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.

Celebrating Laue – comprehensive protein structures

In the last of our three posts celebrating Max von Laue and Laue diffraction, Prof John Helliwell tells us how the technique has impacted on protein crystallography.

What is it?

To get a comprehensive picture of a protein structure in its functional state(s) can lead to seeking a time-resolved ‘movie’. But the X-ray data collection may be too slow to capture the interesting moment of function. As an alternative to use of monochromatic radiation the use of polychromatic X-ray synchrotron radiation leads to much quicker diffraction data collection. Moreover the sought after hydrogenation and bound water details may be incomplete as hydrogen scatters X-rays weakly. But this latter challenge can be overcome by use of neutron diffraction, as deuterium scatters neutrons as strongly as e.g. carbon. But neutron beams are much weaker in intensity than synchrotron X-ray beams.

As in the X-ray case above though the polychromatic neutron emission from either a nuclear reactor or a pulsed source can be more efficiently harnessed using Laue diffraction and again leads to shorter exposure times. Research in the 1980s [1,2] led to a much improved understanding of Laue crystallography and greatly assisted accurate Laue diffraction data analysis along with newly developed software.

Concanavalin A is isolated from jack beans; approx 10% by weight of protein is con A. Its role is not known for sure but is implicated in an anti fungal protection strategy for the bean via protein to protein  cross linking involving polysaccharide.

Concanavalin A is isolated from jack beans; approx 10% by weight of protein is con A. Its role is not known for sure but is implicated in an anti fungal protection strategy for the bean via protein to protein
cross linking involving polysaccharide.

Overall this has led to a variety of time-resolved Laue X-ray protein crystallography and neutron Laue crystallography studies [eg 3,4]. As just one example the crystal shown here, ~3mm long, is of the lectin protein concanavalin A, (isolated from jack beans ~1cm, also pictured), and was used both to help develop these modern Laue methods and to reveal new details of its bound water structure at the sugar molecular recognition binding site both at room temperature and at 15K [4].

 

What does the structure look like?

Concanavalin A is a tetramer of 100 kDa

A crystal of jack bean concanavalin A,  saccharide free crystal form.

In a final twist of this story the neutron Laue study of concanavalin A at 15K [4] showed that freeze quench time-resolved studies with neutrons even of large crystals of a protein was possible. In the ten years since this study [4] there have been various further improvements in neutron beam fluxes, measuring apparatus and use of fully deuterated proteins in Europe, Japan and the USA radically changing the scope of neutron macromolecular crystallography to smaller crystals and larger molecular weights of proteins.

[1] D.W.J. Cruickshank, J.R. Helliwell and K. Moffat ‘Multiplicity Distribution of Reflections in Laue Diffraction’. Acta. Cryst. A43, 656-674 (1987).

[2] D.W.J. Cruickshank, J.R. Helliwell and K. Moffat ‘Angular distribution of reflections in Laue diffraction’ Acta Cryst. (1991) A47, 352–373.

[3] J.R. Helliwell, Y.P. Nieh, J. Raftery, A. Cassetta, J. Habash, P.D. Carr, T. Ursby, M. Wulff, A.W. Thompson, A.C. Niemann and A. Hädener “Time–resolved structures of hydroxymethylbilane synthase (Lys59Gln mutant) as it is loaded with substrate in the crystal determined by Laue diffraction” (1998) Faraday Trans. 94(17), 2615–2622.

[4] M.P. Blakeley, A.J. Kalb (Gilboa), J.R. Helliwell & D.A.A. Myles (2004) “The 15-K neutron structure of saccharide free concanavalin A” Proceedings of the National Academy of Sciences USA 101, 16405-16410. PDB code 1XQN.

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.

Chymotrypsin — the first protease structure

Today we have a guest post from Prof Stephen Curry, Professor of Structure Biology at Imperial College London.  Stephen blogs at Occam’s Typewriter and the Guardian and was one of the founding members of the Science is Vital UK campaign group.  He is currently visiting Australia and is giving a series of public lectures, the next in Sydney on 18th August.

SC-Aug2013

Without further ado – here is Stephen’s post.

What does it look like?

4cha-surf

This is a schematic representation of the protein chymotrypsin.

This image, made with PyMOL (using the coordinates from the Protein Data Bank entry 4CHA), shows the overall fold of the polypeptide chain of the molecule, along with a semi-transparent rendering of its surface. The lumpiness of the surface is due to the individual atoms of the protein because the structure has been determined at very high resolution.

What is it?

Chymotrypsin a protease molecule that participates in the digestion in the small intestine of proteins consumed as food. It acts like a molecular pair of scissors but instead of having sharp blades for cutting chymotrypsin uses chemistry. What this means is that it catalyses the cleavage of the peptide bonds between amino acids in selected targets. The three amino acid side-chains of the catalytic centre are shown as sticks in the picture above — from the top these are serine, histidine and aspartic acid. To the right of them you can see a deep pocket that  chymotrypsin uses to grab hold of a large, aromatic side-chain in a target proteins. During this transient embrace, the Ser-His-Asp ‘catalytic triad’ works, along with a water molecule, to destabilise and then break the adjacent peptide bond in the target. Repeated action of chymotrypsin and other proteases in the digestive tract breaks proteins down to their component amino acids so that ultimately they can be used by the body to build up new proteins, or as the starting material for synthesizing other essential molecules.

Where did the structure come from?

The structure of chymotrypsin — published initially by a team led by David Blow — was only the fourth protein structure to be solved by X-ray crystallography. It is a landmark because it was the first protease structure to be worked out by the technique and almost immediately revealed not just the architecture of the polypeptide chain but the detailed chemical mechanism that it deployed to break peptide bonds. The Ser-His-Asp catalytic triad has since been found in many other proteases in related and unrelated structures, providing intriguing molecular examples of convergent and divergent evolution.

Personally, the structure has a special meaning since David Blow was an inspirational figure who in helped me to get into protein crystallography at the outset of my career in 1989. I was fortunate to be able to return the favour many years later when my group solved the structure of the 3C protease from foot-and-mouth disease virus which turns out to be a distant relative of chymotrypsin.

“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).

A different type of donut – beta-sliding clamp

What does it look like?

Beta has a striking resemblance to a donut, but isn’t as tasty. The protein is actually a dimer, with the arrows showing the dimeric interface. Each dimer has three domains comprised of two beta-sheets and two a-helices.

Image generated by VMD (Visual Molecular Dynamics) using the coordinates from the Protein Data Bank (PDB code: 3Q4J)

Image generated by VMD (Visual Molecular Dynamics) using the coordinates from the Protein Data Bank (PDB code: 3Q4J)

What is it?

The Escherichia coli beta-sliding clamp (beta) is an aptly named DNA replication protein. It is clamped onto and slides along DNA, recruiting other replication proteins to the DNA during the different stages of replication. What makes this an interesting protein is the nature of these interactions, as every protein that interacts with b binds in the same pocket by very similar amino acid motifs. On the structure, the consensus amino acid motif (QLDLF) is bound into the pocket. This conserved binding has made the E. coli beta protein a target for novel antibiotics, and many potential antibacterial small molecules have been published.

Where does it come from?

The first crystal structure was solved by X.P. Kong and colleagues in 1992 [1] and since then, many structures of E. coli beta in complex with protein binding partner consensus motifs [2] and small molecule inhibitors [3] have been solved.

 

[1] Kong, X.P., Onrust, R., O’Donnell, M., and Kuriyan, J. (1992). Three-dimensional structure of the b subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell 69(3): 425-437

[2] Wolff, P., Olieric, V., Briand, J.P., Chaloin, O., Dejeagere, A., Dumas, P., Ennifar, E., Guichard, G., Wagner, J., and Burnouf, D.Y. (2011). Structure-based design of short peptide ligands binding onto the E. coli processivity ring. Journal of Medicinal Chemistry 54(13): 4627-4637

[3] Yin, Z., Whittell, L.R., Wang, Y., Jergic, S., Lui, M., Harry, E.J., Dixon, N.E., Beck, J.L., Kelso, M.J., and Oakley, A.J. (2014). Discovery of lead compounds targeting the bacterial sliding clamp using a fragment-based approach. Journal of Medicinal Chemistry 57: 2799-2806

“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.

Cold as Ice… with ‘Maxi’ sacrifice

What is it?

This is the structure of the antifreeze protein (AFP) Maxi. Maxi is found in winter flounders, fish that live in the cold North Atlantic ocean and the protein stops these fish from freezing. AFPs bind to ice crystals, and stop them from growing further. Usually, AFPs have residues on their outer surface that are responsible for interacting with ice crystals. However, Maxi is the first AFP that has its ice binding residues on the INSIDE of the protein.

What does it look like?

Unlike most globular folded proteins, that fold up tightly and repel waters from the centre of the protein, the Maxi protein is full of a layer of water molecules (http://www.rcsb.org/pdb/explore/explore.do?structureId=4KE2). Maxi has two rod shaped coils (yellow and pink) that pack loosely, loose enough to allow a single layer of water molecules to fill the spaces (blue spheres). The waters act as a sort of glue, holding the four helices of the Maxi together. These water molecules also ‘leak’ out of the ends of the gap between the helices, helping the protein to attach to a forming ice crystal and then stopping its growth.

Image of the hyperactive type I antifreeze protein, Maxi (pdb code 4EK2) generated by Pymol (http://www.pymol.org/)

Image of the hyperactive type I antifreeze protein, Maxi (pdb code 4EK2) generated by Pymol (http://www.pymol.org/)

Where did the structure come from?        

Maxi_2The structure was published in science in February 2014 by Peter Davies and colleagues (http://www.sciencemag.org/content/343/6172/795) (DOI: 10.1126/science.1247407).

Myoglobin: (Don’t) hold your breath!

A short break from our rainbow coloured crystal structures, as today would be John Kendrew’s birthday.  John Kendrew received a Nobel Prize with Max Perutz for being the first to determine a protein structure – and it was this one!

What does it look like?

Myaglobin

Cartoon representation of oxymyoglobin isolated from the sperm whale (PDB ID 1MBO1). Myoglobin is shown in cartoon representation (blue) in complex with its iron haem group (green) and molecular oxygen (red spheres.) The image was generated using the molecular graphics software PyMOL.

What is it?

Ever wonder why seals and whales can dive for up to an hour on a single breath, but you can’t make to the other end of the swimming pool? The answer, in part, is myoglobin.

Myoglobin is a specialised oxygen carrier protein, similar to the better-known haemoglobin. Whilst haemoglobin circulates in the blood and facilitates oxygen transport, myoglobin is present in the skeletal muscle (i.e. those muscles that allow you to move) where it enables oxygen storage.

Oxygen transport in the body relies on the fact that oxygen binding proteins pick up and hold onto oxygen when they are in an environment where oxygen concentration is high (e.g. the lungs), and later give up that oxygen when they are in an environment where oxygen concentration is low (e.g. the brain, exercising muscles) thus delivering it to where it is needed. Haemoglobin and myoglobin both deliver oxygen, but they differ in how low the oxygen concentration (more precisely the oxygen partial pressure) of the local environment has to be before they will release their precious cargo. Haemoglobin can give up oxygen quite readily, but myoglobin only releases oxygen when the local concentration of oxygen is much lower; this difference effectively allows myoglobin to store oxygen reserves in the muscles ready for future periods of activity.

Seals, whales and other deep diving sea mammals have a much higher concentration of myoglobin in their muscles than humans. This is crucial for their ability to take long underwater dives. Typically when proteins are very highly concentrated in one place, they start to clump together preventing them from functioning properly. Deep diving sea mammals however have evolved variants of myoglobin that have a very highly charged surface that effectively makes them less “sticky” and allows them to remain at high concentrations without a loss of function 2.

Where did the structure come from?

The crystal structure of myoglobin was determined by John Kendrew and colleagues using protein that they isolated from sperm whales 3,4. It was the first protein structure to be solved by protein crystallography. Kendrew subsequently shared the 1962 Nobel Prize for Chemistry with Max Perutz “for their studies of the structures of globular proteins.”

  1. S. Phillips. JMB, 142:531-554 (1980)
  2. S. Mirceta et al., Science 340, 1234192 1-8 (2013)
  3. J. Kendrew et al., Nature 181, 662-666 (1958)
  4. J. Kendrew et al., Nature 185, 422-427 (1960)