The Joy and Pain of Structural Biology

The British Crystallographic Association is, as its name implies, the main organisation supporting crystallograpy and crystallographers in the UK. Theirs is a multi-disciplinary science, and the different needs of the eclectic group of people who call themselves crystallographers are met by the Association's special interest groups: among them, the Biological Structures Group for structural biology. The annual meeting of this group, known as the Winter Meeting, takes place in December, often just a few working days before Christmas. The BSG Winter Meeting has featured on this blog on several occasions: in 2016, it was held at Birkbeck and celebrated the work of one of our distinguished emeritus professors, Steve Wood.

The 2017 meeting, held in the University of Cambridge's famous Cavendish Laboratory, had a rather unusual theme. The organisers asked each of the invited speakers to talk about the ups and downs of their scientific career - the 'joy and pain' of the meeting title - by focusing on one challenging or important piece of work, perhaps described in a single published paper. Not every speaker managed to keep to just one paper, but all the talks gave useful and at times inspiring insights into how structural biology is done.

First of all, however, Malcolm Longair, head of the Cavendish Laboratory from 1997 to 2005 and perhaps the only astrophysicist to address the Biological Structures Group, gave a short history of the university's Physics department that was based there and its links to structural and molecular biology. That early history was quite extraordinary; many of the most important advances in atomic and nuclear physics, including the discoveries of the electron and the neutron and the first controlled nuclear disintegrations, were made there. A lab photo taken in 1932 includes no fewer than nine Nobel laureates.

Crystallography, in those early years, was thought of as part of physics; J.D. Bernal and his group joined the lab in 1931, and the younger Bragg became head of the department in 1938. The rest, as Longair said, was history: seeds of the discipline we now know as structural molecular biology were sown in Bragg's time with Perutz and Kendrew's work on globin structure as well as Watson and Crick's on that of DNA. By the time those studies reached their triumphant conclusion, however, the crystallographers were no longer strictly part of the Cavendish. The 'Unit for Research on the Molecular Structure of Biological Systems’, set up by the Medical Research Council, moved out of the main lab in 1957 into a building known as the 'MRC Hut'. This was the first home of the MRC Laboratory for Molecular Biology (MRC-LMB) at Cambridge with its enduring reputation for excellent structural biology research.

The next speaker, Cambridge University's Tom Blundell, began by describing his early career in 'the Other Place': Dorothy Hodgkin's lab at Oxford, where he had shared some of the glory of the insulin structure. He had considered talking about insulin at this meeting, but, he explained, "Dorothy had had the pain of trying to solve the structure for 30+ years... I had the joy of a paper in Nature!" The story he told instead was his group's own: solving the structures of proteins involved in DNA repair. This was a long story, taking in 15 years' worth of papers in Nature (2002, 2010) and Science (2017) and culminating in the 'great joy' of discovering inhibitors validated against an important protein target for oncology.

DNA damage taking the form of simultaneous breaks in both DNA strands (double-strand breaks) are common but can lead to cell death or cancer. Fortunately, they are easily repaired in healthy cells, mainly through the mechanism of non-homologous end joining (NHEJ). Blundell's group have studied the proteins involved in this complex mechanism for many years. It is a three-stage process, in which the component proteins assemble on the DNA molecule either side of the break; the ends are 'pruned' by adding or removing nucleotides to restore the original sequence and finally, the ends are joined through DNA ligation. One of the proteins involved is a kinase, DNA-PKcs, that exists as a single polypeptide chain of 4128 amino acids. Blundell's group published the structure of this huge molecule in 2010 (PDB 3KGV) and it is still the longest single-chain protein to have been solved by X-ray crystallography. Blundell explained that the chain folds into a flexible, circular 'cradle' like structure that can support the DNA double helix, with the ligation taking place inside. The mechanism requires proteins to work as 'stages, scaffolds and steps' to hold the complex together for repair, and his group has solved structures of many other components including the Ku70-Ku80 heterodimer that recognises and binds to the break, initiating the repair, and a nuclease named Artemis with 'a nice pocket for drug discovery'.

Two talks on structural biology as applied to drug discovery followed. The first was by Pamela Williams from Astex Pharmaceuticals, a company founded by Blundell with Harren Jhoti in 1999 that has just registered its first drug - a kinase inhibitor, Kisqali® (ribociclib) - for clinical use in breast cancer. Williams' talk highlighted another protein family that is just as important in pharmacology as the kinases: cytochromes P450. We have about 50 different P450 subtypes in our livers, and they catalyse reactions that modify drug molecules so they can be more easily removed from our bodies. A handful of these - the subtypes known as 1A9, 2C9, 2C19 and 3D6 - metabolise most prescription drugs. Human (and all eukaryotic) P450s are monotopic membrane proteins with flexible active sites, which allow them to bind a wide variety of substrates but which make the structures hard to solve. Williams' involvement with P450 structural biology began with the first mammalian structure, rabbit cytochrome 2C5, and she joined Astex from California to work on the first human structure, the subtype 2C9. This was published in 2003 (PDB 1OG2); a large number of other human structures have followed, yielding useful insights into drug metabolism.

Ben Bax, who studied for his PhD under Tom Blundell at Birkbeck, has just moved to the University of York after eighteen years at the pharma company GlaxoSmithKline (GSK). His talk described work at GSK to determine the structures of bacterial DNA gyrases. These are members of a large class of enzymes called topoisomerases that catalyse topological transitions in DNA; the gyrase, which catalyses DNA supercoiling, is the target of the widely used quinolone family of antibiotics (e.g. ciprofloxacin). However, quinolone resistance is increasing, mainly through mutations at specific amino acid positions of the target gyrase. GlaxoSmithKline is investing heavily in the development of novel gyrase inhibitors based on oligonucleotides, and Bax' structural biology group has contributed a large number of still unpublished structures of the enzyme with and without inhibitors or DNA bound to this work.

Janet Thornton, emeritus director of the European Bioinformatics Institute, is one of the best known figures in British bioinformatics. Her talk, on what she termed an 'accidental' paper, took the audience back to the basic principles of protein structure. In the late 70s, when she started her career, there were only about fifteen protein structures known but scientists were already examining those structures to determine characteristic patterns. Many of these first structures determined had major inaccuracies, and discovering and correcting these was a major task for early structural biologists. The Ramachandran Plot, now half a century old, was one of the first tools to be developed to gauge the quality of a protein structure, and it is still widely used. Thornton's 'accidental' (and very highly cited) paper described the program PROCHECK, which runs this and other checks on a structure to give a comprehensive assessment of its quality. A PROCHECK record for each structure in the PDB is linked from the database PDBsum.

The final talk provided delegates with a rare opportunity to hear a new Nobel Laureate - in this case, the Laboratory of Molecular Biology's own Richard Henderson - tell the story behind some of his ground-breaking research. Henderson shared the 2017 Chemistry Nobel, for "developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution", with Joachim Frank and Jacques Dubochet. He chose to talk about one structure that he had in some senses made his own: that of bacteriorhodopsin, a proton pump found in Archaea that captures light energy as photons and that has many structural and mechanistic similarities with the G-protein coupled receptors, although the exact evolutionary relationship is unclear. Henderson's studies of this important molecule started in the 1970s with structures that were just about detailed enough to show the cylindrical helices. It took him over 15 years'effort with collaborators in Berlin, Berkeley and elsewhere to improve the technology enough to solve the so-called 'phase problem' and obtain an atomic-resolution structure by electron diffraction. The rest, again, is history.

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.