Pumping life

Publiceret April 2012

The name PUMPKIN may suggest a research centre focused on American Halloween traditions or the investigation of the growth of vegetables – however this would be misleading. Researchers at PUMPKIN, short for Centre for Membrane Pumps in Cells and Disease, are in fact interested in a large family of membrane proteins: P-type ATPase pumps. This article takes the reader on a tour from Aarhus to Copenhagen, from bacteria to plants and humans, and from ions over protein structures to diseases caused by malfunctioning pump proteins. The magazine Nature once titled work published from PUMPKIN ‘Pumping ions’. Here we illustrate that the pumping of ions means nothing less than the pumping of life.

“P-type ATPases are membrane pumps responsible for the transport of various cations and lipids in all kingdoms of life and their proper function is absolutely necessary for survival,” explains Poul Nissen, director of the PUMPKIN centre that spreads over several departments at Aarhus University and the University of Copenhagen. Poul became interested in P-type ATPases when he returned back to Denmark from his postdoctoral stay at Yale University where he worked on unraveling the structure and function of the ribosome in the laboratory of the 2009 Nobel Prize laureate Thomas A. Steiz. “The main reason why I decided to focus on membrane pumps was the long-standing research tradition of studying these proteins at both Aarhus and Copenhagen Universities, and the fact that I could see great interesting projects lying in front of us,” says Poul.

2012-2 Poul Nissen
Poul Nissen, director of the PUMPKIN centre

The long and successful tradition of investigating membrane pumps in Denmark started when the first P-type ATPase - the sodium-potassium pump - was discovered at Aarhus University in 1957 by Jens Christian Skou, who was later awarded one half of the 1997 Nobel prize in Chemistry ‘for the first discovery of an ion-transporting enzyme’. In his preparation of finely ground crab nerve membranes, J.C. Skou identified a membrane pump that uses the energy derived from hydrolysis of ATP to maintain a stable difference in the concentration of potassium and sodium across biological membranes. Shortly after, other P-type ATPases and their crucial roles for all cells were identified and investigated. Because of their indispensable role in many life processes it is perhaps not surprising that many members of the P-type ATPase family have been implicated in the onset of various diseases. Consequently, P-type ATPases are clinically important drug targets e.g. in the treatment of congestive heart failure or gastric ulcers, and they encompass promising targets for a broad range of novel drugs.

Research at the PUMPKIN centre is highly interdisciplinary, making use of the knowledge, expertise and equipment from many groups spanning over a number of institutions. P-type pumps are therefore studied from different perspectives, starting with basic research on their structure and function and ending up transferring this knowledge to biotechnology and drug discovery. This broad approach involves the use of a great variety of experimental techniques such as X-ray crystallography, biochemical studies, bioinformatics and cell biology.

A defining feature of research at the PUMPKIN centre is a high level of collaboration and interaction between different institutions and researchers with various backgrounds. But how can one manage to coordinate such a big research centre spread over many different institutions? “The key to success here is the integrity and good chemistry among participants,” believes Poul. Everybody at the centre can confirm that collaboration, openness and an extremely friendly atmosphere really are the key defining features at PUMPKIN. All international students and researchers are surprised and impressed by this relaxed and friendly approach, and maybe this is one of the reasons why the environment at PUMPKIN has become truly international and English is often spoken more frequently than Danish. “Research cultures of many countries are mixed and the best of each contribution is exposed and grasped by others,” describes Poul the international environment at PUMPKIN – and you can see he really enjoys leading a big international group. “International students are often a great inspiration for others thanks to their enthusiasm to learn new things, their curiosity and hard work attitude,” he adds. And Poul evidently knows how to attract them – more than half on the young researchers (PhD students and postdocs) at PUMPKIN are foreigners, from countries as close as Germany, Sweden or the Baltics or as far away as China. “We always make sure that there are great projects with great questions that the students can identify with,” Poul explains. “And we never forget to focus on the education, talent development and career opportunities of our young participants.”

2012-2 Pumpkin Logo
The PUMPKIN logo, designed by Helene P. Brovang and Morten Kjeldgaard, expresses not only the subject of interest of the centre but also its approaches and organization. Its four corners represent the four different states that a P-type ATPase pump repeatedly undergoes within its functional cycle (left) and also symbolize the interdisciplinary approach (middle) and cross-institutional organization (right) of PUMPKIN with a high level of collaboration and interaction; twisted angles are a symbol of a broader perspective of research that is ranging from detailed structures of pumps at an atomic level to their roles in human physiology of health and disease.

One of the most important breakthroughs for the PUMPKIN centre took place in 2007, not even one year after its inauguration and exactly 50 years after J.C. Skou discovered the sodium-potassium pump. Scientists from the centre published three individual articles in a 2007 December issue of the highly prestigious journal Nature (and earned the front cover illustration!), describing fundamental new insight on the structure and function of three different P-type ATPases. All these structures, together with many mutational, biochemical and physiological studies that followed, allow researchers at PUMPKIN to gain more information about the functional cycle of these pumps, their important structural features and physiological roles. And what are the goals and plans for PUMPKIN’s bright future? “It would be great to have crystal structures and functional studies for all types of P-type ATPases,” says Poul. “At the same time we are now more and more interested in larger and more complex networks of pumps and their interaction partners where the vision is to get to a bigger picture integrating our knowledge from structural biology, biochemistry, molecular cell biology and physiology.” It looks like Danish Universities will certainly keep their leading position in the field of P-type ATPase research that was initiated by J.C. Skou 55 years ago.

The Green Branch of PUMPKIN

Twenty-four years. That is the expertise Michael Palmgren has in research on proton pumping P-type ATPases from plants. Back in 1988 as PhD student at the University of Lund in Sweden, he published his first paper ‘Modulation of plasma-membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty-acids and phospholipase A2.’ Since then a lot has changed. On the personal side, Michael has become professor at the University of Copenhagen and a group leader at PUMPKIN. On the subject side, oat has been outcompeted by the model plant Arabidopsis thaliana, and research is performed on various aspects of P-type ATPases. “The reason why we focus on plasma membrane proton ATPases,” Michael explains, “is that they have been present in plants since plants evolved. They create a membrane potential which enables transport of other ions across the membrane. Proton transport is simply among the most important actions in plants.”

2012-2 Michael Broberg Palmgren

Michael Broberg Palmgren, leader of the PUMPKIN
branch in Copenhagen.

Besides proton pumps, the research group works also on pumps called flippases. Flippases (a subfamily of P-type ATPases) are thought to transport lipids between the two leaflets of a biological membrane. This process would provide the starting point for vesicle formation, a process important in endo- and exocytosis.

How do the flippases and proton pumps function at the atomic level? How are they regulated? What are their physiological roles? These questions drive Michael and 16 others at PUMPKIN in Copenhagen. Alex Green Wielandt is one of them. He is at the beginning of his 3 year PhD studies, and uses a technique where he introduces proton pumps into artificial lipid vesicles called liposomes. Once the pump is captured in the vesicle’s membrane, Alex can adjust the experimental settings according to his needs for studying the pumps. He generated mutant proton pumps in which specific amino acids are changed. Together with a variety of chemicals, he measures the efficiency of proton transport and hopes to trace that back to a mechanism of how protons are transported inside the pump. “I am making good progress and the first results are really promising,” he says, and adds with a smile: “Since I became a father in November last year, I work even faster so that I’m back at home early.”

In a workplace where most people are between 25 and 35 years old, announced pregnancies and births are often celebrated. Merethe Mørch Frøsig is working on the regulation of flippases and is also a PhD-student at PUMPKIN. “I brought a big cake and fruits on the day I told my colleagues about my pregnancy. Det var hyggeligt!” she smiles. Her project will not pause during the time she takes maternity leave. “I’ve great colleagues who will continue parts of my projects while I’m on leave so that nothing is lying still. This is very important in an environment as dynamic as the PUMPKIN centre.”

2012-2 Pumpkin Researchers
Researchers at PUMPKIN at the annual meeting in August 2011. Since its foundation, PUMPKIN considerably grew in size and consists now of 12 individual research groups that attract more and more students, postdocs and visiting scientists every year.

From basic science to business: Pcovery

“I think it has been fun,” says Morten Buch-Pedersen with his energetic voice, “what we have done in the last 3 years.” Morten is co-founder and CEO of Pcovery, a spin-off from PUMPKIN. “We saw a clear commercial potential for drugs targeting P-type ATPases existing in fungi, but not animals and humans. The medical need for a selective fungicide is enormous.”

Box 1: Heterologous expression

Heterologous means ‘derived from a different organism'. Molecular biologists use this term to describe that they copied a genetic sequence from one organism to another. Often the genetic sequence contains a gene that codes for a protein. If the gene is translated into a functional protein in the new organism, the process is called heterologous expression.

 

Having a PhD in Biology and having worked as postdoc at PUMPKIN, Morten had not had any business classes, when he started Pcovery together with Claus Olesen, Michael B. Palmgren, Poul Nissen and Anne-Marie Lund Winther back in 2009. “Well, I never managed to get a permanent position in the university anyways,” he jokes, “so I decided to go into another competitive field.” With Poul and Michael from PUMPKIN on the company’s advisory board, Pcovery got a seal of quality right away from the start. The company’s aim is ambitious but significant: to develop a chemical compound that inhibits the plasma membrane proton pump of fungi. This chemical compound would not have any target in the human body, as we do not have this kind of pump.

2012-2 Morten Buch Pedersen
Morten Buch-Pedersen, co-founder
and CEO of Pcovery

Work in the field of molecular biology requires not only creative people, but also proper equipment and facilities, such as classified laboratories. Obviously, most small start-ups cannot raise the money for this. Thus, an indispensable help for small biotech companies comes from universities, explains Morten: “We rent equipment and other things we need from the University of Copenhagen. Also our offices are located on the university’s campus.”

Despite the proximity, PUMPKIN’s spin-off is a company completely independent from the Centre of Excellence. With direct investments in Pcovery, “Novo Seed and Østjyst Innovation have funded our projects,” Morten emphasizes. Referring to people who see themselves launching a company he says: “If you believe in it you have to know that it is hard work to get it started and also to keep it running. But as a famous sports brand puts it: Just do it – it is a great experience!”

2012-2 proton pumpkin
The X-ray crystallographic structure representing an active form of the proton pump in complex with Mg-AMPPCP (an analogue of ATP). Ten transmembrane helices are shown in orange, green and brown; the nucleotide-binding domain (N) in red; the phosphorylation domain (P) in blue; and the actuator domain (A) in yellow. Mg-AMPPCP is shown as a ball-and-stick representation. The grey box depicts the approximate location of the plasma membrane. From: Pedersen BP, Buch-Pedersen MJ, Morth JP, Palmgren MG, Nissen P (2007) Crystal structure of the plasma membrane proton pump. Nature 450:1111-1114

From basic science to novel cancer therapies: Inhibition of the calcium pump

Research on the use of inhibition of the calcium ATPase in the fight against prostate cancer is a great example of how a successful collaboration between laboratories and people with different backgrounds leads to the transformation of basic research into potential medical applications. So how can the inhibition of one calcium transporter be useful in the treatment of prostate cancer?

2012-2 Jesper Just Møller

Jesper Vuust Møller, group
leader at PUMPKIN.

The problem with slowly proliferating cancer types (such as prostate cancer) is that they are resistant to standard chemotherapeutic methods that are targeted against rapidly dividing cells. Of course there is an extensive search for strategies how to overcome this problem, and, as Jesper V. Møller explains: “One of these strategies is to selectively inhibit a protein whose proper function is absolutely necessary for the survival of cells. And this is where the calcium ATPase comes into play.” Professor emeritus and dr. med. Jesper V. Møller from the Department of Biomedicine at Aarhus University has been involved in research on the structure and function of the calcium pump since the 1970s. Although he has retired several years ago, Jesper still works in his office every day from the early morning – sometimes even when he is on vacation. “Science is a part of my life,” he laughs.

But why is the proper function of the calcium pump absolutely necessary for life? “As you might have guessed from its name, the calcium pump maintains a stable concentration of calcium inside cells and their organelles that is required for a great number of processes ranging from fertilization and cell division to vision or muscle contraction,” Jesper explains. A disturbance in the calcium balance caused by inhibition of the calcium pump is simply so stressful that it results in cell death. “To use this inhibition in the fight against cancer, this toxic effect has to be selectively targeted towards cancer cells,” emphasizes Jesper. To achieve this, a team of scientists and oncologists from the John Hopkins University Hospital lead by Samuel Denmeade and John T. Isaacs designed an inhibitor molecule that can be activated by a specific enzyme expressed only by cancer cells. “Without the precise knowledge of the calcium pump structure and the characteristics of the binding sites for inhibitors, it would be impossible to design these new specific drugs,” explains Jesper. Thanks to the successful combination of X-ray crystallography, bioinformatics, biochemistry and physiological studies in cells and tissues, a novel prodrug acting on the calcium pump is now being tested in phase I clinical trials in patients with a late stage of prostate cancer. Fingers crossed!

Box 2: Vesicles and flippases

2012-2 Vesicles

The flippase sits in the membrane, transporting selected lipid types (here shown with a blue head) from the outside to the inside leaflet of a membrane. The accumulation of these lipids on the inside leaflet of the membrane causes vesicle budding. These vesicles function as carriers for diverse compounds that are taken into the cell (depicted here) or extruded.

Frontier research on the sodium-potassium pump

As previously mentioned, Aarhus University has a long tradition in studying the Na+,K+-ATPase, commonly known as the sodium-potassium pump. In late 2007, on the 50th anniversary of its discovery by Danish Nobel laureate J.C. Skou, the PUMPKIN center published an article in Nature describing the structure of the Na+,K+-ATPase from pig kidneys. This work has been cited hundreds of times in other papers, leaving no doubt about its high significance and the new possibilities it has opened for both fundamental science and medical research. So what does the pump do that makes it so important?

The sodium-potassium pump creates electrochemical gradients of sodium and potassium across animal cell membranes. It does so by hydrolyzing an ATP molecule to pump 3 Na+ out of the cell and 2 K+ in. Many other transporters utilize this gradient as a driving force for their own transport processes. Because one more charge is pumped out than taken in, the pump also establishes an electrical gradient across the membrane – the resting potential. In excitable cells like neurons and muscle cells, this resting potential is regularly disrupted by events called action potentials. Neurons, for example, use action potentials to communicate with one another. During action potentials, the balance of intra- and extracellular sodium and potassium is disrupted, which the sodium-potassium pump then restores. Once its work is finished, the neuron has returned back to normal and is ready for transmission of another signal.

The sodium-potassium pump structure allows us to gain invaluable insight on how the protein performs this truly fascinating job at the atomic level. Moreover, knowing the structure of a protein also makes it possible to rationally design both inhibitors and activators for it. And in the case of the sodium-potassium pump such drugs could have a huge market: the pump is associated with diseases ranging from migraine to hypertension to rapid-onset Parkinsonism. PUMPKIN researchers have recently solved a structure of the sodium-potassium pump in complex with ouabain, a potent inhibitor, and are at the forefront of structure-based drug design for this fascinating target.

2012-2 Michael Voldsgaard Clausen

Michael Voldsgaard Clausen,
PhD student.

At PUMPKIN we are also doing research on the sodium-potassium pump beyond crystal structures. “I study different isoforms of the Na+,K+-ATPase by doing experiments on frog eggs,” says Michael Voldsgaard Clausen. Michael, a PUMPKIN researcher, works in the electrophysiological section of PUMPKIN’s Aarhus branch. His research involves injecting mRNA encoding various isoforms of the sodium-potassium pump into oocytes of Xenopus laevis, the African clawed frog. When the pump is expressed, Michael uses specialized equipment to obtain an electrical readout from the whole cell. He then uses these readouts to compare activity between the different isoforms. “This method allows very versatile biochemical characterization of the pump,” Michael explains. “You can measure total enzyme kinetics. You can compare isoform transition rates between individual steps in the functional cycle. You can assess the effects of various mutations on pump activity. The amount of information electrophysiological studies can provide us is truly astounding. And the best part is that the whole time the pump is located in the oocyte’s membrane, surrounded an environment very close to the native one. This reduces experimental artifacts to a minimum.” Like other PUMPKIN researchers, Michael often stays late into the night to execute a critical experiment, the results of which are later to be shown with pride at a presentation. As other PUMPKIN members, Michael still finds time to enjoy life away from the lab – when playing guitar in the band “Pyroclastic Flow” and conquering the world’s tallest peaks.

Copper pumps: mysterious, yet crucial

One of PUMPKIN’s most recent breakthroughs has been the structure of a copper pumping P-type ATPase. Although they are less well known than the sodium-potassium pump and the calcium pump, copper pumps are nonetheless vital for our wellbeing. This is underscored by the fact that they are found in organisms ranging from bacteria to humans.

So what do these proteins do? As the name implies, they transport copper from the cell’s cytoplasm either into various cellular compartments or to extracellular space. Although cells need copper to function properly, the paradox is that copper is extremely toxic in free form and therefore it must be bound to buffering agents such as glutathione at all times. Copper buffering is in fact so efficient that there is on average less than one free copper ion per cell! However, if copper concentration were to exceed the cell’s buffering capacity, the unbound copper would start to cycle between oxidation states, generating massive amounts of free radicals and unleashing total destruction upon the cell. Therefore, intracellular sensors constantly keep track of copper levels and put the pumps to work when required.

Humans have two copper pumps. If mutations cause them to malfunction, this leads to the crippling Menkes’ and Wilson’s diseases. Our structure of a copper pump allows a better understanding of how specific disease-causing mutations affect the proteins and whether the effect of at least some of these mutations could be reversed.

Many pathogenic bacteria such as Mycobacterium tuberculosis require copper pumps for virulence. This is because bacteria need to survive a ‘copper burst’ once swallowed by macrophages in order to carry on the infection. If the bacterial pumps were to be inactivated by an inhibitor, this could lead to a significant decrease in pathogen survival in macrophages, and ultimately to an easy victory over the disease. This is where PUMPKIN’s copper pump structure may be of great use in structure-based drug design. Such drugs would also prevent bacteria from acquiring resistance towards copper/silver surfaces and sprays, which are gaining popularity as a measure against multidrug-resistant bacteria in hospitals.

2012-2 Pontus Gourdon

Pontus Gourdon, postdoc.

Pontus Gourdon, a postdoc at PUMPKIN’s Aarhus branch, was one of the scientists spearheading the effort to determine the copper pump structure. “Once we obtained our first crystal hits,” he recalls, “We started to optimize them – making them larger, thicker, and better diffracting. We did this by varying the crystallization conditions one small step at a time. As you never know which least expected component will give you better results, we tried to make our optimization approach as systematic and unbiased as possible. This of course meant we had to test a large number of crystallization conditions – 16800 to be exact – but in the end it was all worth it.” By ‘it was all worth it’ Pontus is referring to the fact that the copper pump structure was published in the highly prestigious journal Nature, a not-so-uncommon achievement of the PUMPKIN center.

The pipeline

So how do we actually get all our membrane protein structures?

First of all, it’s important to obtain our target proteins in sufficient amounts. This typically involves either purifying it from a large batch of natural source tissue (just like Jens Christian Skou did back in his day with the sodium-potassium pump from crab nerve) or expressing it heterologously. Both options have their pros and cons. Once a particular protein expression system has been proven to work, a purification protocol needs to be established. The first step here is to lyse the cells and separate the membrane fraction, where our target proteins reside. Afterwards, the membrane fraction is solubilized with a detergent. Now that our target protein is in solution, we can separate it from other membrane proteins, a process which usually involves multiple rounds of various chromatography techniques. Once the protein has been estimated to be of about 95% purity, we can start to crystallize.

2012-2 Crystallization

A. A protein crystallization setup B. The phase diagram in protein crystallization. In a successful crystallization experiment, the protein follows the red trajectory.

During the usual crystallization procedure, a small drop of protein solution is mixed with an equally small drop of precipitant solution and placed over a large reservoir of precipitant-like solution in a tightly sealed chamber. With time, some of the water from the protein-precipitant drop will evaporate to the large reservoir volume, causing the protein’s concentration in the drop to rise. This hopefully causes the protein to land in the nucleation zone. There small protein clusters form, onto which new protein molecules attach in an orderly fashion. This continues while the concentration drops until it hits the border of the undersaturated area. At that point, growth stops and thus a crystal is born. A lot of luck is involved in successfully crystallizing a protein; choosing the right combination of precipitants, additive chemicals, temperature, protein concentration and so forth may take a very long time. There also have been many jokes about things like moon phases affecting crystal growth. Once good enough crystals have been obtained, they are frozen in liquid nitrogen. This is to prevent damage from energetic X-rays during data collection.

Afterwards, the crystals are taken to a synchrotron – a facility where very bright X-rays are generated. There the crystals are mounted between the X-ray beam trajectory and a photon detector and shot at. X-ray diffraction then occurs in the crystal and some of the diffracted beams hit the detector and are recorded as spots (Figure 7). Many such images are taken while the crystal is rotated. Once finished with that, these images are fed into a program that measures the intensity of each of the spots. This is then combined with the phases of the diffracted beams, another crucial piece of information which needs to be determined separately. The program can then calculate the electron density of the protein which constitutes the crystal. With that done, we build our polypeptide chain into this density. Finally, after some fine-tuning of both the electron density and polypeptide chain we obtain a final model of the protein structure in atomic detail. Voilà!

Box 3: Sample preparation

There are multiple ways of treating the target protein before crystallization trials. One special technique developed at PUMPKIN is HiLiDe - a procedure with the goal of creating a high lipid, high detergent environment for membrane proteins prior to crystallization. This serves as a reasonable imitation of the protein's natural surroundings. It has been a huge success at our center and has been highly useful in multiple projects. New methods are another important way how PUMPKIN contributes to the global scientific community.

To be continued…

PUMPKIN has gone a long way since its foundation. Great discoveries have been made, including the determination of the structures of several extremely important P-type ATPases. Young scientists have been – and still are – trained in cutting edge methods relevant to the field of membrane protein research. And yet much remains to be done. Many discoveries lie ahead in the fundamental aspects of P-type ATPase function and regulation, including research on whole interaction networks of pumps. Furthermore, many clinical applications of PUMPKIN’s research are waiting to be developed. In the future, we will know more about the exact molecular mechanism behind pumping life and how we can control it selectively.