Phox-homology (PX) domain-containing proteins are a family of 49 cytoplasmic and membrane associated proteins that are central to processes as diverse as membrane remodeling, intracellular trafficking, cell signaling and cytokinesis. This thesis examines the structure and function of several related PX proteins SNX17, SNX27 and SNX31. These proteins form a sub-family called the PX-FERM proteins, because of the presence of the defining PX domain and a C-terminal 4.1/ezrin/radixin/moesin (FERM) domain. SNX17 is the founding member of the family and is an endosomally-associated protein that binds to specific motifs in the cytoplasmic region of transmembrane receptors (cargo). It drives cargo recycling to the plasma membrane and thus acts as a release valve for several cargos (e.g. amyloid precursor protein (APP)), shuttling them away from lysosomal degradative pathways. Aberration in SNX17 function has a severe physiological impact on receptor turnover. The exact mechanisms regulating these cargo-sorting processes remain elusive. This thesis presents the molecular characterization of SNX17, its interaction with biological membranes and cargo molecules and its relationship to other PX-FERM proteins.
Chapter 2 defines SNX17 as the founding member of a novel sub-family of PX proteins called the PX-FERM proteins. Structural predictions and sequence analyses show that the SNX17 C-terminal domain resembles an atypical FERM domain. SNX17 is closely related to the PX proteins SNX27 and SNX31, altogether forming the PX-FERM sub-group. Thermodynamic characterization of PX-FERM proteins reveals that the FERM domain of PXFERM proteins interacts directly with Asn-Pro-Xaa-Tyr/Asn-Xaa-Xaa-Tyr (NPxY/NxxY) peptide motifs derived from various cargos, suggesting that all PXFERM proteins may be involved in controlling intracellular sorting of cargo such as APP. Chapter 2 also reveals the mechanistic basis for endosomal localization of the PX-FERM proteins via binding the endosomal phosphoinositide lipid PI3P. Analysis of SNX17 and SNX27 by small angle X-ray scattering (SAXS) shows that the molecules adopt non-self-assembling, compact modular structures in solution. In addition each of these proteins unexpectedly binds to small GTPase molecules utilizing the FERM domain. Such multimodal interactions place the PX-FERM proteins at a hub of endosomal sorting and signaling pathways. The potential role of PX-FERM proteins as adaptors in orchestrating signaling cascades at the endosome is discussed in detail in Chapter 3.
Chapter 4 describes the mechanism of sequence (NPxY/NxxY) dependent endosomal cargo recycling by PX-FERM proteins. The crystal structure of the SNX17 FERM-P-selectin complex reveals a novel atypical architecture of the FERM domain and the molecular basis for recognition of these essential sorting sequences. Importantly, PX-FERM proteins share a promiscuous ability to bind a wide array of putative cargo molecules, including receptor tyrosine kinases (RTKs). Based on SAXS-reconstructions a model is proposed that describes the coordinated molecular interactions of PX-FERM proteins with membrane, cargo and regulatory proteins required for the recycling and signaling outcomes.
The data presented in Chapter 5 demonstrate a novel differential membrane binding preference between the PX-FERM proteins. This difference in binding to membrane phosphoinositides (PIs) may partly explain their varied intracellular localization under certain physiological conditions, in particular for the SNX27 molecule, which can be recruited to the cell surface under certain stimuli. Isothermal titration calorimetry (ITC) assessment of the PI binding preference of SNX27 shows that the FERM domain of SNX27 mediates the interaction with plasma membrane-enriched PIs that are phosphorylated at more than one position, in stark contrast to SNX17 which binds exclusively to PI3P. This chapter provides a mechanistic explanation for this variation in PI specificity, and the potential role of this lipid interaction in SNX27 translocation to the plasma membrane is discussed.
Chapter 6 summarizes attempts to purify and crystallize PX-FERM proteins and their various truncations. Although to date crystallization experiments have failed to deliver crystals diffracting to high resolution, exciting preliminary crystals for the full length SNX17 molecule have been obtained that with further optimization may yield diffraction quality crystals for elucidation of a high-resolution crystal structure. Such structures will not only reveal how individual binding sites are related to each other, but may also provide insights into possible conformational changes and regulation by inter-domain interactions.
The concluding Chapter 7 presents a final summary of the work presented, and also discusses the role of the PX-FERM proteins in the broader context of cellular trafficking and membrane organisation both in health and disease. In particular potential regulatory interactions of the PX-FERM proteins are outlined, incorporating some preliminary unpublished data that will establish new directions for this work into the future.
Overall, this thesis defines a novel sub-family of PX molecules called the PX-FERM proteins, demonstrates the endosomal membrane attachment mechanism adopted by PX-FERM proteins, and explains and how once at the early endosomes PX-FERM proteins specifically engage sorting motifs present in the cargo molecules to direct their cell surface recycling. The structural arrangement of PX-FERM multiple domains in solution highlight a rigid modular architecture primed for multiple membrane and protein interactions synergistically.