Structural and Biochemical Characterisation of the NHERF1-CFTR Interaction

Abstract

NHERF1 is a PDZ domain containing scaffold protein which facilitates the formation of a number of important protein complexes. PDZ domains are globular protein recognition modules which bind to short linear motifs, generally found at the extreme C-termini of integral membrane proteins. Via its two PDZ domains, NHERF1 is able to bind to the C-termini of a range of membrane receptors and channels, including that of the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a chloride ion channel, loss of function mutations of which cause the autosomal recessive disease cystic fibrosis. The most common cystic fibrosis causing mutation is the deletion of phenylalanine at position 508 (F508del), which causes protein-misfolding and subsequent targeting of CFTR for degradation. NHERF1 overexpression has been shown to increase both the plasma membrane localisation and activity of F508del CFTR and, as such, the interaction between these two proteins is of particular therapeutic interest. The structure the extended PDZ2 domain of NHERF1 in complex with the PDZ binding motif of CFTR was determined using X-ray crystallography. This structure provided a new insight into the structural determinants of this interaction. In particular, the structure revealed that specific side chain mediated interactions within the binding pocket enable the recognition of the arginine at the -2 position of the PDZ binding motif. This contradicts previous studies, which have suggested that the loss of this interaction is the sole determinant for the lower binding affinity exhibited NHERF1 PDZ2 as compared to PDZ1. As such, this work suggests that instead a variety of dynamic and allosteric effects dictate the specificity and affinity of the PDZ domain motif interaction. Further to this, this study employed a range of novel techniques to further characterise the interaction between NHERF1 and CFTR. iBox PAK4cat in-cellulo crystallisation is based on the phenomenon that serine/threonine kinase PAK4 and its endogenous inhibitor, iBox, form cytoplasmic rod-type protein crystals when transfected into mammalian cells. The in-cellulo crystal structure of PAK4cat bound to iBox revealed that iBox-PAK4cat molecules form a hexagonal array within the crystal lattice, with channels 80 Å in diameter that run the length of the crystal. The work presented in this study sought to explore whether it is possible to incorporate other proteins into this cavity and, as such, determine whether iBox PAK4cat crystals can be utilised for other experimental purposes. A novel in cellulo iBox PAK4cat protein protein interaction screening assay was developed. This assay enabled further characterisation of the NHERF1 CFTR PDZ domain motif interaction in a cellular context. The results from this study corroborated the finding from the crystal structure, indicating that the arginine at the -2 position of the CFTR PDZ binding motif is specifically recognised by the binding pocket of NHERF1 PDZ2. Finally, the PDZ domains of NHERF1 were employed in a proof of concept study into the use of iBox PAK4cat crystals as the basis for the development of a novel in-cellulo platform for the crystallisation of guest proteins. While ultimately this work did not enable determination of the in-cellulo structures of the NHERF1 PDZ domains, the results generated from this study have given a useful insight into the iBox-PAK4cat system and will inform future optimisation studies.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Robert Ford (Supervisor) & Martin Pool (Supervisor)