The client-binding domain of the cochaperone Sgt2 has a helical-hand structure that binds a short hydrophobic helix

Abstract

The correct targeting and insertion of tail-anchored (TA) integral membrane proteins (IMP) is critical for cellular homeostasis. The mammalian protein SGTA, and its fungal homolog Sgt2 (Sgt2/A), binds hydrophobic clients and is the entry point for targeting of ER-bound TA IMPs. Here we reveal molecular details that underlie the mechanism of Sgt2/A binding to TA clients. We establish that the Sgt2/A C-terminal region is conserved but flexible, sufficient for client binding, and has functional and structural similarity to the DP domains of Sti1. A molecular model for Sgt2/A-C reveals a helical hand forming a hydrophobic groove, consistent with a higher affinity for TA clients with hydrophobic faces and a minimal length of 11 residues. Finally, we show that a hydrophobic face metric improves the predictions for TA localization in vivo. The structure and binding mechanism positions Sgt2/A into a broader class of helical-hand domains that reversibly bind hydrophobic clients.

The STI1‐domain is a flexible alpha‐helical fold with a hydrophobic groove

“…It is worth noting that low resolution structural studies have suggested that dimeric Sgt2 and HIP position their STI1-domains on opposite ends of a dimer molecule in the absence of client. 22,83 Still, given the noted flexibility in these proteins, 2,20 the possibility of cooperation remains, with the molecular details an open question.…”

“…[17][18][19] For the mammalian SGTA and its homologs, including yeast Sgt2, the STI1-domain directly binds to clients. 20 SGTA homologs play a number of roles, with the best characterized involving the targeting of tailanchored (TA) proteins to the ER membrane as a member of the Guided Entry of TA protein (GET) pathway. [21][22][23][24][25] SGTA has been suggested to play a role in the degradation of mislocalized membrane proteins in conjunction with the protein Bag6.…”

The Molecular Biodiversity of Protein Targeting and Protein Transport Related to the Endoplasmic Reticulum

“…Despite similar hydrophobicity thresholds of yeast Sgt2 and Get3, faster binding kinetics were assumed to increase the efficiency of Sgt2 in the rejection of unsuitable cargo [ 152 , 156 , 196 ]. Looking for features in yeast TMHs that allow better discrimination between different TA proteins, it was recently revealed that a so-called hydrophobic helical face geometry (the clustering of hydrophobic residues on one side of a TMH) was more successful in predicting the destined organelle than the overall hydrophobicity [ 205 ]. Strikingly, the finding was reflected by the results of a modeled structure and the binding behavior of yeast Sgt2.…”

“…How the binding and release of cargo is regulated without nucleotide binding and hydrolysis has yet to be determined. Recent computational modeling supported by biochemical data identified a similar hand structure for the binding of at least 11 residues, preferably containing clusters of leucine residues [ 205 , 208 ]. Since a typical TA-TMH has 18–20 amino acids, it was speculated that the two C-termini in a SGTA homodimer may occupy the full length of the signal, similar to the binding mode of calmodulin.…”

“…By contrast, human SGTA appears to be more ordered with a higher content of glutamine residues, which may explain the differences in client binding, despite a similar hydrophobicity of the binding groove. Here, the binding to a more ordered structure causes lower entropic costs which may set a lower hydrophobicity threshold and, indeed, human SGTA was found to be less selective [ 205 ]. These data match with other findings indicating that SGTA contributes to the biogenesis of TA proteins with TMHs of high and low hydrophobicity [ 161 ].…”