Neil McDonald: Projects

Cell surface receptor activation and oncogenic deregulation

To understand how extracellular signals are received at the cell membrane and trigger intracellular signalling pathway activation, we study the receptor tyrosine kinase (RET). RET is crucial for embryonic and adult development and its mutation underlies three human diseases (Hirschsprung's disease, kidney agenesis and cancer).

We are interested in how RET becomes activated in response to binding a bipartite ligand that is comprised of a glial cell line derived neurotrophic factor (GDNF) family ligand and a GDNF family receptor a (GFRα) co-receptor. To visualise this interaction, we have reconstituted several vertebrate RET ternary complexes containing both ligand and co-receptor, permitting a hybrid structural approach. The downstream consequence of RET ligand occupancy is the trans-phosphorylation of internal tyrosine sites within the tyrosine kinase catalytic domain. A careful analysis of the kinetics of tyrosine auto-phosphorylation for wild type and oncogenic RET mutants using label-free mass spectrometry has revealed surprising differences in the order of auto-phosphorylation.

We are continuing to characterise how regions flanking the core RET kinase domain contribute to ligand-dependent activation as well as how they are perturbed in an oncogenic context.

Our earlier structural work on RET identified a folding bottleneck that could be eliminated by removal of just two amino acids. Removing these residues in a panel of RET Hirschsprung's (HSCR) disease mutants restored cell surface expression to most of these mutants instead of their being retained within the endoplasmic reticulum as immature forms.

Recently we carried out a small biased siRNA screen to identify components of the RET maturation pathway that could rescue HSCR mutants and influence cell surface levels of wild type RET. Characterising factors that control RET maturation and export will help improve our understanding of how thresholds of wild type and pathological RET signalling are set.

Polarity signalling assemblies at the plasma membrane

Protein kinase C iota (PKCι) is a serine-threonine kinase that affects many cellular processes, including growth, proliferation, and motility. PKCι associates with two discrete polarity complexes; one containing the polarity proteins Par-3 and Par-6 (PAR complex) and the other contains Crumbs, Stardust and PatJ (the Crbs complex). Both complexes are found in vertebrates and invertebrates where they are crucial for maintaining apical-basal polarity.

Loss of cell polarity is often associated with aggressive epithelial cell cancers, and PKCι is frequently found in abnormally high abundance in epithelial cancers. PKCι is therefore a validated oncogene and potential drug target.

This year we reported the discovery and mode of action of potent PKCi-selective chemical inhibitors (Kjaer et al., 2013; Biochem J. 451(2): 329-42). These inhibitors were identified in collaboration with the Cancer Research Technology (CRT) Discovery Lab and the Protein Phosphorylation Group.

Prior to publication, few isoform-specific chemical biology tools were available to inhibit PKCι catalytic activity. We determined a crystal structure of PKCι bound to a representative compound, CRT0066854, revealing how it binds within the nucleotide cleft and displaces a crucial Asn-Phe-Asp motif found in many AGC kinases (Figure 1A).

CRT0066854 inhibits phosphorylation of the PKCι substrate LLGL2 in cell lines and exhibits phenotypic effects in a range of cell-based assays. This compound has been sent out to numerous academic laboratories for use as a chemical biology tool to modulate PKCι/PKCζ activity in vitro and in vivo in a variety of cancer models. During the inspection of the PKCι-CRT0066854 inhibitor structure, we noticed an invariant motif on the surface of PKCi (Figure 1B).

In collaboration with the Protein Phosphorylation Group, we explored a possible role for this RIPR motif in protein-protein interaction leading to the discovery that this motif is required to engage the PKCι substrate LLGL2 (Linch et al., 2013; Sci Signal. 6(293): ra82). Surprisingly, PKCι mutants associated with human cancer were found that target this motif highlighting the importance of PKCι substrate recruitment.

Figure 1

Figure 1. A. Close-up of the CRT0066854 inhibitor binding-site within the PKCι nucleotide-binding pocket (cyan). Inhibitor is shown in orange and dashed lines indicate hydrogen-bonds between protein and inhibitor. A bound water molecule and an iodide ion are shown as spheres. B. Schematic of the PKCι kinase domain structure bound to CRT0066854 highlighting the location of the RIPR motif which is required for the engagement of specialised PKCι substrates such as LLGL2. (Click to view larger image)

Structural biology of the XPF/MUS81/FANCM endonuclease family

Mammalian endonucleases play key roles in several DNA repair pathways including nucleotide excision repair (NER), interstrand crosslink repair (ICL), recombination and replication fork repair, and thereby help maintain genomic integrity.

We have been studying the XPF family of structure-specific endonucleases as potential translational research projects. Our rationale is that understanding endonuclease catalytic mechanism and identifying a chemical inhibitor that could block NER and ICL, may render a wider range of tumour cells sensitive to DNA-damaging agents. The human XPF family comprises at least three active hetero-dimeric endonuclease complexes; XPF-ERCC1, MUS81-EME1 and the largely uncharacterised MUS81-EME2 complex. A fourth complex, FANCM-FAAP24, belongs to the XPF family but does not exhibit endonuclease activity.

This year we have reported the identification and structure of a DNA-binding winged helix domain within human MUS81 that had been missed in previous studies (Fadden et al., 2013; Nucleic Acids Res. 41(21): 9741-52). We show that this domain can bind dsDNA and plays a role in positioning the scissile bond within junction substrates (Figure 2A).

We also recently reported the crystal structure of a C-terminal fragment of FANCM complexed to its FAAP24 partner protein and a short dsDNA oligonucleotide (Coulthard et al., 2013; Structure. 21: 1648-58). In this structure, the FANCM (HhH)2 hairpins are buried within an interface with the FANCM pseudo-nuclease domain. The dsDNA trajectory is distinct from our previous archeal XPF-dsDNA structure suggesting that in order to engage both the FAAP24 (HhH)2 hairpins and the FANCM pseudo-nuclease domain it must deviate substantially from linear B-DNA.

Collaborations with the Genetic Recombination and Architecture and Dynamics of Macromolecular Machines Groups at the Clare Hall Laboratories have validated functionally important roles for the FAAP24 (HhH)2 and FANCM pseudo-nuclease domain consistent with our structure and led to an EM structure using full length FANCM-FAAP24 complex.

We are also continuing to work collaboratively with CRT to discover chemical inhibitors of human XPF-ERCC1 using our published fluorescence-based assay (Bowles et al., 2012; Nucleic Acids Res. 40: e101).

Figure 2

Figure 2. A. Identification of a DNA-binding winged helix domain within Mus81. Previous studies had missed the presence of this domain within Mus81. This structure was determined by NMR. B. Crystal structure of a C-terminal fragment of FANCM complexed to its FAAP24 partner protein. The FANCM (HhH)2 hairpins are buried whilst FAAP24 (HhH)2 domain binds dsDNA. The trajectory of the dsDNA relative to the FANCM pseudo-nuclease domain is distinct from our previous archeal XPF-dsDNA structure. For dsDNA to engage both the FAAP24 (HhH)2 hairpins and the FANCM pseudo-nuclease domain it must be bent from a linear B-DNA conformation. (Click to view larger image)

Selected publications

Ivanova M, Saiu P, Thompson BJ & McDonald NQ. Structural insights into PDZ-mediated interaction of Pals1 and Crumbs suggest a stable apical polarity recruitment platform. (2015) Accepted Act Cryst D.

Goodman KM, Kjær S, Beuron F, Knowles PP, Nawrotek A, Burns E, Purkiss AG, George R, Santoro M, Morris EP & McDonald NQ. RET recognition of GDNF-GFRa1 ligand by a composite binding site promotes membrane-proximal self-association (2014) Cell Reports. In Press.

Plaza-Menacho I, Barnouin K, Goodman K, Martínez-Torres RJ, Borg A, Murray-Rust J, Mouilleron S, Knowles PP, McDonald NQ. Oncogenic RET kinase domain mutations perturb the autophosphorylation trajectory by enhancing substrate presentation in trans. (2014) Mol Cell ;53(5):738-51

Coulthard R, Deans AJ, Swuec P, Bowles M, Costa A, West SC, McDonald NQ. Architecture and DNA Recognition Elements of the Fanconi Anemia FANCM-FAAP24 Complex. (2013) Structure;21(9):1648-58.

Fadden AJ, Schalbetter S, Bowles M, Harris R, Lally J, Carr AM,McDonald NQ. A winged helix domain in human MUS81 binds DNA and modulates the endonuclease activity of MUS81 complexes. (2013) Nucleic Acids Res. Vol. 41 Issue 21, p9741-52.

Linch M, Sanz-Garcia M, Soriano E, Zhang Y, Riou P, Rosse C, Cameron A, Knowles P, Purkiss A, Kjaer S, McDonald NQ, Parker PJ. A cancer-associated mutation in atypical protein kinase cι occurs in a substrate-specific recruitment motif. (2013) Sci Signal. 6(293):ra82

Kjær S, Linch M, Purkiss A, Kostelecky B Knowles PP, Rosse C, Riou P, Soudy C, Kaye S, Patel B, Soriano E, Murray-Rust J, Barton C Dillon C, Roffey J, Parker PJ & McDonald NQ. Adenosine-binding motif mimicry and cellular effects of a thieno[2,3-d]pyrimidine-based chemical inhibitor of atypical protein kinase C isozymes. (2013) Biochemical Journal 451, p329-342.

Mouilleron S, Wiezlak M, O'Reilly N, Treisman R, McDonald NQ. Structures of the Phactr1 RPEL Domain and RPEL Motif Complexes with G-Actin Reveal the Molecular Basis for Actin Binding Cooperativity. (2012) Structure. 20(11):1960-70.

Bowles M, Lally J, Fadden A, Mouilleron S, Hammonds T & McDonald NQ. A fluorescence based incision assay for human XPF-ERCC1 activity identifies important elements of DNA junction recognition. (2012) Nucleic Acid Research. 40(13):e101

Mouilleron S, Langer CA, Guettler S, McDonald NQ, Treisman R. Structure of a Pentavalent G-Actin•MRTF-A Complex Reveals How G-Actin Controls Nucleocytoplasmic Shuttling of a Transcriptional Coactivator. (2011) Sci Signal.;4(177):ra40

Boeda B, Knowles PP, Briggs D, Murray-Rust J, Soriano E, Garvalov BK, McDonald NQ, Way M. Molecular recognition of the Tes LIM2-3 domains by the actin-related protein Arp7A. (2011) J Biol Chem. 286(13):11543-54.

Kjaer S, Hanrahan S, Totty N, McDonald NQ. Mammal-restricted elements predispose human RET to folding impairment by HSCR mutations. Nat Struct Mol Biol. 2010;17(6):726-31

Kostelecky B, Saurin AT, Purkiss A, Parker PJ, McDonald NQ. (2009) Recognition of an intra-chain tandem 14-3-3 binding site within PKCepsilon. EMBO Rep. 2009 Sep;10(9):983-9.

Riou P, Kjaer S, Garg R, Purkiss A, George R, Cain RJ, Bineva G, Reymond N, McColl B, Thompson AJ, O'Reilly N, McDonald NQ, Parker PJ & Ridley AJ. 14-3-3 proteins interact with a hybrid prenyl-phosphorylation motif to regulate Rnd proteins. Cell (2013) ;153(3):640-53.

Neil McDonald

neil.mcdonald@crick.ac.uk
+44 (0)20 379 61958

  • Qualifications and history
  • 1991 PhD in Crystallography, Birkbeck College, UK
  • 1991-1994 Postdoctoral Fellow (Lucille P. Markey Scholar), Columbia University, USA
  • 1994 Established Structural Biology Lab at the Imperial Cancer Research Fund, UK
  • 1996 Reader (Associate Professor), Birkbeck College, London, UK
  • 1999 Principal Scientist, Imperial Cancer Research Fund, London. (in 2002 the Imperial Cancer Research Fund became London Research Institute, Cancer Research UK)
  • 2006 Professor of Structural Biology, Institute of Structural Molecular Biology, Birkbeck College, London UK
  • 2015 Group Leader, the Francis Crick Institute, London, UK