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Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography	Structures of the human SPAK and OSR1 conserved C-terminal (CCT) domains Karen T. Elvers , Magdalena Lipka-Lloyd , Rebecca C. Trueman , Benjamin D. Bax , Youcef Mehellou Chembiochem DOI: 10.1002/cbic.202100441 Diamond Proposal Number(s): [20147] Show Abstract ▼ Abstract: STE20/SPS1-related proline/alanine-rich kinase (SPAK) and Oxidative Stress Responsive 1 (OSR1) kinase are two serine/threonine protein kinases that regulate the function of ion co-transporters through phosphorylation. The highly conserved C -terminal (CCT) domains of SPAK and OSR1 bind to RFx[V/I] peptide sequences from their upstream With No Lysine Kinases (WNKs), facilitating their activation via phosphorylation. Thus, the inhibition of SPAK and OSR1 binding, via their CCT domains, to WNK kinases is a plausible strategy for inhibiting SPAK and OSR1 kinases. To facilitate structure-guided drug design of such inhibitors, we expressed and purified human SPAK and OSR1 CCT domains and solved their crystal structures. Interestingly, these crystal structures show a highly conserved primary pocket adjacent to a flexible secondary pocket. We also employed a biophysical strategy and determined the affinity of SPAK and OSR1 CCT domains to short peptides derived from WNK4 and NKCC1. Together, this work provides a platform that facilitates the design of CCT domain specific small molecule binders that inhibit SPAK- and OSR1-activation by WNK kinases, and these could be useful in treating hypertension and ischemic stroke.	Nov 2021
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	BTN3A1 discriminates γδ T cell phosphoantigens from non-antigenic small molecules via a conformational sensor in its B30.2 domain Mahboob Salim , Timothy J. Knowles , Alfie T. Baker , Martin S. Davey , Mark Jeeves , Pooja Sridhar , John Wilkie , Carrie R. Willcox , Hachemi Kadri , Taher E. Taher , Pierre Vantourout , Adrian Hayday , Youcef Mehellou , Fiyaz Mohammed , Benjamin E. Willcox Acs Chemical Biology DOI: 10.1021/acschembio.7b00694 Diamond Proposal Number(s): [14692] Show Abstract ▼ Abstract: Human Vγ9/Vδ2 T-cells detect tumour cells and microbial infections by recognising small phosphorylated prenyl metabolites termed phosphoantigens (P-Ag). The type-1 transmembrane protein Butyrophilin 3A1 (BTN3A1) is critical to the P-Ag-mediated activation of Vγ9/Vδ2 T-cells, however, the molecular mechanisms involved in BTN3A1-mediated metabolite sensing are unclear, including how P-Ag are discriminated from non-antigenic small molecules. Here, we utilised NMR and X-ray crystallography to probe P-Ag sensing by BTN3A1. Whereas the BTN3A1 Immunoglobulin Variable domain failed to bind P-Ag, the intracellular B30.2 domain bound a range of negatively-charged small molecules, including P-Ag, in a positively-charged surface pocket. However, NMR chemical shift perturbations indicated BTN3A1 discriminated P-Ag from non-antigenic small molecules by their ability to induce a specific conformational change in the B30.2 domain that propagated from the P-Ag binding site to distal parts of the domain. These results suggest BTN3A1 selectively detects P-Ag intracellularly via a conformational antigenic sensor in its B30.2 domain, and have implications for rational design of antigens for Vγ9/Vδ2 -based T-cell immunotherapies.	Sep 2017
I04-1-Macromolecular Crystallography (fixed wavelength)	Structural insights into the activation of MST3 by MO25 Youcef Mehellou , Dario R. Alessi , Thomas J. Macartney , Marta Szklarz , Stefan Knapp , Jonathan M. Elkins Biochemical And Biophysical Research Communications 431 (3), 604 - 609 DOI: 10.1016/j.bbrc.2012.12.113 PMID: 23296203 Diamond Proposal Number(s): [8421] Show Abstract ▼ Abstract: The MO25 scaffolding protein operates as critical regulator of a number of STE20 family protein kinases (e.g. MST and SPAK isoforms) as well as pseudokinases (e.g. STRAD isoforms that play a critical role in activating the LKB1 tumour suppressor). To better understand how MO25 interacts and stimulates the activity of STE20 protein kinases, we determined the crystal structure of MST3 catalytic domain (residues 19–289) in complex with full length MO25β. The structure reveals an intricate web of interactions between MST3 and MO25β that function to stabilise the kinase domain in a closed, active, conformation even in the absence of ATP or an ATP-mimetic inhibitor. The binding mode of MO25β is reminiscent of the mechanism by which MO25α interacts with the pseudokinase STRADα. In particular we identified interface residues Tyr223 of MO25β and Glu58 and Ile71 of MST3 that when mutated prevent activation of MST3 by MO25β. These data provide molecular understanding of the mechanism by which MO25 isoforms regulates the activity of STE20 family protein kinases.	Feb 2013

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