So changing this to aspartate (Asp, E) gives greater flexibility and favors monomerization (PDB entries 1zeg, 1zeh or 1kei)
There are also forms to make it slower-acting so you don’t have to inject is as frequently/ Levemir is covalently bound to a fatty acid – this makes it stick to albumin (a protein that’s at really high concentrations in the blood). So the albumin competes with the receptors, leading to longer action. Degludec is a similar version but with stabler hexamers too
Dorothy solved the structure of the pig (porcine) version of insulin. It only differs from the human version by a single amino acid at the C-terminus of the B chain. Human receptors still recognize it so, even before recombinant expression technology, animal versions of insulin could be purified and used to treat diabetics.