University of Cambridge
Biophysical and computational insights into the stability of RhoB and its cancer-associated variants
Abstract
dc:description.abstractRhoB is a small GTPase, part of the Rho family of the Ras superfamily of small G proteins, that act as cellular signalling switches. RhoB is involved in regulating the cytoskeleton, angiogenesis, cell death, endosome trafficking and DNA damage repair. Unlike other small GTPases such as K-Ras, RhoA and Cdc42, RhoB is understudied in terms of its structure, dynamics, and role in physiology and disease. Moreover, RhoB has been found to mediate both tumour promoting and tumour suppressing effects. Furthermore, recent genome sequencing projects have identified RHOB mutations in bladder cancer that could affect its GTPase function. This research project aimed to provide molecular insights into wildtype RhoB and its bladder cancer variants using molecular dynamics (MD) simulations and nuclear magnetic resonance (NMR) spectroscopy, plus other biophysical and biochemical techniques. Information of this sort should improve our knowledge of fundamental biology in health and disease about RhoB, and could inform further therapeutic research. In vitro thermal stability studies revealed that RhoB is much less stable than related GTPase superfamily members, and bladder cancer mutations further affected its melting temperature. To investigate the structural basis of its decreased stability and dynamics compared to other GTPases, solution NMR experiments were performed on RhoB. The sequential assignment of signals from backbone amide sites enabled 15N nuclear spin relaxation measurements to be collected, which verified that regions of RhoB are flexible by reporting on the motions of individual residues at different timescales. The relaxation experiments demonstrated that RhoB followed a similar dynamics pattern to the previously published Cdc42 order parameters; these proteins are mostly rigid on the ps – ns timescale, with localised motion at the N-terminus and in the switch regions. However, more RhoB peaks could be identified as belonging to the switch I region than in any of the published backbone assignments for Cdc42. This suggested that the relaxation properties of backbone amide sites in the switch I region of RhoB may be affected more by rapid local motions (on the ps – ns timescale) than conformational exchange line broadening (in the µs – ms timescale) that conceals this region in Cdc42 spectra. MD simulations with GROMACS were employed to complement the experimental NMR dynamics and biophysical data collected on RhoB. The simulations were applied to predict how different Rho family proteins (RhoB, Cdc42 and RhoA) begin to unfold in silico. MD simulation trajectory analysis indicated that RhoB was more mobile and displayed larger root mean square deviation (RMSD) excursions than the other Rho GTPases at multiple temperatures. Accordingly, RhoB exhibited higher root mean square fluctuations (RMSF) in various functional regions of the small GTPase; for example, an increased amplitude of motion was observed in the switch I region of RhoB than for the equivalent region in Cdc42 – sequence differences could be influencing this increased mobility. This work has helped pinpoint and improve our understanding of the sources of instability in RhoB and across Rho GTPases in general, which may potentially have physiological and therapeutic relevance.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2025
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Hall, Morwenna
- Advisors dc:contributor.advisor
-
- Broadhurst, Bill
- Nietlispach, Daniel
Subjects
dc:subject × 9Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.131606
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/405369