Give me a resin

What is it?

Is there a more evocative Christmas smell than the fragrance of a fresh pine tree? Mulled wine with cinnamon spices or a roasting turkey are close contenders for the prize, but for literary purposes let’s opt for the heady scent of a Christmas Fir. That rich winter wonderland terpentine-like citrus aroma is the product of several molecules (read more here http://www.compoundchem.com/2014/12/19/christmastrees/), the most significant of which is pinene.

Pinene is a terpene found in the resin of pine trees, and as well as generating the distinctive coniferous scent, it is also a potent inhibitor of the human Cytochrome P450 2B enzyme. CYP450 proteins are a superfamily of enzymes essential for hormone, cholesterol and vitamin D synthesis and metabolism. They also assist in the clearance of toxins from the body via the liver. So, if you ever you needed a “resin” not to eat your Christmas tree…

What does it look like?

Pinene

Researchers determined the crystal structure of (+)–α-pinene bound to CYP450 2B6 to better understand how this pine tree molecule can bind, inhibit, and alter the enzyme1. This is important to learn about how the enzymes generally interact with a diverse range of substrates.

(+)-α-pinene binds tightly at the CYP450 2B6 active site. The CYP450 2B6 active site is remarkably flexible and moves and shifts to mould around the pinene molecule as it binds.

Where did the structure come from?

This structure is of human CYP450 2B6 and is PDB ID 4I91.

References

  1. Wilderman et al., Journal of the American Chemical Society 2013: 135: (10433-10440)

A little helper – Elf3

What is it?

E74-like factor-3 (Elf3) is a transcription factor. This means that it is a protein involved in the expression of genes. Elf3 works with other proteins to regulate genes involved in inflammation and cancer; it is has been found to be abnormally expressed in some lung and breast cancers.

In order to ensure proper gene expression, transcription factor activity is highly regulated. Accordingly Elf3 is held in an “off” state and must be specifically turned on before it can bind to DNA to start gene expression. The interaction of Elf3 with DNA is also highly specific, again to avoid abnormal gene expression that may lead to disease.

In order to learn how Elf3 recognises, binds and activates gene expression, researchers solved the crystal structure of the Elf3 bound to DNA from one of its target genes.

What does it look like?

ELF3This structure is of a specific part of mouse Elf3, the so-called E-twenty-six (Ets) domain, bound to DNA from mouse Type II TGF-b receptor promoter. Promoter DNA sequences provide a “start to read me from here” instruction to transcription factors so this structure provides lots of information about how the two molecules talk to each other.

Elf3 (green) is shown bound to DNA (red double helix)

Where did the structure come from?

This structure is PDB ID 3JTH1.

References

  1. Agarkar et al., Journal of Molecular Biology, 2010. 397, 278-289

Oh deer…

Roisin McMahon gets us into the jollity of the season….

Did Rudolph go to school?

No. He was Elf-taught.

What do reindeers hang on their Christmas trees?

Horn-aments!

How come you never head about the tenth reindeer, Olive?

Because Olive, the other reindeer, used to laugh and call him names

What is it?

Today’s festive crystal structure is of reindeer β-lactoglobulin. β-lactoglobulin is a whey protein present in the milk of many but not all mammals. It is not found in human milk. Despite extensive investigation, the function of β-lactroglobulin is not yet known. It may be a food source, or it may function as a carrier protein as it is known to bind hydrophobic molecules.

UntitledWhat does it look like?

β-lactoglobulins are part of the lipocalin superfamily. They have a 8 stranded β-barrel fold with one α-helix. Hydrophobic molecules such as retinol or palmitic acid can bind within the centre of the barrel.

Where did the structure come from?

This structure is of reindeer β-lactoglobulin. The protein was purified from reindeer milk. (Q. How do you milk a reindeer? Answers on a post card please as I really wish I had a punch line for this one.) It is PDB ID 1YUP1.

References

  1. Oksanen et al., Acta Crystallographica Section D 2006. 62:1369-1375

Jokes from here http://www.whychristmas.com/fun/cracker_jokes.shtml and here http://www.bjentertainments.co.uk/dan/xmas%20jokes.htm

From crystal to structure in 84 years

What is it?

In 1926 Jack Bean urease was the first protein to be crystallised 1, earning a place in the history books for this humble plant protein and a Nobel Prize for James B. Sumner (see yesterday’s post for more). Besides establishing that proteins could be crystallised, Sumner’s work was remarkable for demonstrating that proteins could be isolated and purified whilst retaining enzymatic activity. Despite this landmark advance in protein biochemistry and crystallography, it was a further 84 years before the crystal structure of Jack Bean Urease was determined, in what must surely be the longest running crystallography endeavour in history2.

Where did the structure come from?

Urease is an enzyme in plants, bacteria and fungi that helps to break urea into ammonia and carbon dioxide. Whilst other urease structures were known – for example Helicobacter pylori urease, the structure of which helped to explain how this bacterium can survive in the acid environment of the human stomach3 – the structure of Jack Bean urease was only determined in 2010.

What does it look like?

Urease

Plant ureases are single chain proteins. Jack Bean urease is a T-shaped molecule of four domains (coloured here in red, blue, yellow and pink.) In common with other ureases, Jack Bean Ureases binds two nickel ions (blue spheres) in its active site; these are required for enzymatic breakdown of urea.

This structure is Protein Data Bank ID 3LA4

References

  1. Sumner, 1926: J. Biol. Chem. 69:435-441
  2. Balasubramanian & Ponnuraj. 2010 J. Mol. Biol. 400:274-283
  3. Ha et al., 2001. Nature Structural Biology 8: 505-509

Superbug superpowers

What is it?

For this, the second installment in mechanisms of bacterial antibiotic resistance, (read the first here) we turn to Penicillin Binding Protein 2a (PBP2a) from methicillin resistant Staphylococcus aureus or MRSA.

Fuelled by sensationalist headlines such as “Superbug crisis worse than feared” and “MRSA out of control and getting stronger” MRSA is now synonymous with the most feared of resistant bacterial infections. But what do we know about how it can side-step antibiotic action?

As the name indicates, MRSA is resistant to the β-lactam antibiotic methicillin. Methicillin – like other β-lactams – mimics a substrate of a penicillin binding protein (PBP). Bacterial PBPs are necessary for cell wall assembly, and antibiotics such as methicillin disrupt cell wall formation by inhibiting normal substrate binding to PBPs.

MRSA can resist methicillin because they have acquired a mecA gene that produces a PBP variant –PBP2a – that simply doesn’t bind β-lactam antibiotics very well. As a result, the PBPs evade methicillin inhibition and continue to build cell walls normally.

Where did the structure come from?

This is the structure of a mecA gene encoded PBP2a from MRSA strain 27r [1].

What does it look like?
superbugPBP2a is a large multi-domain protein; the transpeptidase domain (blue) and penicillin binding domain (yellow) work together to assist proper cell wall synthesis. Comparison of methicillin-sensitive and methicillin-resistant PBPs revealed that methicillin-resistant PBP2a has evolved a distorted active site in its penicillin-binding domain that disrupts interaction with β-lactams, protecting the protein, and thus the bacterium, from antibiotic inhibition.

This structure is Protein Data Bank ID 1VQQ

 

 

Reference:

[1] Lim and Strynadka. Nature Structural Biology 9: 870-876, 2002

Bacteria: How do they resist?

What is it?

When is the last time you were prescribed antibiotics? Aren’t they simply amazing? Painful, unpleasant, potentially life-threatening infections cleared up in a matter of days: pneumonia, cellulitis, abscesses, urinary tract infections, bacterial meningitis, etc. Extraordinary!

But what about when the drugs don’t work? Antibiotic resistance is one of the most significant threats to global public health that we face today, and fear for the future.

Bacteria have evolved a myriad of ways to fight back against our attempts to kill them with antibiotics. This includes production of β-lactamase, a class of enzymes that are able to degrade antibiotics containing a β-lactam ring. β-lactam antibiotics include penicillin, carbapenems and monobactams.

Where did the structure come from?

blactamaseThis is the structure of a β-lactamase from Staphylococcus aureus. S. aureus is responsible for a variety of skin infections and is a major contributor to post-surgical infections in hospitals.

What does it look like?

S. aureus β-lactamase is a small protein made up of two closely associated domains (coloured green and blue.) The enzyme active site is located in the interface between the two domains, and contains 3 catalytically important residues (serine, lysine and glutamate, highlighted in yellow) that are responsible for hydrolysis of the β-lactam ring.

This structure is Protein Data Bank ID 3BLM

References

  1. Herzberg & Mout. 236:694-701, 1987
  2. JMB. 217:701-719, 1991

Caffeine hit: Adenosine A2A receptor

What is it?

Espresso, cold press, café latte, flat white? Percolator, aeropress, capsule, instant? Single origin, blend, single estate? How do you like your daily coffee hit to be delivered?

Regardless of how you chose to consume your caffeine, its stimulant properties are the product of a common mode of action: namely the blockade of adenosine receptors in the brain. Adenosine receptor blockade disrupts the normal action of adenosine, a neurotransmitter with a role in wakefulness and locomotion. An additional knock on effect of reduced adenosine signaling is the release of adrenaline from the adrenal gland. Together these outcomes lead to many of the effects that we associate with caffeine consumption such as increased alertness, shakiness and an elevated heart rate.

Adenosine receptors have also been implicated in a number of neurodegenerative diseases including Parkinson’s disease and Alzheimer’s. Chemicals similar to caffeine but with improved properties and increased specificity of action have the potential to be used as treatments for these debilitating diseases.

Where did the structure come from?

This is the structure of the adenosine-A2A receptor in complex with caffeine 1. The adenosine A2A receptor is a G-protein coupled receptor (GPCR). GPCRs are a family of membrane proteins that respond to a variety of extracellular chemical compounds to activate a range of specific responses inside the cell.

adenosineWhat does it look like?

The structure of the adenosine A2A receptor is typical of a GPCR i.e. it has seven transmembrane spanning segments. Caffeine (yellow) binds within a cavity formed between the membrane spanning segments. Examining how caffeine binds and blocks the adenosine A2A receptor can help to inform the future design of new adenosine A2A receptor drugs.

This structure is Protein Data Bank ID 3RFM

 

References

1. Dore et al., Structure 19:1283-1293, 2011

 

 

Melatonin: Are you feeling sleepy?

What is it?

What time did you go to bed last night? Chances are that it was sometime after darkness had fallen. “Of course it was,” I hear you cry, but have you ever really thought about just how much your body is in sync with the daily cycles of light and darkness? All organisms are affected by diurnal light/dark patterns. In vertebrates, melatonin – the so-called “hormone of darkness” – is a chemical that acts to regulate our circadian rhythm in response to light levels. Melatonin levels vary over the day increasing as light levels fall (acting to make you sleepy at night) and falling again as light returns (helping you to wake up in the morning) and keeping us synchronised with daily – and seasonal – changes in daylight. This becomes all too apparent when we suddenly try to force a new sleeping pattern by crossing time zones; “jet lag” is in part the result of a sudden mismatch between established melatonin cycles and different environmental light patterns and it can take up to a week for the body to re-sync melatonin cycles with the new environment.

Melatonin is produced in a two-step process from serotonin. Serotonin N-acetyltransferase is the first enzyme in this pathway and its activity varies over the day in order to concurrently regulate melatonin production.

Where did the structure come from?

This structure is of sheep serotonin N-acetyltransferase 1.

What does it look like?

melatoninSerotonin N-acetyltransferase is a globular protein made up of a central eight-stranded b sheet flanked by five a-helices. Serotonin binds within a funnel structure formed by the sheet and three sections of the protein that converge to form the roof of the funnel. The serotonin-binding site, and a second binding site for a co-factor necessary for the chemical modification of serotonin are located at the end of this funnel within the protein interior.

This particular structure of the enzyme (Protein Data Bank ID 1CJW) was determined in complex with a substrate analogue that mimics a catalytic intermediate of the serotonin modification process. It provided crucial information about the mechanism of this bedtime enzyme.

References

1. Hickman et al., Cell 97: 361-9, 1999

Haemoglobin: Bloody protein!

Today to celebrate what would have been Max Pertuz’s 100th birthday, we examine the structure of haemoglobin for which Perutz shared – with John Kendrew – the 1962 Nobel Prize for Chemistry.

What is it?

Haemoglobin is an iron-containing oxygen carrier protein that transports oxygen from the lungs to elsewhere in the body and which when associated with that oxygen gives our blood its characteristic bright red colour.

Where did the structure come from?

To examine the structure of haemoglobin, Perutz and colleagues isolated the protein from horse blood which is abundant in haemoglobin1; horses typically have a haemoglobin concentration of ~150 g per litre of blood. By comparison in the modern day lab scientists producing recombinant protein ( via a process of co-opting bacteria or other cells to produce large amounts of proteins that they don’t normally produce) usually have to work very hard, growing tens of litres of culture to produce just milligrams (i.e. one thousandth of a gram) of protein for experiments. Hence, in these early days of protein science, horse blood provided a ready source for Perutz and co. to study.

What does it look like?

haemoglobin-finalExtensive investigation of the structure of haemoglobin has revealed much about the intricacies of oxygen binding and release. Haemogoblin is made up of four subunits, each of which contains an iron-containing heme group. Remarkably, binding of oxygen to iron in one subunit directly increases the propensity of another of the subunits to bind oxygen, meaning that as the protein binds oxygen it becomes easier to bind more oxygen. This so-called co-operative binding is pivotal to the efficiency with which haemoglobin can be loaded with oxygen and was the first time such an allosteric phenomenon was observed in proteins.

This structure is of human deoxyhaemoglobin 2 i.e. haemoglobin without oxygen bound. Each of the four subunits are coloured differently, with the oxygen binding heme groups show in black. This structure is Protein Data Bank ID 2HHB

References

1. Perutz, Rossmann, Cullis, Muirhead, Will & North. 1960 Nature 185: 416-422

2. Fermi, Perutz, Shaana and Fourme. 1984 J. Mol. Bio. 175: 159-174

Botox: Toxic medicine

What is it?

Botulinum toxin A –- more commonly known today as Botox – is a neurotoxin produced by the bacterium Clostridium botulinum. It acts on nerves at the point at which they meet the muscles that they control, where it disrupts transmission of chemical messenger molecules causing muscular paralysis. Botulinum toxin is one of the most potent human toxins and C. botulinum infection causes botulism, a serious paralytic illness. Thankfully botulism is rare. More common today is the deliberate localised injection of the toxin for cosmetic purposes, where its paralytic properties can be used to banish wrinkles, laughter lines, and the ability of professional actors to convincingly convey normal human facial expressions. Botox also has a number of important medical applications and is now routinely used to treat eye misalignment (strabismus), excessive sweating (hyperhidrosis), migraine and urinary incontinence, with no side effects upon acting ability yet reported.

What does it look like?

botoxBotulinum toxin A is made up of three functional domains which each play a distinct role in delivery and action of the toxin. The receptor-binding domain (yellow and red) is important for entry into the nerve cells where the toxin is initially encapsulated within a small membrane bordered compartment called an endosome. Next the toxin’s translocation domain (green) induces a pore in the compartment to allow transport of the enzymatic domain (blue) out of the endosome and into the cytoplasm of the cell where it cuts target proteins in the nerve to block chemical messenger release and induce paralysis. This image was generated using PDB ID 3BTA1 and the molecular graphics software PyMOL.

Where did the structure come from?

The crystal structure of botulinum toxin A was first published 1 in the scientific journal Nature in 1998, using toxin isolated from liquid cultures of Clostridium botulinum.

1. Lacy et al., Nature (1998) 5: 898-902