Specificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD.

To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.

Proteolysis of Rab32 by Salmonella GtgE induces an inactive GTPase conformation.

Rab GTPases are central regulators of intracellular vesicular trafficking. They are frequently targeted by bacterial pathogens through post-translational modifications. Salmonella typhimurium secretes the cysteine protease GtgE during infection, leading to a regioselective proteolytic cleavage of the regulatory switch I loop in the small GTPases of the Rab32 subfamily. Here, using a combination of biochemical methods, molecular dynamics simulations, NMR spectroscopy, and single-pair Förster resonance energy transfer, we demonstrate that the cleavage of Rab32 causes a local increase of conformational flexibility in both switch regions. Cleaved Rab32 maintains its ability to interact with the GDP dissociation inhibitor (GDI). Interestingly, the Rab32 cleavage enables GDI binding also with an active GTP-bound Rab32 in vitro. Furthermore, the Rab32 proteolysis provokes disturbance in the interaction with its downstream effector VARP. Thus, the proteolysis of Rab32 is not a globally degradative mechanism but affects various biochemical and structural properties of the GTPase in a diverse manner.