Shining light on the 3D structures of membrane proteins

A symposium was held at Birkbeck on August 10, 2016 to honour one of the college’s most distinguished structural biologists, Professor Bonnie Wallace. This was organised by a postdoctoral member of her group, Lee Whitmore, and her long-term colleague and collaborator Dr Bob Janes from Queen Mary, University of London to celebrate her 65th birthday. It featured speakers from five continents, all of whom had collaborated with her or worked in her lab at some point in their careers.

Wallace joined what was then the Department of Crystallography at Birkbeck in 1990 after holding several research positions in her native USA. Her research on the structure and function of membrane proteins has won her several prestigious awards, including the Biochemical Society’s triennial AstraZeneca award in 2010. This is given for outstanding research in a UK or Irish laboratory that leads to the development of a new method or reagent. She has made significant contributions to both the development of circular dichroism spectroscopy as a tool for investigating the structures of proteins (including membrane proteins) at less than atomic resolution, and to studies of membrane protein structures using crystallography and electron microscopy. Her studies of voltage-gated sodium channel structures have led to some important insights about their functions in health and disease.

The symposium was divided into three sessions, with the first devoted to circular dichroism spectroscopy and the second two to membrane proteins. A general report of the day has been published on the Biological Sciences website; here, to fit in with the remit of the PPS course, I concentrate on the sections on membrane proteins.

The first talk was on electron microscopy, given, appropriately enough, by one of the pioneers of the field: Richard Henderson from the MRC Laboratory of Molecular Biology in Cambridge. Throughout most of the 1970s and 1980s he and his collaborator, Nigel Unwin, worked on the development of electron microscopy techniques for the study of protein structures. Most of their work involved the proton pump, bacteriorhodopsin, which is found in very high concentrations in the purple membranes of Halobacteria. At the beginning, this work was very time-consuming: it took them a year to locate the C-terminus of the protein, and another to determine the binding site of its ligand, retinal.

The first near atomic resolution structures of this protein were obtained in the mid-1990s. At about that time, too, he switched the focus of his interest from the structures of ‘2D crystals’ of bacteriorhodopsin to those of ‘blob-like’ single particles: isolated protein chains or, more often, membrane-embedded protein complexes. The list of biologically and medically important complexes to have been solved using this technique is now growing rapidly, and includes rotary ATP synthase (see the previous post on this blog); the next complex in the electron transport chain, known as respiratory complex I; and gamma secretase, which is a potential drug target for Alzheimer’s disease.

Molecular simulation and modelling techniques have developed alongside those of structural biology and for almost as long. Mark Sansom, a professor of structural bioinformatics at the University of Oxford, described simulations of membrane proteins. He started his talk describing a program to visualise and analyse the pores through the centres of membrane proteins that was written by Oliver Smart (now at the EBI) when he was a postdoc in Wallace’s group. This program, HOLE, is relatively simple but is still widely used. Sansom’s current work uses molecular dynamics to model the membrane bilayer with numbers of embedded proteins, focusing particularly on interactions between those proteins and the lipids of the membrane.

Not surprisingly, there were several talks about the ion channels that have been a focus of so much of Wallace’s more recent research: voltage-gated sodium channels. Hugh Hemmings from Weill Cornell Medicine, New York, USA described how these channels have become useful targets for anaesthetic drugs. General anaesthesia is a drug-induced coma characterised by unconsciousness, immobility and amnesia; an effective anaesthetic will achieve all these and a wide variety of molecules have been employed to greater or lesser effect since the nineteenth century. Many of these target proteins involved in the release of neurotransmitters by pre-synaptic nerves, including ion channels; sodium channels were first proposed as anaesthetic targets in the late 1970s but fell out of favour for several decades. Interest in this mechanism of anaesthesia has revived with the use of the bacterial proteins – a focus of Wallace’s structural studies – as a model system. Hemmings’ current studies focus on the mechanism through which volatile anaesthetics such as isoflurane inhibit the passage of sodium ions through these channels.

Crystal structure of the NavMs voltage-gated sodium channel pore depicted in ribbon motif and viewed from the membrane normal direction. each of the four monomers in the tetrameric structure is depicted in a different colour (from Naylor et al., 2016 - Wallace lab paper). The transmembrane sodium pathway run through the middle of the structure, from top to bottom.

Lin Field of Rothamsted Research, Harpenden, UK, described research leading to a very different application of sodium channel blockers: as insecticides. Insects cause an immense amount of crop damage worldwide, but non-specific insecticides might be toxic either to humans or to beneficial insects such as bees. The mechanism of the pyrethroid class of insecticides was unknown when the first members of this class were patented, but they are now known to bind to voltage-gated sodium channels and prevent their closure. Structural studies of these proteins have shown how mutations that are known to lead to pyrethroid resistance can prevent the molecules from binding, and why these compounds have very little effect on the very similar mammalian channels. Researchers hope that these studies are taking us nearer to the development of ideal, ‘designer insecticides’ that are only harmful to pest species.

Further talks were given by Wallace’s first Ph.D. student at Birkbeck, Declan Doyle, who is now at the University of Southampton; by Per Bullough from the University of Sheffield; and by Dame Carol Robinson, the first woman to be appointed as a full professor of chemistry at the University of Oxford. The symposium ended with a summary and vote of thanks from Janes, who stressed that it did not mark Wallace’s retirement: she still loves science and has many questions to answer. I hope that I will be blogging innovative research from the Wallace lab for many years to come.

Wallace’s research has been described in this blog on several previous occasions – see in particular this post from April 2013 and this one from November 2010. The use of cryo-electron microscopy to determine atomic resolution structures of proteins is covered in depth in our Techniques in Structural Molecular Biology course, which is one of the options for the second year of the Structural Molecular biology MSc.

From Genome to Proteome: BCA Winter Meeting 2012

The British Crystallographic Association is the main UK organisation supporting the science of crystallography in all its forms. Every year, its Biological Structures Group holds a meeting in the run-up to Christmas to discuss and celebrate recent developments in structural biology research. In 2012, this Winter Meeting was held at the MRC Laboratory of Molecular Biology at the University of Cambridge.

The LMB, as it is usually known, is one of the birthplaces of modern structural and molecular biology. It moved into its current building in 1962, the year when four of its most famous scientists were awarded two Nobel Prizes for some of the most important discoveries in twentieth-century biology: James Watson and Francis Crick or the structure of DNA, and Max Perutz and John Kendrew for the very first three-dimensional structures of proteins (myoglobin and haemoglobin, respectively).

It was appropriate, therefore, that the theme of this year's Winter Meeting was "From Genome to Proteome". The basic molecular processes that underlie all of life - DNA replication, transcription of DNA into RNA and translation of RNA into protein - are all, now, quite well understood. These processes are all very complicated and require numerous proteins, many of which interact together to form complexes and "molecular machines" that are quite large, at least in molecular terms. Scientists presenting at the meeting discussed recent, innovative studies of the structures of many of these proteins and the nucleic acids that they interact with. Many of these processes will be discussed in some detail in section 8 of the PPS course, "The Protein Lifecycle".

The meeting programme was divided into three sections, corresponding respectively to DNA synthesis and repair, RNA transcription and protein translation.

DNA Replication and Repair

DNA synthesis and repair are not even mentioned in the famous Central Dogma of Molecular Biology (put very simplistically, DNA makes RNA makes protein) but they are, of course, essential for it. The first speaker in this session, and therefore in the meeting as a whole, was Luca Pellegrini from the University of Cambridge. He described structural studies of the first part of this process: the initiation of DNA synthesis. In all organisms, this process involves an enzyme called primase, which is found at the DNA replication fork - the point at which the strands of the original DNA helix divide so that a new strand can be synthesised on each of the template strands. Pellegrini and his group have solved the structure of several of the subunits of yeast primase, alone and bound to part of the DNA polymerase Pol alpha, and are using these structures to deduce the precise mechanism of this vitally important process.

Then Neil Kad of the University of Essex described the techniques he has developed for visualising individual molecules, and how he is applying them to the study of DNA repair by nucleotide excision. Briefly, this technique involves stretching a single molecule of DNA between two positively charged silica beads, and tagging individual molecules of DNA-binding proteins using fluorescent quantum dots so that their binding to and progress along this DNA "tightrope" can be monitored. He has discovered that although single subunits of the Uvr DNA repair protein complex may bind DNA and search it for errors, a complex between the subunits UvrA and UvrB is required for quick and efficient searching.

Schematic diagram of a "DNA tightrope" with labelled proteins bound. (c) Neil Kad, from the Kad Lab homepage

Transcription

The spliceosome is a "molecular machine" comprised of protein and small nuclear RNA (snRNA) subunits that found only in eukaryotes and that catalyses the removal of introns from the messenger RNA precursor molecules that are initially transcribed from DNA. Chris Oubridge, a member of Kiyoshi Nagai's group at the MRC Laboratory of Molecular Biology in Cambridge (and therefore one of the "home team") described an atomic resolution structure of a complex known as U1 that forms a major part of the soliceosome. This "small nuclear ribonucleoprotein" (snRNP) comprises the snRNA molecule U1 bound to ten proteins. This technically challenging exercise in X-ray crystallography is yielding important insights into the function and mechanism of this important part of the spliceosome.

Structure of the U1 ribonucleoprotein, from Kiyoshi Nagai's web pages at the MRC-LMB.

Another interesting presentation in the Translation section was given by David Lilley from the University of Dundee, who described the structures of kink turns in RNA molecules, and how these structural motifs interact with proteins.

Translation

Since the modern Laboratory o Molecular Biology was constituted as the "Unit for Research on the Molecular Structure of Biological Systems'" in 1947, nine Nobel prizes have been awarded to scientists working there. Its most recent laureate, Venki Ramakrishnan, shared the 2009 chemistry prize with Tom Steitz from the US and Ada Yonath from Israel for determining the first atomic resolution structure of the ribosome. Israel Sanchez from Ramakrishnan's lab at the LMB gave a presentation on the mechanism by which stop codons, which give the signal to terminate protein synthesis, are decoded on the ribosome. This process, which occurs when one of the stop codons (UAA, UAG and UGA in the standard genetic code) binds to the ribosomal A site, is still less well understood than the process through which "sense" codons are decoded into amino acids. Sanchez and his colleagues are studying the structure and function of ribosomes bound to modified RNA in which the uridine in the first position of a stop codon has been substituted by pseudo-uridine. They have discovered that the decoding centre of the ribosome is more flexible than they had originally thought, an insight that may help the understanding of the termination of protein synthesis further.

The final speaker was Birkbeck's own Cara Vaughan. She discussed some of her recent research using a combination of X-ray crystallography and electron microscopy to decipher the assembly of the kinetochore. This is a structure that forms in eukaryotic cells during cell division and that links the dividing chromosome to the mitotic spindle. Vaughan's research concerns a protein called Hsp90 that activates many signalling proteins. This protein is a member of a class of proteins termed the chaperones, which are generically involved in the folding, unfolding and activation of other proteins. Vaughan and her co-workers have solved the structure of two interacting proteins found in yeast, Sgt1 and Skp1, which togethe3r seem to hold Hsp90 in an open conformation that enables other kinetochore proteins to bind.

Image of a dividing eukaryotic cell. The chromosomes are shown in blue, the microtubules of the mitotic spindle in green, and the kinetochores in pink. Image from Wikimedia Commons.

The annual Winter Meeting is the most high profile event organised by the Biological Structures Group of BCA. The association as a whole organises many other events, including, this year, the annual European Crystallographic Meeting. ECM 28 will be held at the University of Warwick from 25-29 August 2013; it will provide an opportunity for British and European crystallographers to celebrate the origin of their science with the discovery of X-ray diffraction by father and son William Henry and William Lawrence Bragg, almost exactly a hundred years ago.