Roger Williams

Roger Williams

Electron cryo-microscopy and hydrogen/deuterium exchange mass spectrometry of DNA damage repair complexes and Structural studies of assembly of phosphoinositide 3-kinase-related complexes on membranes, using cryo-electron tomography and hydrogen/deuterium exchange mass spectrometry
Group Leader page

Project 1: Electron cryo-microscopy and hydrogen/deuterium exchange mass spectrometry of DNA damage repair complexes

ATM and ATR are members of the phosphoinositide-3-kinase-like protein kinases family (PIKKs). These enzymes are activated in response to DNA damage and they are master kinases linking DNA damage to cell cycle progression and cell death. The enzymes are part of complexes that sense DNA breaks and phosphorylate a wide range of targets in the DNA damage response. Cancer cells rely on this pathway to survive genomic instability, and this has led to an interest in inhibiting these enzymes as a cancer therapeutic strategy.

The project involves understanding how these kinases are activated in response to DNA damage, with an emphasis on the interactions that they make with protein complexes on DNA. The project involves electron cryo-microscopy and hydrogen/deuterium exchange mass spectrometry of large enzyme complexes. The work builds on the experience of the group with other members of the PIKK family and the large-scale mammalian cell culture that has been optimised in the group for these enzymes. The structural work will be carried out in parallel with mammalian cell biology and in vitro enzymology. The hydrogen/deuterium exchange mass spectrometry will provide insight into the interactions that the enzyme complexes make in DNA repair. This technique has been optimised in the group to provide single-residue resolution.

Structural work will take advantage of the extensive electron cryo-microscopy facilities in the LMB.


References:

Baretić D, Pollard HK, Fisher DI, Johnson CM, Santhanam B, Truman CM, Kouba T, Fersht AR, Phillips C, Williams RL (2017)
Structures of closed and open conformations of dimeric human ATM.
Sci Adv. 10;3(5):e1700933. PMID 28508083.

Baretić, D., Berndt, A., Ohashi, Y., Johnson, C.M., and Williams, R.L. (2016)
Tor forms a dimer through an N-terminal helical solenoid with a complex topology.
Nat Commun 7, 11016.

Masson GR, Burke JE, Williams RL (2016)
Methods in the Study of PTEN Structure: X-Ray Crystallography and Hydrogen Deuterium Exchange Mass Spectrometry.
Methods Mol Biol. 1388:215-230. PMID: 27033079

Baretić, D., and Williams, R.L. (2014)
PIKKs--the solenoid nest where partners and kinases meet.
Curr Opin Struct Biol 29:134-142. PMID 25460276


Project 2: Structural studies of assembly of phosphoinositide 3-kinase-related complexes on membranes, using cryo-electron tomography and hydrogen/deuterium exchange mass spectrometry

The phosphatidylinositol 3-kinase (PI3K) known as VPS34 is the primordial member of the PI3K family of enzymes that include both lipid and protein kinases. These enzymes function in large protein complexes, and a subset of these enzymes, including class I PI3Ks, VPS34 and mTOR assemble in complexes that are active on cellular membranes. We are using single-particle cryo-electron microscopy, cryo-electron tomography and hydrogen/deuterium exchange mass spectrometry to understand the structures of these complexes, how they assemble on membranes and how they are regulated on membranes by other membrane-associated proteins. These enzyme complexes have key roles in ageing, neurodegeneration, autophagy, phagocytosis, endocytosis and immunity.

The project involves determining how these complexes are recruited to cellular compartments where they are activated by association with G-proteins and/or receptor tyrosine kinases. Assembly of these complexes on membranes is fundamental to their functions and understanding this process could be fundamental for pharmaceutical development. The structural work will be carried out in parallel with mammalian cell biology and in vitro assays. The HDX-MS provides unequalled insight into the interactions that the enzyme complexes make with membranes and G-proteins. This technique has been optimised in the group to provide single-residue resolution and is central to innovative approaches that we have implemented to develop allosteric inhibitors.


References:

Rostislavleva, K., Soler, N., Ohashi, Y., Zhang, L., Pardon, E., Burke, JE, Masson, GR, Johnson, C., Jan Steyaert, J., Ktistakis, NT, Williams, RL. (2015)
Structure and flexibility of the endosomal Vps34 complex reveals the basis of its function on membranes.
Science 350:aac7365. PMID 2645021

Ohashi Y, Soler N, García Ortegón M, Zhang L, Kirsten ML, Perisic O, Masson GR, Burke JE, Jakobi AJ, Apostolakis AA, Johnson CM, Ohashi M, Ktistakis NT, Sachse C, Williams RL (2016). Characterization of Atg38 and NRBF2, a fifth subunit of the autophagic Vps34/PIK3C3 complex.
Autophagy 12: 2129-2144. PMID: 27630019

Baretić D, Berndt A, Ohashi Y, Johnson CM, Williams RL (2016).
Tor forms a dimer through an N-terminal helical solenoid with a complex topology.
Nat Commun. 7:11016. PMID: 27072897

Baretić D. and Williams RL. (2014)
PIKKs--the solenoid nest where partners and kinases meet.
Curr Opin Struct Biol 29:134-142. PMID 25460276