Steve Gamblin: Projects

Chromatin modification enzymes

Epigenetic processes are an integral part of the molecular mechanisms that define the gene expression program unique to different cell types. Chromatin modification enzymes are key components of epigenetic networks, and are precisely targeted to nucleosomes associated with specific genes, where they place chemical modifications on histone side chains. Effector proteins are then recruited to these distinct patterns of marks via their recognition domains (Figure 1). We are interested in the molecular basis of the targeting and regulation of modification enzymes. The repressive Polycomb (PcG) and activating Trithorax (TRX) proteins regulate the expression of fundamental genes that determine cell fate. Essential to their function, the PcG group protein EzH2, and TRX group MLL1, have histone lysine methylation activity. This activity is derived from their catalytic SET domains. In both enzymes the SET domain activity is dependent on the assembly of multi-protein complexes. We are interested in how the interactions within these complexes target and regulate the enzymes and the potential implications this has for future therapeutic approaches.

PcG proteins are essential for stem cell maintenance and embryonic development. Three families of complexes containing PcG proteins have been identified to date in Drosophila: Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2) and PhoRC. One aspect of our work on the molecular mechanisms underlying epigenetic regulation is the functional and structural characterisation of the Polycomb Repressive Complex 2. We have previously shown (Margueron, et al., 2009) that the aromatic cage of the EED component specifically binds histone tails carrying trimethyl-lysine residues associated with repressive chromatin marks, and that this leads to the allosteric activation of the methyltransferase activity of PRC2. Mutations in EED that prevent binding to repressive trimethyl-lysine marks abolish the activation of PRC2 in vitro and in Drosophila reduce global H3K27 methylation and interfere with development. Our work shows the functional and structural basis for epigenetic self-renewal and leads us to conclude that PRC2 readout of H3K27me3 is key to the propagation of this repressive mark.

Figure 1

Figure 1. A key part of epigenetic signalling networks is the site-specific modification of histones by modification enzymes. These marks then recruit effector proteins with complimentary recognition domains.

AMP-activated protein kinase

AMP-activated protein kinase (AMPK) regulates cellular metabolism in response to the availability of energy, and is therefore a target for type-2 diabetes treatment. It senses changes in the ratio of AMP/ATP by binding both species in a competitive manner. Thus, increases in the concentration of AMP activate AMPK resulting in the phosphorylation and differential regulation of a series of downstream targets that control anabolic and catabolic pathways. While most studies have focused on understanding the regulation of AMPK by AMP and Mg-ATP, we have recently shown that ADP is also a physiological activator of AMPK.

We have solved a number of high-resolution crystal structures of mammalian AMPK in complex with nucleotides. Structural and solution studies reveal that two sites on the regulatory domain bind AMP, ADP or Mg-ATP, whereas a third site contains a tightly bound AMP that does not exchange. The phosphate groups of AMP/ADP/ATP lie in a groove on the surface of the regulatory fragment, which is lined with basic residues, many of which are associated with disease-causing mutations. Our binding studies indicate that Mg-ATP binds more weakly to these exchangeable sites, providing an explanation as to how micromolar concentrations of AMP and ADP can compete with millimolar concentrations of Mg-ATP.

Our structures revealed that a region extending from the kinase domain, termed the α hook, interacts with AMP/ADP bound in the regulatory fragment. This interaction allows the kinase domain to bind to the regulatory fragment of AMPK, which is refractory to dephosphorylation and inactivation by protein phosphatases. We envisage that Mg-ATP binding at this site leads to the displacement of the α hook and inactivation of AMPK.

AMPK has emerged as an attractive therapeutic target for treating metabolic disorders and a number of direct AMPK activators have been developed. We solved the crystal structure of full length AMPK in complex with these activators. Activator 991 modulates the enzyme by binding to AMPK at an interface formed between the kinase domain and the carbohydrate-binding module. The activator binding site is separate from the nucleotide binding site and raises the possibility that these activators may be mimicking a natural metabolite. Further studies are ongoing to fully understand the molecular basis of cellular energy regulation of AMP-activated protein kinase.

Figure 2

Figure 2. Structure of AMP protein kinase (AMPK) in complex with AMP molecules and an activating drug which binds between the Kinase Domain and the Carbohydrate Binding Domain (Xiao et al 2013). The inset shows details of activating drug binding.

Influenza hemagglutinin

The influenza virus surface glycoprotein hemagglutinin (HA) mediates receptor binding and membrane fusion in influenza infections. The viruses that caused the three influenza pandemics of the twentieth century in 1918, 1957, and 1968 had distinct hemagglutinin receptor binding glycoproteins that had evolved the capacity to recognise human cell receptors. We have determined the structure of the H2 hemagglutinin from the second pandemic, the "Asian Influenza" of 1957 (Figure 3). We can compare it with the 1918 "Spanish Influenza" hemagglutinin, H1, and the 1968 "Hong Kong Influenza" hemagglutinin, H3, and show that despite its close overall structural similarity to H1, and its more distant relationship to H3, the H2 receptor binding site is closely related to that of H3 hemagglutinin.

HA binds to sialic acids that are found in α2,3 or α2,6 linkages to galactose on cell surface glycoproteins and glycolipids. Avian influenza viruses preferentially bind α2,3-linked sialic acids, which predominate in the enteric tract of birds where the viruses replicate. Human influenza viruses preferentially bind to α2,6-linked sialic acids, which are the predominant form found in the human upper respiratory tract. We have been developing a quantitative assay to measure the binding of influenza viruses to immobilized sialic acids. Using instrumentation from Biacore and Fortebio, we are able to measure virus binding in real time to determine the specificity for different sugars. Analysis of different human, avian and swine influenza strains, as well as analysis of certain mutations in specific strains, reveals which viruses are able to bind well to human receptors.

By applying structural and biophysical techniques, we have recently shown how mutations to the sialic acid binding site of the avian H5N1 virus, have allowed it to acquire a strong binding preference for the human receptor over the avian equivalent. The altered binding mode now resembles that previously observed in historically significant pandemic viruses. Characterisation of the recently emerged H7N9 influenza virus hemaglutinin indicates that although it has also acquired higher affinity for the human receptor sialic acid, it has retained high affinity binding of the avian receptor. The retention of avian receptor binding will contribute to its low transmissibility due to sequestration by avian-like receptors in human mucins. Through these studies we are better able to understand the significance of mutations in this rapidly evolving virus.

Figure 3

Figure 3. Ribbons representation of different H2 HA monomers and receptor binding sites.