Coexpression, copurification, crystallization and preliminary X-ray analysis of a complex of ARL2-GTP and PDE delta.

The small GTPase ARL2 (from Mus musculus) and an effector protein, the delta subunit of human cGMP phosphodiesterase (hPDE delta), were coexpressed and copurified from Escherichia coli as a stable complex. Coexpression significantly increased the otherwise low yield of PDE delta production in E. coli. The complex, which contains ARL2 in the activated GTP-bound form, was crystallized in two forms. The first belongs to the monoclinic space group P2(1), with unit-cell parameters a = 48.1, b = 45.7, c = 74.7 A, beta = 94.0 degrees and one complex (39 kDa) in the asymmetric unit. Cryocooled crystals diffract to 2.3 A using synchrotron radiation. The micro-focused X-ray beam at beamline ID13 (ESRF) allowed the use of very small crystals, which helped to overcome twinning and enabled the identification of a molecular-replacement solution. The second form recrystallized from the first one after several months. These crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 44.5, b = 65.4, c = 104.4 A and one complex in the asymmetric unit. They diffracted to 1.8 A using synchrotron radiation.

Structural and biochemical properties show ARL3-GDP as a distinct GTP binding protein.

BACKGROUND: Based on sequence similarities, Arf-like (ARL) proteins have been assigned to the Arf subfamily of the superfamily of Ras-related GTP binding proteins. They have been identified in several isoforms in a wide variety of species. Their cellular function is unclear, but they are proposed to regulate intracellular transport. RESULTS: The 1.7 A crystal structure of murine ARL3-GDP provides a first insight into the structural features of this subgroup of Ar proteins. The N-terminal extension of ARL3 folds into an elongated loop region that is hydrophobically anchored onto the surface by burying 1440 A2. The features observed suggest that ARL3 releases its N terminus and undergoes a beta sheet register shift upon the binding of GTP. The structure and kinetic experiments with fluorescent mGDP demonstrate that tight GDP (but not GTP) binding is achieved in the absence of a magnesium ion. This is due to a lysine residue in the active site, close to the canonical Mg2+ site found in other GTP binding proteins. This is a distinct feature separating ARL2 and ARL3 from Arf proteins. CONCLUSION: The disturbed magnesium binding site and the independence of GDP coordination from the presence of Mg2+ separate ARL2 and ARL3 from Arf proteins. The D sheet register shift, which is similar to that of Arf, that is observed in the present structure, along with the postulated release of the N-terminal extension and the concomitant exposure of a patch of conserved hydrophobic residues in this region suggest that ARL proteins might be localized to target membranes upon exchange of GDP to GTP. Contrary to the situation in Arf, however, the conformational change to ARL-GTP does not require the presence of membranes and might thus be energetically unfavored. Together with the very low affinity described for the interaction of ARL3 with Mg-GTP, this suggests that ARL protein activation requires the presence of effectors stabilizing the GTP coordination rather than guanine nucleotide exchange factors (GEFs).

The delta subunit of rod specific cyclic GMP phosphodiesterase, PDE delta, interacts with the Arf-like protein Arl3 in a GTP specific manner.

Recently, we have shown that the delta subunit of the cGMP phosphodiesterase (PDE delta) interacts with the retinitis pigmentosa guanine regulator (RPGR). Here, using the two-hybrid system, we identify a member of the Arf-like protein family of Ras-related GTP-binding proteins, Arl3, that interacts with PDE delta. The interaction was verified by fluorescence spectroscopy and co-immunoprecipitation. Arl3 features an unusually low affinity for guanine nucleotides, with a KD of 24 nM for GDP and 48 microM for GTP. Fluorescence spectroscopy shows that PDE delta binds and specifically stabilizes the GTP-bound form of Arl3 by strongly decreasing the dissociation rate of GTP. Thus, PDE delta is an effector of Arl3 and could provide a novel nucleotide exchange mechanism by which PDE delta stabilizes Arl3 in its active GTP-bound form.