Conformational control of small GTPases by AMPylation

Abstract

Posttranslational modifications (PTMs) are important physiological means to regulate the activities and structures of central regulatory proteins in health and disease. Small GTPases have been recognized as important molecules that are targeted by PTMs during infections of mammalian cells by bacterial pathogens. The enzymes DrrA/SidM and AnkX fromLegionella pneumophilaAMPylate and phosphocholinate Rab1b during infection, respectively. Cdc42 is AMPylated by IbpA fromHistophilus somniat tyrosine 32 or by VopS fromVibrio parahaemolyticusat threonine 35. These modifications take place in the important regulatory switch I or switch II regions of the GTPases. Since Rab1b and Cdc42 are central regulators of intracellular vesicular trafficking and of the actin cytoskeleton, their modifications by bacterial pathogens have a profound impact on the course of infection. Here, we addressed the biochemical and structural consequences of GTPase AMPylation and phosphocholination. By combining biochemical experiments and NMR analysis, we demonstrate that AMPylation can overrule the activity state of Rab1b that is commonly dictated by binding to guanosine diphosphate or guanosine triphosphate. Thus, PTMs may exert conformational control over small GTPases and may add another previously unrecognized layer of activity control to this important regulatory protein family.

Monoclonal Anti-AMP Antibodies Are Sensitive and Valuable Tools for Detecting Patterns of AMPylation

“…The human Cdc42 1-179aa Q61L (referred to as Cdc42)-encoding DNA was cloned into a modified pGEX-4T-1 vector (GE Healthcare, Chicago, Illinois) as previously described (Barthelmes et al, 2020), resulting in a construct with a N-terminal glutathione S-transferase (GST) tag followed by the Tobacco Etch Virus (TEV) protease cleavage site. As previously described (Schoebel et al, 2009), the human Rab1b 3-174aa (referred to as Rab1b)-encoding DNA was cloned into a modified pMAL vector (New England Biolabs), resulting in a construct with a N-terminal hexahistidine (6xHis) tag, followed by maltose-binding protein (MBP) and the TEV protease cleavage site.…”

“…The human FICD 102-458aa E234G (referred to as FICD)-encoding DNA was cloned into a modified pMAL vector (New England Biolabs), resulting in a construct with a N-terminal 6xHis tag, followed by the HaloTag®, the TEV protease cleavage site and a Strep-tag® II. The Vibrio parahaemolyticus VopS 31-387aa (referred to as VopS)-encoding DNA was cloned into a modified pMAL vector (New England Biolabs) as previously described (Barthelmes et al, 2020), resulting in a construct with a N-terminal 6xHis tag, followed by MBP and the TEV protease cleavage site. The Histophilus somni IbpA 3483-3797aa I3455C (referred to as IbpA)-encoding DNA was cloned into a modified pSF vector (Oxford Genetics Ltd, Oxford, UK), resulting in a construct with a N-terminal decahistidine (10xHis) tag, followed by MBP, the TEV protease cleavage site and a 3xFLAG® tag.…”

“…Human pSer111 Rab1b was a kind gift of Dr. Sophie Vieweg and was produced as published before (Vieweg et al, 2020). Cdc42, VopS, Rab1b, DrrA and AnkX were expressed and purified as previously described (Barthelmes et al, 2020;Ernst et al, 2020;Müller et al, 2010;Schoebel et al, 2009). In brief, plasmids were transformed into chemically competent BL21 (DE3) cells (Cdc42, Rab1b, IbpA) or Lemo21 cells (VopS) or BL21-CodonPlus (DE3)-RIL cells (DrrA, Rab8a, AnkX) or Rosetta 2 cells (BiP, FICD) and protein was expressed in LB medium after induction with 0.5 mM isopropyl-βdithiogalactopyranoside (IPTG) for 20 h at 25 °C (Cdc42, Rab1b) or 20 °C (VopS, DrrA, Rab8a, AnkX, IbpA) or 23 °C (FICD) or 3 h at 37 °C (BiP).…”

Proteolysis of Rab32 by Salmonella GtgE induces an inactive GTPase conformation

“…Nonetheless, the indirect minor impact of GtgE-mediated cleavage on the switch II has a big consequence for the interaction of Rab32 with GDI resulting in the ability to bind to Rab32 cleaved :GDP and Rab32 cleaved :GTP. Recently, it has been shown that bacteria can lock Rab1b in the active state using AMPylation (Barthelmes et al, 2020). Consequently, Salmonella may use proteolysis to force Rab32 into an inactive-like conformation as demonstrated here by NMR.…”

Rab1-AMPylation by Legionella DrrA is allosterically activated by Rab1

“…The main effect of proximity consists in increasing the effective local concentration of the reactants to boost reaction rates and enable reactions that would not yield products in the absence of the concentration effect. To stabilise the DrrA ATase -Rab1b interface, we overexpressed various C-terminally His6-tagged Rab1b constructs (amino acids 3−174; Rab1b 3−174 with a Q67A Rab1b mutation that minimises Rab1b GTP hydrolysis) 22 , 23 bearing BrC6K at different positions (R69 Rab1b , T72 Rab1b , I73 Rab1b and R79 Rab1b ) in the vicinity of the AMPylation site (Y77 Rab1b ), together with an N-terminally Strep-tagged wild type (wt) DrrA ATase construct (amino acids 16−352; DrrA 16−352 ). Co-expression of Rab1b with BrC6K at position 69 Rab1b and wt DrrA ATase resulted in the formation of a covalently crosslinked complex, as confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) shift analysis via α-His6 and α-Strep western blotting (Fig.…”