Structural insights into the dual nucleotide exchange and GDI displacement activity of SidM/DrrA

Abstract

GDP-bound prenylated Rabs, sequestered by GDI (GDP dissociation inhibitor) in the cytosol, are delivered to destined sub-cellular compartment and subsequently activated by GEFs (guanine nucleotide exchange factors) catalysing GDP-to-GTP exchange. The dissociation of GDI from Rabs is believed to require a GDF (GDI displacement factor). Only two RabGDFs, human PRA-1 and Legionella pneumophila SidM/DrrA, have been identified so far and the molecular mechanism of GDF is elusive. Here, we present the structure of a SidM/DrrA fragment possessing dual GEF and GDF activity in complex with Rab1. SidM/DrrA reconfigures the Switch regions of the GTPase domain of Rab1, as eukaryotic GEFs do toward cognate Rabs. Structure-based mutational analyses show that the surface of SidM/DrrA, catalysing nucleotide exchange, is involved in GDI1 displacement from prenylated Rab1:GDP. In comparison with an eukaryotic GEF TRAPP I, this bacterial GEF/GDF exhibits high binding affinity for Rab1 with GDP retained at the active site, which appears as the key feature for the GDF activity of the protein.

Figure 1

Interaction between SidM/DrrA and Rab1. (A) Structure of SidMcd–Rab1(1-175;N121I). SidM/DrrA interacts with the Switch I, Switch II and P-loop regions of Rab1. The mutated residue Ile121 (in sticks) of Rab1 is not involved in the intermolecular interaction. The inset shows that SidMcd is a flat molecule. (B) Interaction between SidM/DrrA and α2 of Rab1. The largely dislocated central region of Switch I forms α2 that is stabilized by mixed hydrophobic and hydrophilic interactions with a cleft of SidMcd. Tyr37 of Rab1 interacts tightly with multiple hydrophobic residues of SidMcd, including A435 that is mutated in this study (Figure 3A). An arrow pointing Ile38 is shown to emphasize that this residue is also involved in tight hydrophobic interaction with SidMcd, and thus is an important residue conferring recognition specificity (see text). (C) Stabilization of the nucleotide-free P-loop. The structures of isolated Rab1:GDP (PDB code: ) and Rab1 in complex with SidMcd are shown in the same orientation. The dotted lines, red sphere and green crosses denote polar interactions, Mg²⁺ and Mg²⁺-chelating water molecules. P-loop is in close contact with bound Mg²⁺-GDP, which is also involved in direct interactions with Switch I (top). On SidMcd binding, P-loop moves into the GDP-binding site and its conformation is stabilized by Switch II involved in multiple interactions with the loop, including the Lys21–Asp63 interaction (bottom).

Figure 2

Basis for the GDF activity of SidM/DrrA. (A) SidM/DrrA and RabGDI bind to the same surface of Rab1/Ypt1. The residues of Rab1 and Ypt1 involved in the intermolecular interaction (<4.0 Å) with SidMcd or RabGDI were identified from the presented structure and the RabGDI–p-Ypt1:GDP structure (PDB entry: ). These residues are indicated on the sequence alignment (top) by the triangles. They are mapped on Rab1 bound to SidMcd or on Ypt1:GDP bound to RabGDI (bottom). They are located on the same surface of Rab1/Ypt1 and largely overlap with each other. (B) Native-gel based GDF activity assay involving SidM/DrrA and GDI1–p-Rab1:GDP only. GDI1–p-Rab1:GDP (5 μM) was incubated with SidM/DrrA at 5, 20, 80 and 160 μM for 5 h at 25°C. Large molar excess of SidM/DrrA is required for considerable GDI1 displacement from p-Rab1. The GDI1–p-Rab1:GDP sample contained residual GDI1 (first lane) because supplemented GDI1 was not completely isolated from the complex. (C) A putative model for simultaneous interaction of p-Rab1 with SidM/DrrA and GDI1. The partly dissociated GDI1–p-Rab1:GDP complex is constructed based on the structures of Ypt1:GDP alone or bound to RabGDI. The flexible C-terminal segment of Ypt1 in the structure of Rab1GDI–p-Ypt1:GDP is indicated by a dotted line. NTD and CTD stand for the N- and C-terminal domain of SidM/DrrA, respectively. The K_d value for the interaction between RabGDI and unprenylated Ypt1:GDP (Pylypenko et al, 2006) is shown to indicate that the interaction between GDI1 and the GTPase domain of Rab1:GDP would be similarly weak. (D) Detection of the SidM/DrrA–p-Rab1 binary complex. In the presence of 0.1 mM EDTA, GDI1–p-Rab1:GDP (15 μM) was incubated for 5 h at 25°C with SidM/DrrA (WT) or a SidM/DrrA mutant (A435E), both at 60 μM. The top band was identified as the binary complex by mass spectrometry and immunoblotting using antibodies against the FLAG tag (linked to SidM/DrrA), GDI1 or Rab1 (data not shown). EDTA does not seem to affect the stability of the GDI1–p-Rab1GDP complex (lanes 1 and 2). Lane 5 represents a negative control using a SidM/DrrA mutant containing A435E mutation that results in reduced affinity for Rab1 as described in Figure 3.

Figure 3

Both GEF and GDF activities of SidM/DrrA are affected by a single mutation on its Rab1-interacting interface. (A) Structure-based design of a SidM/DrrA mutant. SidMcd is in surface representation and the Rab1 residues are represented by sticks. The red arrow indicates that glutamic acid at the position of A435 of SidM/DrrA is sterically incompatible for interacting with Rab1. (B) The A435E mutation reduces the binding affinity between SidMcd and Rab1(1-175):GDP. The ITC run and the deduced K_d are shown. (C) The A435E mutation reduces the GEF activity of SidMcd. In the presence of 0.2 mM GTP, Rab1:mant-GDP was incubated with wild-type SidMcd or SidMcd(A435E) at the indicated concentrations. The decreased fluorescence as a result of the mant-GDP-to-GTP exchange was continuously monitored and used to deduce the k_cat/K_M values (M⁻¹ s⁻¹), as reported previously (Murata et al, 2006). (D) On the native gel, the SidM/DrrA(A435E) mutant exhibits barely detectable GDF activity.

Figure 4

Effects of PC liposomes and GTP on GDI displacement by SidM/DrrA. (A) Effect of PC liposomes. GDI1–p-Rab1:GDP (5 μM) was incubated for 5 h with wild-type or A435E mutant SidM/DrrA at 5, 20 or 80 μM in the presence of 2 mM PC liposomes. After centrifugation, the supernatant and liposome fractions were visualized on a native or denaturing gel. The A435E mutant exhibits barely detectable GDI1 displacement. The right panel shows quantification of the band intensities of released GDI1 (lanes 1–4 in Figures 2B and 4A). Wild-type SidM/DrrA exhibits higher GDF activity compared with its activity in the absence of the liposomes. (B) Effect of the presence of both PC liposomes and GTP. GDI1–p-Rab1:GDP (5 μM) was incubated with wild-type SidM/DrrA (3.5 μM) up to 5 h in the presence of 2 mM PC liposomes and 1 mM GTP. The native gel shows complete displacement of GDI1 from p-Rab1 in 4 h with concomitant enrichment of p-Rab1 in the liposome fraction (denaturing gel). Lane 1 is for control showing GDI1–p-Rab1:GDP complex incubated for 5 h without added SidM/DrrA. (C) GDF activity assay involving the SidM/DrrA(A435E) and SidMcd(A435E) mutants. GDI1–p-Rab1:GDP (5 μM) was incubated with 5 μM of wild-type SidM/DrrA, SidM/DrrA(A435E), wild-type SidMcd or SidMcd(A435E) for 30 min in the presence of 2 mM PC liposomes and 1 mM GTP. Displacement of GDI1 is observable for the two mutants. Regardless of the mutation, full-length protein is more active than the central domain.

Figure 5

TRAPP I has no GDF activity toward RabGDI–p-Ypt1:GDP. Yeast RabGDI–p-Ypt1:GDP (5 μM) was incubated with yeast TRAPP I (5 μM) for 4 h in a buffer solution containing 20 mM Tris−HCl (pH 7.5), 150 mM NaCl in the absence or presence of PC liposomes (2 mM) and GTP (1 mM). On the native gel, RabGDI release is unobservable.

Figure 6

Measurement of the binding affinity between SidMcd and Rab1(1-175) with GDP retained. ITC was performed with increasing concentration of GDP, which should suppress the release of GDP in the two-step reaction shown at the top. The protein samples were prepared in a buffer solution containing 20 mM Tris−HCl (pH 7.5), 100 mM NaCl and 1 mM MgCl₂. A representative ITC run is shown and the K_d values measured at the different GDP concentrations are shown on the plot.