Biogenesis of the multivesicular body (MVB) organelle is an important process for regulation of signalling in the cell. Signal receptors embedded within the outer MVB membrane can be sorted into intralumenal vesicles which bud away from the cytosol to within the MVB preventing further signalling. Sorting of receptors, invagination of the membrane and release of vesicles into the MVB lumen are mediated by the endosomal sorting complexes required for transport (ESCRT) along with a range of accessory proteins including histidine domain protein tyrosine phosphatase (HD-PTP). HD-PTP is a multidomain protein which makes several interactions with ESCRT partners, including ESCRT-0, ESCRT-I and ESCRT-III. This thesis focusses specifically on the interaction between HD-PTP CC domain and Ubap1 (ESCRT-I), and the two interactions of HD-PTP Bro and PRR domains with STAM2 (ESCRT-0) SH3 and Core domains. To address the structure of HD-PTP, multiple techniques were used: X-ray crystallography, which gives high resolution structural information; small angle X-ray scattering (SAXS), which gives low resolution data for large non-crystallisable units in their solution state; and double electron-electron resonance (DEER) spectroscopy, which gives high resolution nanometre-range distance constraints between cysteines labelled with methanethiosulfonate spin label (MTSL). It was shown by X-ray crystallography that HD-PTP has an elongated CC domain, in stark contrast to its homologues ALIX and Bro1 which both have V-shaped CC domains. The CC domain showed limited flexibility both by SAXS and DEER. Further investigation showed that there was no significant conformational change upon binding its ESCRT-I partner Ubap1. The multidomain structure of HD-PTP Bro1-CC-PRR was described by SAXS, showing that these domains form an extended arrangement in solution. In addition, SAXS was also used to analyse the structure of these domains in complex with STAM2 (ESCRT-0), which showed that STAM2 is simultaneously tethered by the Bro1 domain and PRR. The Bro-CC-PRR portion of HD-PTP, has 9 cysteines, so with the aim of measuring local structural information in the CC domain alone, alternative spin labelling methods were investigated. Use of a bromoacrylaldehyde spin label (BASL), instead of MTSL, allowed more selective labelling of surface exposed cysteines, and avoided labelling most of the cysteines in the Bro1 domain. This novel method allowed the shape of the CC domain to be monitored during STAM2 binding and showed that there is no induced conformational change.
|Date of Award
|31 Dec 2017
- The University of Manchester
|Philip Woodman (Supervisor), Lydia Tabernero (Supervisor) & Alistair Fielding (Supervisor)