Munc18: No Munc-eying around with our molecular transport machinery!

What is it?

The transport of chemicals or proteins across the membranes of cells is a vital part of life. Chemicals may need to be released in order to send a signal to the cell next door, or sometimes, a protein must be relocate from inside the cell to sit on the cell membrane so it can do its job.

For this reason, the Sec/Munc18 (SM) family of proteins—involved in fusion between two membranes—are an essential for this molecular transport and play a key part of healthy cell function. These SM proteins are involved in neurotransmitter release in the brain and in the maintenance of glucose levels (i.e. by relocating the glucose transporter to the cell surface so it can pump glucose into the cell).

Within our cells, chemicals or molecules can be isolated from the rest of the cell contents by containing them within small membrane encapsulated vesicles. But the molecular mechanisms behind how proteins, including SM, coordinate the fusion of these vesicle membrane to the outer membrane of the cell, so that neurotransmitters or the glucose transporter can do their thing, is not understood.

Crystal structures of SM proteins alone, and in complex with their partner proteins are helping us to decode the complex mechanisms of the membrane fusion event. And the importance of research in this area is essential for the development of new treatments for neurological diseases or diabetes. Indeed, Randy W. Schekman, Thomas C. Südhof, and James E. Rothman were awarded the 2013 Nobel Prize in Physiology or Medicine for their discoveries on the protein machinery that regulates vesicle traffic, including the SM proteins! You can read more about their Nobel Prize and their research here.

What does it look like?

SM proteins, such as Munc18-1 (or Munc18a) shown below, consist of three separate domains that fold into a U-shaped or arch-shaped architecture. Image generated by Pymol (using the coordinates from the protein data bank (accession code: 3PUJ).

SM proteins, such as Munc18-1 (or Munc18a) shown below, consist of three separate domains that fold into a U-shaped or arch-shaped architecture. Image generated by Pymol (using the coordinates from the protein data bank (accession code: 3PUJ).

Where did the structure come from?

This is the structure of rat Munc18-1 that was solved in complex with a portion of its partner protein, syntaxin (Sx). The structure was published in 2011 PNAS by a team of researchers from the Institute for Molecular Bioscience, UQ, Australia.

A total of 12 SM structures have been determined, either alone or complexed to a truncated protein partner or short peptide sequence. Click here for a review on the current structural information of SM proteins.

Cop this! The protein ‘cop’ (Cop9) with multiple tasks

What is it?

The human signalosome complex (also known as COP9 signalosome or CSN) acts like a police cop within our cells. Like a policeman, it has multiple jobs, from defense to development. In particular, it serves our cell by ‘policing’ the large quantity of proteins in our cells. If certain proteins get out of hand, Cop9 plays a role in their degradation or removal.

Cop9 is vital for maintaining normal cellular function. It coordinates around 20% of all protein degradation in the body. If the protein malfunctions, a whole range of problems can ensue. This may lead to problems during our development. Cop9 has also been linked to breast and liver cancer, making it an attractive drug target for novel cancer therapies.

What does it look like?

Cop9 is massive. It contains eight individual domains (coloured separately below), six of which form a ‘horseshoe’ shape. Due to its massive size, Cop9 can perform a whole lot of different functions, and these individual functions are highly regulated.

The structure of Cop9 with its eight domains coloured separately (NB- its overall dimensions are 173 x 142 x 108Å). Image generated by Pymol (http://www.pymol.org/) (pdb code: 4D10 http://www.pdb.org/pdb/explore/explore.do?structureId=4D10)

The structure of Cop9 with its eight domains coloured separately (NB- its overall dimensions are 173 x 142 x 108Å). Image generated by Pymol (http://www.pymol.org/) (pdb code: 4D10 http://www.pdb.org/pdb/explore/explore.do?structureId=4D10)

Where did the structure come from?

The structure of the human COP9 signalosome was determined by X-ray crystallography. Because it is such a large complex, this was not an easy feat. It required a team of dedicated and expert researchers. The structure was published only a few months ago (August 2014) in Nature (http://www.nature.com/nature/journal/v512/n7513/full/nature13566.html). This new structure provides vital insight into how the complicated protein Cop9 performs its duties.

The Good and Bad of Prions: Proteins that cause Mad Cow’s disease

What is it?

Prion proteins play an important role in insulating our nerves ­­– maintaining the protective myelin sheath that surrounds them.

However, prion proteins can adopt two states: a folded form and a misfolded form. The misfolded form is the ‘bad’ one: it can assemble into a tough fibril, suffocating the neurons in the brain, and after many years, it will eventually cause death.

The misfolded form of the prion protein has been implicated in many human pathologies from Alzheimer’s disease to other neurodegenerative disorders.

This ‘bad’ form of prion is also the infectious agent in both Mad cow’s disease and the human variant of the disease, Creutzfeldt–Jakob disease.

What does the ‘good’ form of Prion look like?

The correctly folded form of the prion protein is made up of alpha helices as shown in yellow and purple below. This structure was first published in Nature Structural Biology in 2001 (http://www.nature.com/nsmb/journal/v8/n9/full/nsb0901-770.html#f1)

Image generated by Pymol (http://www.pymol.org/) using the coordinates from the protein data bank (accession code: 1I4M).

Image generated by Pymol (http://www.pymol.org/) using the coordinates from the protein data bank (accession code: 1I4M).

What does the ‘bad’ form of Prion look like?

When the prion protein misfolds, it can associate with other misfolded proteins, growing end to end to form a tough fibril. In 2008, we saw the first glimpse of how the misfolded protein can assemble together into this tough fibril that resembles something like an accordion.

You’ll notice this form of the protein consists mostly of beta strands (shown as red arrows). The hydrophobic parts beta strands of this misfolded protein enable the proteins to ‘velcro’ together into these large fibrous structures. These fibrils cannot be broken down, and instead form large deposits in the brain.

This structure was published in Science in 2008 (http://www.sciencemag.org/content/319/5869/1523).

Image generated by Pymol (http://www.pymol.org/) using the coordinates from the protein data bank (accession code: 2RNM).

Image generated by Pymol (http://www.pymol.org/) using the coordinates from the protein data bank (accession code: 2RNM).

‘Bad’ prions and Mad cow’s disease

Mad cow disease (also known as bovine spongiform encephalopathy) is a fatal disease that affects the central nervous system of adult cattle. It’s called Mad cow’s disease because the cow begins to act very strangely, eventually losing the ability to walk, and ultimately dies.

The misfolded form of the cow prion protein causes the disease. It is very unusual to have an infectious agent that is a protein and not a bacteria or a virus! But indeed, these misfolded prion proteins are highly infectious: just a small amount of a misfolded prion protein in the body can corrupt a whole bunch of surrounding ‘good’ prion proteins.

As the cow prion protein is very similar to the human prion protein, eating infected meat could mean that this Mad cow’s disease spreads to humans. Unfortunately, prions are not destroyed even if the beef or material containing them is cooked or heat-treated. But the parts of the cow with the highest concentration of this protein (brain and spinal cord) can be removed to reduce the chance of disease spreading to humans. Fortunately, to date, very few people have been diagnosed with the human variant of the disease (Creutzfeldt–Jakob disease)

For more information about human spongiform encephalopathy and Mad cow’s disease see: http://www.betterhealth.vic.gov.au/bhcv2/bhcarticles.nsf/pages/Creutzfeldt_Jakob_disease

 

Cone snail toxins as painkillers – The structure of an alpha-conopeptide

This post will discuss conotoxins as a source of painkillers, as we continue to celebrate National Pain Week in Australia.

What is it?

A Textile cone snail (Conus textile) taken at Cod Hole, Great Barrier Reef, Australia.  Photographer: Richard Ling

A Textile cone snail (Conus textile) taken at Cod Hole, Great Barrier Reef, Australia. Photographer: Richard Ling

Cone snails (marine molluscs of the genus Conus) immobilise their prey by using a cocktail of peptides. These conopeptides or conotoxins from the venom of cone snails are active against receptors and ion channels.  Conopeptides are over 100 times more potent than morphine, and they lack many of the side effects of current treatments. One conopeptide (x- MVIIA which is marketed as Prialt or ziconotide) is currently available for the treatment of chronic pain, although many more are in trials.

These drugs still need to be injected, however, researchers are aiming to develop these compounds into an orally active pill or tablet form.

Here we are looking at the structure of an alpha-conotoxin (PnIB) from Conus pennaceus. This toxin blocks the nicotinic acetylcholine receptor at nerve cells and muscles.

What does it look like?

Conpeptides such as PnIB are small peptides (10 to 30 amino acids) and have one or more disulfide bonds. The structure of PnIB has two disulfide bonds (yellow) that constrain and stabilize the small peptide. The structure is further stabilized by 21 intramolecular, 3 intermolecular, and 35 water-associated hydrogen bonds

snailsImage generated by Pymol (http://www.pymol.org/) using the coordinates from the protein data bank (accession code: 1AKG).

Where did it come from?

The 1.1 Å resolution crystal structure of alpha-conotoxin PnIB was determined by researchers at the University of Queensland’s Institute for Molecular Bioscience and published in Biochemistry in 1998.

Ion Channels & Chronic Pain – Structure of the Voltage-Gated Sodium Channel

What is it?

We have all felt pain when we cut our finger, or burn ourselves. This ‘feeling’ comes from the action of sodium channels sitting in the membranes of our nerves. Voltage-gated sodium channels (Nav) are essential the electrical impulses that transmit signals throughout our nervous system.

Pain is not always a bad thing in the body. It can act as a warning signal – reminding us to remove our hand away from the fire to prevent further damage to our body. But for some people, the pain never goes away. This type of pain is termed chronic pain. For these sufferers, currently available painkillers can become ineffective, leaving them with little options to manage their pain.

When we have a local anaesthetic, our sodium channels are being blocked. Because of their role in pain transmission, sodium channels are being investigated for new analgesic drug development. That is, scientists aim to develop new powerful painkillers that lack the addiction and drowsiness side effects, but give relief to sufferers.

Humans have nine different sodium channels. The Nav1.7 sodium channel is the one that plays a key role in pain transmission. This was identified when researchers found that people with mutations in the Nav1.7 sodium channel did not feel pain (termed congenital indifference to pain).

The structure of a sodium channel in humans has not yet been determined, but we have gained information from the structure of a bacterial sodium selective channel.

What does it look like?

The bacterial sodium channel shown below is less complex than the human channel. But the structure does give us some information as to how drugs (anaesthetics and toxins) bind to the channel.

This is a side view of the bacterial sodium channel that sits in the membrane of the nerve cell:

sodium_1This is a view from the bottom of the channel, showing the ‘central pore’ that sodium ions can move through:

sodium_2Image generated by Pymol (http://www.pymol.org/) using the coordinates from the protein data bank (accession code: 3RVY) (http://www.rcsb.org/pdb/explore/explore.do?structureId=3RVY)

How do we use this structure to find more effective pain treatments?

Researchers are investigating toxins from venomous animals (snakes, spiders and cone shells) and plants as a source of painkilling molecules. This is because venoms have evolved over millions of years to target sodium channels – as this is the fastest way to incapacitate their prey.

Researchers from Australia (University of Queensland’s Institute for Molecular Bioscience) have found a number of new molecules that are potential analgesics from these venoms. For example, they have found a venom used by a centipede to paralyse prey contains a molecule more effective than morphine in blocking pain. This molecule is selective for Nav1.7 over the eight other channels. This selectivity is key – otherwise, we will get side effects because the other channels play important roles in heart and muscle function.

Where did the structure come from?

The crystal structure of a voltage-gated sodium channel from Arcobacter butzleri in its ‘closed’ conformation with four activated voltage sensors was determined to 2.7Å resolution and published in Nature in 2011.

How does the channel open and close?

In 2012, the structure of an ‘open’ bacterial form of the sodium channel was solved providing insight into how the channel opens and closes. For a movie of this see: http://www.nature.com/ncomms/journal/v3/n10/extref/ncomms2077-s3.mpg

In the next post, we will discuss some more about these toxins as potential analgesics.

“Let’s talk about Sex” hormones – Part 2: The Androgen Receptor & Testosterone

What is it?

The androgen receptor is activated by testosterone, a steroid responsible for ‘male’ characteristics. Testosterone is also important for regulating muscle, blood cell formation and sexual function. Testosterone travels around the blood stream via carrier proteins, and can pass through the membrane of cells. Once inside the cell, testosterone is further activated before binding to the androgen receptor.

The androgen receptor, like the oestrogen receptor, acts to turn on expression of a number of different genes involved in ‘male’ characteristics and anabolic function. The oestrogen and androgen receptors are very similar in structure and function.

Males have much higher levels of testosterone, and females are much more sensitive to this hormone. Not only does testosterone play a role in maturation of the male sex organs, it is important for general health, such as prevention of osteoporosis. It also has a positive effect on muscle mass and strength, leading to the sometimes inappropriate use of anabolic steroids in athletes.

Interestingly, falling in love has been shown to initially decrease testosterone levels in males, and increase testosterone levels in females. However, these gender differences in testosterone levels are not maintained for a long period of time in the relationship!

What does it look like?

The structure of human androgen receptor binding domain (blue) bound to testosterone (yellow) (pdb code: 2AM9) was determined in 2006 to 1.65 angstroms.

Image generated by Pymol (http://www.pymol.org/) (pdb code: 2AM9).

Image generated by Pymol (http://www.pymol.org/) (pdb code: 2AM9).

Where does the structure come from?

The structure described above was published in 2006 in Protein Science. For a detailed list of all available crystal structures of the androgen receptor, bound to differing ligands, see http://www.proteopedia.org/wiki/index.php/Androgen_receptor/.

“Let’s talk about Sex” hormones – Part 1: The Oestrogen Receptor

What is it?

Oestrogen is a steroid hormone that circulates in the blood stream and can pass through the membrane of our cells. Here, oestrogen can bind to and ‘activate’ the oestrogen receptor.  Once activated, the oestrogen receptor moves to the nucleus of the cell, where is acts as a transcription factor – that is, it can turn on the expression of certain genes that are important for development.

Oestrogen is known as the female sex hormone, as it stimulates growth of the female sex hormones, breasts and pubic hair, as well as maintaining the menstrual cycle. Oestrogen also plays an important role in mental health: low levels of oestrogen are correlated with low mood. In males, oestrogen is important for sperm maturation and also for a healthy libido.

The oestrogen receptor is expressed widely in different tissues – from the ovaries to the kidneys, brain and heart. It is expressed in both males and females, although is present in much higher levels in females. Whilst oestrogen has a beneficial role in development and cell growth, it unfortunately can also cause cancer cells to proliferate. In fact, the oestrogen receptor has been implicated many cancers: from breast to ovarian, colon and prostate cancers. Oestrogen is thought to drive increased cell division, eventually disrupting DNA repair, and cell death, thereby leading to tumour formation. This has lead to the development of drugs for the treatment of cancer, including Tamoxifen, that block the action of oestrogen at the oestrogen receptor.

What does it look like?

The structure of human estrogen receptor bound to estradiol (pdb code: 1A52) was determined in 1998 to 2.8 angstroms. The receptor functions as a dimer between two proteins (shown in pink). Each protein binds one molecule of estrogen (yellow).

Image generated by Pymol (http://www.pymol.org/) (pdb code: 1A52).

Image generated by Pymol (http://www.pymol.org/) (pdb code: 1A52).

Where does the structure come from?

The structure of the oestrogen receptor was first published in PNAS in 1998 (http://www.pnas.org/content/95/11/5998.long).

“Roll out the Barrel” Structure of the LptD-LptE translocon complex from bacteria

What is it?

Lipopolysaccharide (LPS) is an essential component of the bacterial cell wall and protects bacteria against antibiotics. The LPS translocon complex, consisting of two proteins (LptD:LptE ) is critical for transport and insertion of these protective LPS molecules in the outer wall. Thus, targeting these essential proteins in the bacterial cell membrane may be novel mechanism for developing antibiotics against multi-drug resistant bacteria.

What does it look like?

LptD (pink) forms a novel 26-stranded β-barrel, which is reportedly the largest β-barrel protein structure known to date. LptE (aqua) adopts a roll-like structure located inside the barrel of LptD. Together these proteins form a unique two-protein ‘barrel and plug’ architecture. LPS is passed sequentially from LptC to LptA and then to the LptD:LptE complex, and is finally inserted into the outer membrane of the bacterium.

For more information about this structure, and a movie showing the LptD:LptE complex, see http://www.diamond.ac.uk/Home/News/LatestNews/18-06-14.html

Image generated by Pymol (http://www.pymol.org/) using the coordinates from the protein data bank (accession code: 4N4R)

Image generated by Pymol (http://www.pymol.org/) using the coordinates from the protein data bank (accession code: 4N4R)

Where did the structure come from?        

This structure of LptD:LptE complex from Salmonella typhimurium was determined using the Diamond Synchrotron Light Source and published in Nature in June 2014. Understanding the mechanism of LPS insertion into the outer membrane of bacteria may help develop novel antibiotics.

How Sweet! The structure of the Glucose Transporter 1

What is it?

The human glucose transporter 1 (GLUT1) sits in the membrane of the cell, and is important for supplying glucose to the brain and other organs. This protein catalyses the transport of glucose along its concentration gradient across the membrane. It is important for the uptake of glucose required to drive respiration in all cells in our body.

What does it look like?

The glucose transporter consists of twelve TM helices that create a central groove for transporting glucose across the membrane. This structure of the transporter was only published this month (June 2014) and shows GLUT1 in its inward-open conformation (i.e. the transporter is open to the inside of the cell). The protein itself is highly dynamic, constantly changing conformation in order to transport glucose. In order to determine the crystal structure of this protein, researchers had to mutate the protein to ‘lock’ it in one conformation.

Image generated by Pymol (http://www.pymol.org/) using the coordinates from the protein data bank (pdb code: 4pyp)

Image generated by Pymol (http://www.pymol.org/) using the coordinates from the protein data bank (pdb code: 4pyp)

Where did the structure come from?           

The structure was determined to 3.2 Å and was published in Nature in June 2014.

The structure of the glucose transporter is important as it helps us to map disease related mutations (for example, mutations in GLUT1 are linked to ‘De Vivo’ disease that causes seizures in patients).

P-glycoprotein: It’s as simple as ABC

What is it?

P-glycoprotein is a member of the ABC transporter family of proteins. ABC transporters are one of the oldest families of proteins and are present in bacteria, fungi and mammals. They have likely evolved as a defence mechanism to protect our cells from toxic substances. These ABC transporters, such as P-glycoprotein, sit in the membrane of the cell and play a role in pumping toxic or foreign substances out of the cell. The ABC transporters have been linked to cystic fibrosis, tumour resistance to chemotherapy, and multi-drug resistance.

What does it look like?

The structure of P-glycoprotein (pdb codes: 3G5U, 3G60 and 3G61) was determined in 2009 to 3.8 angstroms. The protein is split into four domains: two transmembrane domains (yellow) that span from inside the cell to outside the cell and, two nucleotide binding domains (purple) that bind ATP to drive the pump. For an excellent movie on the pumping motion of P-glycoprotein see https://www.youtube.com/watch?v=T8dZwSPr8i8.

Image generated by Pymol (http://www.pymol.org/) (pdb code: 3G5U).

Image generated by Pymol (http://www.pymol.org/) (pdb code: 3G5U).

Where does the structure come from?

The structure of P-glycoprotein was published in 2009 in Science. There are many structures of other ABC transporter family members, including the recent structure of an bacterial E.coli ABC transporter (pdb code: 4PL0), that highlights the transition of ABC transporters from inward open to outward open states.

You can read more about P-glycoprotein at: http://www.rcsb.org/pdb/101/motm.do?momID=123