The 2013 meeting was billed as both a "final" event in the centenary year of the Braggs' landmark discoveries and part of the build-up to the International Year of Crystallography, but these were not the only anniversaries highlighted there. 2013 also marked the sixtieth anniversary of the publication of the structure of DNA. The 2013 Winter Meeting was held in King's College London, which played a very important part in that discovery: Maurice Wilkins and Rosalind Franklin, who obtained the X-ray diffraction patterns that led to the discovery of the double helix, were based there. (Wilkins shared the Nobel Prize for this discovery with Watson and Crick; Franklin died in 1958, four years before that prize was awarded.) And the first precise physical model of the double helix is still on display in the college.
Maurice Wilkins' original DNA model
The first researcher to speak at the meeting was Birkbeck's own Professor Bonnie Wallace. Her work on the structures of voltage gated sodium channels has been described on this blog before, most recently in April 2013. These proteins are responsible for the transport of ions in and out of cells, an essential signalling mechanism in all multi-cellular organisms. Their structures, however, are among the most intractable of all membrane proteins (PPS section 11, to be released in May, covers this fast moving field). Wallace has used a combination of X-ray crystallography, spectroscopy and molecular dynamics to explore the structure and mechanism of sodium channels in bacteria. The bacterial sodium channel is simpler than the mammalian equivalent, consisting of a tetramer in which helices from each monomer line the pore. The Wallace group's most recent strucure (PDB 3ZJZ) shows the position of the C-terminal domain of these channels for the first time. This domain consists of a coiled coil formed by one helix from each monomer that is linked to the rest of the protein by a flexible region. Moving the coiled coil up and down causes a conformational change that allows the channel to open and close.
The technique of rational or structure-based drug design, which involve modelling the interactions between a library of potential ligands and a protein binding site, has proved particularly successful in the design of anti-viral drugs. Several inhibitors of HIV protease and of influenza virus neuraminidase that were designed in this way have become very successful drugs. David Stuart from the University of Oxford and the Diamond synchrotron gave a talk illustrating how structure-based in silico techniques are now being applied to design drugs against another virus family: the Picornaviruses. Members of this large family are responsible for a diverse range of diseases, ranging in humans from polio to the common cold. The foot-and-mouth virus, which affects livestock and which devastated parts of the UK countryside in 2001, is also a member of this family.
One of the viruses studied in Stuart's goup is a human picornavirus that causes similar symptoms to the foot-and-mouth virus and that represents a serious threat to public health in East Asia. The disease is known as hand foot and mouth virus, and the virus as CAV16: like all picornaviruses, it consiss of a single strand of RNA enclosed within an icosahedral (20-sided) protein capsid. The intact virus particles are very fragile and diffraction patterns must be captured before the particles disintegrate in the X-ray beam. Stuart and his Chinese collaborators have used one of the microfocus beamlines at Diamond to take snapshots of the virus structure at several points during its life cycle. One of these is of an "uncoating intermediate" that shows one of the viral proteins (VP1) emerging from the capsid so that it can be embedded in the membrane of a host cell Ren et al., 2013). Stuart and his co-workers are now designing compounds to bind to these intermediate structures and prevent the virus from entering its human host cells.
All cells, whether prokaryotic or eukaryotic, contain long molecules of DNA that must be packaged in order to fit into the confined space available. Fortunately for developers of anti-bacterial drugs (and users of antibiotics) bacterial cells package DNA using a different mechanism from mammalian ones. In bacteria, enzymes called topoisomerases bind to, cut and re-join double-stranded DNA so that it can be unwound or untangled ahead of replication. Ivan Laponogov, a postdoctoral research assistant at King's College, described recent work in his group on the structure of one of these enzymes. Bacterial topoisomerase II ia a target for an important class of antibiotics, the fluoroquinolones, but resistance to these drugs is increasing.
These enzymes are powered by ATP and act as "clamps", capturing one double-helical strand of DNA and passing it through a break in another to remove supercoils and knots in the nucleic acid structure. The structure presented at this meeting was the first of a complete topoisomerase dimer bound to DNA in the "open clamp" position. This structure was solved with and without a fluoroquinolone drug (levofloxacin) bound. The structure with drug bound showed that molecule intercalating between DNA bases at the point where the nucleic acid would be cleaved, preventing that cleavage. The structure without the drug showed the DNA in a different position; the position of a functionally important magnesium ion also changed between the structures.
Many essential cellular processes involve a post-translational modification in which poly-(ADP ribose) or PAR is added to amino acid side chains, and the processing of this molecule involves a wide variety of enzymes. Inhibitors of one of these, poly-(ATP ribose) polymerase or PARP, have recently been developed as drugs against cancer. David Leys from the University of Manchester described his work on the structure of another enzyme in the PAR life cycle: poly-ADP-ribose glycohydrolase (PARG), which catalyses the removal of PAR from proteins.
Mammalian PARG enzymes have three domains, a N-terminal regulatory region and two C-terminal domains forming the catalytic region; the equivalent bacterial enzymes lack the N-terminus. Leys and his groups first solved structures of a bacterial PARG bound to ADP-ribose (PDB 3SIG) and to a known inhibitor with a similar structure. They found that a C-terminal helix in the protein was clamped around the terminal ribose of PAR, enabling the release of a single ADP-ribose from the polymer. This basic mechanism is similar in the mammalian enzyme. More recently, the Leys group has solved the structure of PARG bound to an intact PAR substrate (PDB 4L2H); modelling studies based on this structure suggest that the enzyme acts predominantly as an exo-glycohydrolase, that is, it catalyses the removal of one residue at the end of the polymer chain. Understanding the structure and mechanism of these enzymes should enable us to develop small-molecule inhibitors of PARG, and these may one day rival the PARP inhibitors as anti-cancer drugs.
A hundred years on from the "invention" of crystallography and sixty years on from the structure of DNA, these elegant, fascinating and complex structures presented at one meeting give a snapshot of recent progress in structural biology. Furthermore, each of these structures has already provided insights into human disease that may yet lead to the development of useful drugs.