Structural and functional insights into the E3 ligase, RNF126

RNF126 is an E3 ubiquitin ligase that collaborates with the BAG6 sortase complex to ubiquitinate hydrophobic substrates in the cytoplasm that are destined for proteasomal recycling. Composed of a trimeric complex of BAG6, TRC35 and UBL4A the BAG6 sortase is also associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. Here we solve the solution structure of the RNF126 zinc finger domain in complex with the BAG6 UBL domain. We also characterise an interaction between RNF126 and UBL4A and analyse the competition between SGTA and RNF126 for the N-terminal BAG6 binding site. This work sheds light on the sorting mechanism of the BAG6 complex and its accessory proteins which, together, decide the fate of stray hydrophobic proteins in the aqueous cytoplasm

Structures of Get3, Get4, and Get5 Provide New Models for TA Membrane Protein Targeting

The GET pathway, using several proteins (Gets 1–5 and probably Sgt2), posttranslationally conducts tail-anchored (TA) proteins to the endoplasmic reticulum (ER). At the ER, TA proteins are inserted into the lipid bilayer and then sorted and directed to their respective destinations in the secretory pathway. Until last year, there was no structural information on any of the GET components but now there are ten crystal structures of Get3 in a variety of nucleotide-bound states and conformations. The structures of Get4 and a portion of Get5 also emerged in 2010. This minireview provides a detailed comparison of the GET structures and discusses their mechanistic relevance to TA protein insertion. It also addresses the outstanding gaps in detailed molecular information on this system, including the structures of Get5, Sgt2, and the transmembrane complex comprising Get1 and Get2

Structural complexity of the co-chaperone SGTA: a conserved C-terminal region is implicated in dimerization and substrate quality control.

BACKGROUND: Protein quality control mechanisms are essential for cell health and involve delivery of proteins to specific cellular compartments for recycling or degradation. In particular, stray hydrophobic proteins are captured in the aqueous cytosol by a co-chaperone, the small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA), which facilitates the correct targeting of tail-anchored membrane proteins, as well as the sorting of membrane and secretory proteins that mislocalize to the cytosol and endoplasmic reticulum-associated degradation. Full-length SGTA has an unusual elongated dimeric structure that has, until now, evaded detailed structural analysis. The C-terminal region of SGTA plays a key role in binding a broad range of hydrophobic substrates, yet in contrast to the well-characterized N-terminal and TPR domains, there is a lack of structural information on the C-terminal domain. In this study, we present new insights into the conformation and organization of distinct domains of SGTA and show that the C-terminal domain possesses a conserved region essential for substrate processing in vivo. RESULTS: We show that the C-terminal domain region is characterized by α-helical propensity and an intrinsic ability to dimerize independently of the N-terminal domain. Based on the properties of different regions of SGTA that are revealed using cell biology, NMR, SAXS, Native MS, and EPR, we observe that its C-terminal domain can dimerize in the full-length protein and propose that this reflects a closed conformation of the substrate-binding domain. CONCLUSION: Our results provide novel insights into the structural complexity of SGTA and provide a new basis for mechanistic studies of substrate binding and release at the C-terminal region

Solution structure of the SGTA dimerisation domain and investigation of its interactions with the ubiquitin-like domains of BAG6 and UBL4A

BACKGROUND: The BAG6 complex resides in the cytosol and acts as a sorting point to target diverse hydrophobic protein substrates along their appropriate paths, including proteasomal degradation and ER membrane insertion. Composed of a trimeric complex of BAG6, TRC35 and UBL4A, the BAG6 complex is closely associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. METHODOLOGY AND PRINCIPAL FINDINGS: SGTA consists of an N-terminal dimerisation domain (SGTA_NT), a central tetratricopeptide repeat (TPR) domain, and a glutamine rich region towards the C-terminus. Here we solve a solution structure of the SGTA dimerisation domain and use biophysical techniques to investigate its interaction with two different UBL domains from the BAG6 complex. The SGTA_NT structure is a dimer with a tight hydrophobic interface connecting two sets of four alpha helices. Using a combination of NMR chemical shift perturbation, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) experiments we have biochemically characterised the interactions of SGTA with components of the BAG6 complex, the ubiquitin-like domain (UBL) containing proteins UBL4A and BAG6. We demonstrate that the UBL domains from UBL4A and BAG6 directly compete for binding to SGTA at the same site. Using a combination of structural and interaction data we have implemented the HADDOCK protein-protein interaction docking tool to generate models of the SGTA-UBL complexes. SIGNIFICANCE: This atomic level information contributes to our understanding of the way in which hydrophobic proteins have their fate decided by the collaboration between SGTA and the BAG6 complex

Structural and Functional Insights into Small, Glutamine-Rich, Tetratricopeptide Repeat Protein Alpha

SGTA is a co-chaperone that interacts with molecular chaperones and steroid receptor complexes and plays an important role in various cellular pathways. It consists of three structural domains with individual functions, an N-terminal dimerisation domain (SGTA_NT) that assists protein sorting pathways, a central tetratricopeptide repeat (TPR) domain that interacts with heat-shock proteins and a C-terminal glutamine rich region that binds hydrophobic substrates. A range of biophysical techniques has been employed to characterize its structure and to investigate its interactions with binding partners. SGTA interacts with the androgen receptor and other steroid receptor complexes and has been shown to be linked to hormonally induced disease states. Therefore, a full structure of SGTA and further investigation of its function as a molecular co-chaperone could provide us with useful insights into the mechanisms of related pathologies. This review describes how some structural features of SGTA have been elucidated, and what this has uncovered about its function as a co-chaperone. A brief background on the structure and function of SGTA is given, highlighting its importance to biomedicine and related fields. The current level of knowledge and what remains to be understood about the structure and function of SGTA is summarised, discussing the potential direction of future research