Molecules that Walk

The Department of Biological Science’s contribution to Science week 2016 kicked off on 11 April with a lecture by Dr Anthony Roberts, a young Principal Investigator who arrived at Birkbeck in 2014. Anthony received his B.Sc. from Imperial College in London and his Ph.D. from the University of Leeds, and spent four years as a postdoc at Harvard in the USA before moving here to start his own research group as a Sir Henry Dale Fellow of the Wellcome Trust and Royal Society.

Anthony began his lecture by explaining that he was going to talk about molecules that have the capacity to produce directed movement – or to ‘walk’ – and their importance for human health. These molecules are all proteins, and the context in which they move is the interior of living cells. Both the proteins he studies, kinesin and dynein, ‘walk’ on a network of highways conceptually not unlike the transport system that we use to move around London. These cellular highways are filaments called microtubules, which, unlike our roads and railway tracks, are able to self-assemble and also to self-destruct.

The ability to move is one of the fundamental properties of life, and scientists and philosophers have been studying it for millennia. Muscles were identified as the organs of movement in antiquity, but it was not until the mid-twentieth century that the molecules involved in muscle contraction could be identified. The Hungarian physiologist Albert Szent-Györgyi discovered the muscle proteins now named actin and myosin using very simple equipment during the Second World War.

These proteins have similarities with kinesin and dynein, although historically they have been easier to study due to their abundance in muscle; actin forms fibrils and the enzyme myosin binds to and ‘walks’ along these filaments. This process, like all movement, requires energy, and this is obtained from the cell’s power source, the small molecule adenosine triphosphate (ATP). The part of the myosin molecule that binds to actin, which is called its head, breaks a phosphate bond in this molecule to liberate energy and power the walking motion; many of these ‘power strokes’ together cause the muscle fibre to contract.

Ideally, we would want to watch this, or any other form of molecular motion, in real time, but this is impossible because molecules are far too small: smaller than the wavelength of light, so they cannot be viewed in a light microscope. Studies of molecular structure require techniques like X-ray crystallography and electron microscopy, both of which have been used to study motor molecules.

However, neither of these techniques can do more than generate still images. Movement can only be inferred by taking lots of snapshots of the molecules at different points during the movement cycle, rather like the earliest movies. We have now built up a complete picture of actin and myosin that is detailed enough for the positions of individual atoms to be seen clearly.

Not all movement in nature, however, uses muscles. Single-celled organisms – the ‘animalcules’ observed by pioneer microscopist Antonie van Leeuwenhoek in the 1670s – have directed movement, as do bacteria, and these have neither muscles nor nervous systems. And directed movement also occurs inside cells. A good example of this is the division of replicated DNA between daughter cells during cell division.

The interior of all cells is a viscous mixture, crowded with molecules; it is possible for small molecules to move from one part of a cell to another through diffusion, but this process would be impossibly slow for larger ones. Motor proteins, on the other hand, can carry ‘cargo’ molecules across cells remarkably quickly and efficiently. Motor proteins can traverse a distance of 0.1 mm – the length of a large animal cell – in two minutes, which in terms of lengths per second is approximately three times faster than a car.

Both the motor proteins studied in Anthony’s lab, kinesin and dynein, ‘walk’ along microtubules inside cells. These filaments typically form with one end towards the centre of the cell, and its nucleus, and the other towards the cell periphery, and the motor proteins move in opposite directions: dynein towards the nucleus, and kinesin towards the cell edge.

Any kind of directed movement by molecules is challenging for several reasons. Motor molecules have no equivalent of our nervous systems for controlling movement, and they are far too small to be held on their tracks by gravity; instead, they grip the microtubules using chemical forces. They experience negligible inertia, and are constantly buffeted by other molecules in the cell. It would therefore be catastrophic for the whole of a walking molecule to leave its path at once.

The structure and function of conventional kinesin are now fairly well understood. It consists of two identical protein chains, and each chain has two major domains separated by a short linker. The larger domain of each chain coils together to form a single long stalk; the smaller domain is globular and attaches to the microtubule, so the molecule looks rather like a single leg with two feet. Each of the feet is an enzyme that generates the energy for the motion by breaking down ATP to form ADP and release a phosphate group, and it cycles between ATP-bound, ADP-bound and empty states.

The step between ADP-bound and empty is a bottleneck that can be relieved when the foot attaches to the microtubule in a particular position, ensuring that the whole molecule moves in the correct direction. The trailing foot is released from the microtubule and the cycle begins again once ATP has bound to the front foot, triggering a conformational change in the whole molecule.

The core of kinesin is similar in structure to myosin, suggesting that these two proteins have a common ancestor. The other microtubule-bound motor protein, dynein, has a different origin. Although we still know comparatively little about it, it was actually the first of the microtubule-bound motor proteins to be discovered: this was in the 1960s, when it was found as the protein that generates the force that allows protozoa and sperm cells to swim. Anthony’s group, however, has been studying how it functions inside cells to move ‘cargo’ – often nucleic acids or other proteins – from the edges of the cell towards its interior. It also helps to pull the duplicated genetic material between the two halves of the cell during cell division.

Dynein is a much larger and more complex molecule than the other motor proteins. Its structure, like those other proteins, has several components: in this case, a stalk, a ring and a tail, with a linker between the stalk and the ring. Much of what we know about this large structure has come from electron microscopy, and more recently X-ray crystallography.

The structure of dynein; the stalk is shown in yellow and the linker in magenta.

Anthony’s group and others have developed a model in which the main mechanical element is the linker, which bends and straightens to displace the cargo-bound end of the structure along the microtubule in the direction of travel. The image shown here is a still from an animated model of how dynein generates movement, which remains speculative in places and is helping to stimulate new experiments in these areas. It is also incomplete, as it only shows one half of the molecule: we do know that dynein, like kinesin, is a biped, but exactly how its ‘feet’ are coordinated remains at the frontier of our knowledge.

Anthony ended his talk by discussing some actual and potential medical applications of studies of walking molecules. Some commonly used anti-cancer drugs, including taxol, work by stabilising microtubules to prevent motion and therefore stop cancer cells from dividing. Molecules that interact with motor proteins are also being studied as potential treatments for neurodegenerative diseases and for some types of heart disease. One such compound is a myosin activator, omecamtiv mecarbil, which is showing promise as a treatment for heart failure. And we are likely to discover further applications as we learn more about these fascinating walking molecules.

Crystallography: from Chocolate to Drug Discovery

Birkbeck has already established lecture series in honour of some of its most distinguished alumni. Until 2016, however, Rosalind Franklin – co-discoverer of the DNA structure and perhaps the most widely recognisable of its ‘famous names’ – was missing from the list of honourees. This gap has now been filled; the annual Rosalind Franklin lecture forms part of the college’s Athena SWAN programme and will always be given by a distinguished woman scientist. And fittingly, the inaugural lecture, which was part of Science Week 2016, was devoted to Rosalind Franklin’s own discipline, crystallography. Elspeth Garman, Professor of Molecular Biophysics at Oxford University, gave an entertaining and illuminating lecture to a large audience that included Rosalind’s sister, the author Jenifer Glynn.

Garman began her lecture by showing a short video that she had produced for OxfordSparks.net that used a ‘little green man’ to illustrate the method of X-ray crystallography that is used to obtain molecular structures from crystals. The rest of the lecture, she said, would simply go through that process more slowly. She started by showing some beautiful examples of crystals. All crystals are formed from ordered arrays of molecules. They can be enormous, such as crystals of the mineral selenite in a cave in Mexico that measure over 30’ long or too small to be visible with the naked eye.

In the early decades of crystallography, structures could only be obtained from crystals of the smallest, simplest molecules: the first structure of all, published in 1913 by the father-and-son team of W.H. and W.L. Bragg, was of table salt. When they were jointly awarded the Nobel Prize for Physics in 1915, the younger Bragg was a 25-year-old officer in the trenches on the Western Front. His record as the youngest Nobel Laureate was unbroken until Malala Yousafzai’s Peace Prize in 2014.

The Braggs’ discoveries paved the way for studies of the structures of many, many substances: including the chocolate of the lecture title. Few of the audience can have known that chocolate exists in six different crystal forms, or that only one of these (Form V) is good to eat. The process of ‘tempering’ – a series of heating and cooling steps – is used to ensure that it solidifies in the correct form.

Garman then moved on to talk about her own field of protein crystallography. Proteins are the ‘active’ molecules in physiology, and they are formed from long, linear strings of 20 different ‘beads’ (actually, small organic molecules known as amino acids). Chemists can quite easily find out the sequence of these beads in a protein, but it is impossible to work out from this the way that the string will fold up into a definite structure ‘like a piece of wet spaghetti’. And it is this structure that places different units with different chemical properties on the surface or in the interior of the protein, or near each other, and that therefore determines what the protein will do.

Protein crystallography only became technically possible in the mid-twentieth century, and even then it was a painfully slow and complex process that could only be used to study the smallest, simplest proteins. Dorothy Hodgkin, also a professor at Oxford, won her Nobel Prize in Chemistry in 1964 for the structures of two biologically important but fairly small molecules: penicillin, with 25 non-hydrogen atoms and vitamin B12, with 80. She is perhaps better known for solving the structure of insulin, the protein that is missing or malfunctioning in diabetics. This has 829 non-hydrogen atoms; in contrast, the 2009 Chemistry Nobel Prize was awarded for the structure of the ribosome, the large (by molecular standards) ‘molecular machine’ that synthesises proteins from a nucleic acid template. The bacterial ribosome used for the Nobel-winning structural studies is well over 300 times larger than insulin, with over a quarter of a million atoms.

Protein structures are not only beautiful to look at and fascinating to study, but they can be useful, particularly for drug discovery. Many useful drugs have already been designed at least partly by looking at a protein structure and working out the kinds of molecule that would bind tightly to it, perhaps blocking its activity. Some viral proteins have been particularly amenable to this approach. Rosalind Franklin did some of the first research into virus structure when she was based at Birkbeck, towards the end of her tragically short life, and her student Aaron Klug cited her inspiration in his own Nobel lecture in 1982. X-ray crystal structures were used in the design of the anti-flu drugs Relenza™ and Tamiflu™ and of HIV protease inhibitors, and more recently still structures of the foot and mouth virus are helping scientists develop new vaccines for tackling this potentially devastating animal disease. The foot-and-mouth virus structure even made the front page of the Daily Express.

The equipment that Dorothy Hodgkin and her contemporaries used to solve protein structures in the 1960s and 1970s looks primitive today. Now, almost every step of protein crystallography has been automated. Powerful beams of X-rays generated by synchrotron radiation sources, such as the UK’s Diamond Light Source in Oxfordshire, allow structures to be determined quickly from the smallest crystals. It is even possible to control some of these machines remotely; Garman has operated the one at Grenoble from her sitting room. Yet there is one step that has changed remarkably little. It is still almost as difficult to get proteins to crystallise as it was in the early decades. Researchers have to select which of a large number of combinations of conditions (temperature, pH and many others) will persuade a protein to form viable crystals. Guesswork still plays a large part and some researchers seem to be ‘better’ at this than others: Garman adds the acronym ‘GMN’ or ‘Grandmother’s maiden name’ to her list of conditions to reflect this.

Yet, with every step other than crystallisation speeded up and automated beyond recognition, the trickle of new structures in the 70s and even 80s has become a torrent. Publicly available structures are stored online in the Protein Data Bank, which started in 1976 with about a dozen structures: it now (May 2016) holds over 118,000. Protein crystallography as a discipline is thriving, but there are many challenges ahead. We are only now beginning to tackle the 70% or so of human proteins that are only stable when embedded in fatty cell membranes and are therefore insoluble in water. It is possible to imagine a time when it is possible to solve the structure of a single molecule, with no more need for time-consuming crystallisation. And, hopefully, women scientists will play at least as important a role in the second century of crystallography as they – from Quaker Kathleen Lonsdale, who developed important equations while jailed for conscientious objection during World War II, through Franklin and Hodgkin to Garman and her contemporaries – have in the first.