Structural basis for Arl3-specific release of myristoylated ciliary cargo from UNC119.

Access to the ciliary membrane for trans-membrane or membrane-associated proteins is a regulated process. Previously, we have shown that the closely homologous small G proteins Arl2 and Arl3 allosterically regulate prenylated cargo release from PDEδ. UNC119/HRG4 is responsible for ciliary delivery of myristoylated cargo. Here, we show that although Arl3 and Arl2 bind UNC119 with similar affinities, only Arl3 allosterically displaces cargo by accelerating its release by three orders of magnitude. Crystal structures of Arl3 and Arl2 in complex with UNC119a reveal the molecular basis of specificity. Contrary to previous structures of GTP-bound Arf subfamily proteins, the N-terminal amphipathic helix of Arl3·GppNHp is not displaced by the interswitch toggle but remains bound on the surface of the protein. Opposite to the mechanism of cargo release on PDEδ, this induces a widening of the myristoyl binding pocket. This leads us to propose that ciliary targeting of myristoylated proteins is not only dependent on nucleotide status but also on the cellular localization of Arl3.

Structural analysis of the Ras-like G protein MglA and its cognate GAP MglB and implications for bacterial polarity.

The bacterium Myxococcus xanthus uses a G protein cycle to dynamically regulate the leading/lagging pole polarity axis. The G protein MglA is regulated by its GTPase-activating protein (GAP) MglB, thus resembling Ras family proteins. Here, we show structurally and biochemically that MglA undergoes a dramatic, GDP-GTP-dependent conformational change involving a screw-type forward movement of the central ß2-strand, never observed in any other G protein. This movement and complex formation with MglB repositions the conserved residues Arg53 and Gln82 into the active site. Residues required for catalysis are thus not provided by the GAP MglB, but by MglA itself. MglB is a Roadblock/LC7 protein and functions as a dimer to stimulate GTP hydrolysis in a 2:1 complex with MglA. In vivo analyses demonstrate that hydrolysis mutants abrogate Myxococcus' ability to regulate its polarity axis changing the reversal behaviour from stochastic to oscillatory and that both MglA GTPase activity and MglB GAP catalysis are essential for maintaining a proper polarity axis.

The interplay between RPGR, PDEδ and Arl2/3 regulate the ciliary targeting of farnesylated cargo.

Defects in primary cilia result in human diseases known as ciliopathies. The retinitis pigmentosa GTPase regulator (RPGR), mutated in the most severe form of the eye disease, is located at the transition zone of the ciliary organelle. The RPGR-interacting partner PDEδ is involved in trafficking of farnesylated ciliary cargo, but the significance of this interaction is unknown. The crystal structure of the propeller domain of RPGR shows the location of patient mutations and how they perturb the structure. The RPGR·PDEδ complex structure shows PDEδ on a highly conserved surface patch of RPGR. Biochemical experiments and structural considerations show that RPGR can bind with high affinity to cargo-loaded PDEδ and exposes the Arl2/Arl3-binding site on PDEδ. On the basis of these results, we propose a model where RPGR is acting as a scaffold protein recruiting cargo-loaded PDEδ and Arl3 to release lipidated cargo into cilia.

Structural insights into the small G-protein Arl13B and implications for Joubert syndrome.

Ciliopathies are human diseases arising from defects in primary or motile cilia. The small G-protein Arl13B (ADP-ribosylation factor-like 13B) localizes to microtubule doublets of the ciliary axoneme and is mutated in Joubert syndrome. Its GDP/GTP mechanistic cycle and the effect of its mutations in patients with Joubert syndrome remain elusive. In the present study we applied high resolution structural and biochemical approaches to study Arl13B. The crystal structure of Chlamydomonas rheinhardtii Arl13B, comprising the G-domain and part of its unique C-terminus, revealed an incomplete active site, and together with biochemical data the present study accounts for the absence of intrinsic GTP hydrolysis by this protein. The structure shows that the residues representing patient mutations R79Q and R200C are involved in stabilizing important intramolecular interactions. Our studies suggest that Arg79 is crucial for the GDP/GTP conformational change by stabilizing the large two-residue register shift typical for Arf (ADP-ribosylation factor) and Arl subfamily proteins. A corresponding mutation in Arl3 induces considerable defects in effector and GAP (GTPase-activating protein) binding, suggesting a loss of Arl13B function in patients with Joubert syndrome.

Regulation of dynamic polarity switching in bacteria by a Ras-like G-protein and its cognate GAP.

The rod-shaped cells of the bacterium Myxococcus xanthus move uni-directionally and occasionally undergo reversals during which the leading/lagging polarity axis is inverted. Cellular reversals depend on pole-to-pole relocation of motility proteins that localize to the cell poles between reversals. We show that MglA is a Ras-like G-protein and acts as a nucleotide-dependent molecular switch to regulate motility and that MglB represents a novel GTPase-activating protein (GAP) family and is the cognate GAP of MglA. Between reversals, MglA/GTP is restricted to the leading and MglB to the lagging pole defining the leading/lagging polarity axis. For reversals, the Frz chemosensory system induces the relocation of MglA/GTP to the lagging pole causing an inversion of the leading/lagging polarity axis. MglA/GTP stimulates motility by establishing correct polarity of motility proteins between reversals and reversals by inducing their pole-to-pole relocation. Thus, the function of Ras-like G-proteins and their GAPs in regulating cell polarity is found not only in eukaryotes, but also conserved in bacteria.

Dimeric plant RhoGAPs are regulated by its CRIB effector motif to stimulate a sequential GTP hydrolysis.

RopGAPs are GTPase-activating proteins (GAPs) for plant Rho proteins (ROPs). The largest RopGAP family is characterized by the plant-specific combination of a classical RhoGAP domain and a Cdc42/Rac interactive binding (CRIB) motif, which, in animal and fungi, has never been found in GAPs but in effectors for Cdc42 and Rac1. Very little is known about the molecular mechanism of the RopGAP activity including the regulatory role of the CRIB motif. Previously, we have shown that they are dimeric and form a 2:2 complex with ROPs. Here, we analyze the kinetics of the GAP-mediated GTP hydrolysis of ROPs using wild-type and mutant RopGAP2 from Arabidopsis thaliana. For an efficient GAP activity, RopGAP2 requires both the catalytic Arg159 in its GAP domain indicating a similar catalytic machinery as in animal RhoGAPs and the CRIB motif, which mediates high affinity and specificity in binding. The dimeric RopGAP2 is unique in that it stimulates ROP·GTP hydrolysis in a sequential manner with a 10-fold difference between the hydrolysis rates of the two active sites. Using particular CRIB point and deletion mutants lead us to conclude that the sequential mechanism is likely due to steric hindrance induced by the Arg fingers and/or the CRIB motifs after binding of two ROP molecules.

Characterization of interactions of adapter protein RAPL/Nore1B with RAP GTPases and their role in T cell migration.

Using a model of integrin-triggered random migration of T cells, we show that stimulation of LFA-1 integrins leads to the activation of Rap1 and Rap2 small GTPases. We further show that Rap1 and Rap2 have distinct roles in adhesion and random migration of these cells and that an adapter protein from the Ras association domain family (Rassf), RAPL, has a role downstream of Rap2 in addition to its link to Rap1. Further characterization of the RAPL protein and its interactions with small GTPases from the Ras family shows that RAPL forms more stable complexes with Rap2 and classical Ras proteins compared with Rap1. The different interaction pattern of RAPL with Rap1 and Rap2 is not affected by the disruption of the C-terminal SARAH domain that we identified as the alpha-helical region responsible for RAPL dimerization in vitro and in cells. Based on mutagenesis and three-dimensional modeling, we propose that interaction surfaces in RAPL-Rap1 and RAPL-Rap2 complexes are different and that a single residue in the switch I region of Rap proteins (residue 39) contributes considerably to the different kinetics of these protein-protein interactions. Furthermore, the distinct role of Rap2 in migration of T cells is lost when this critical residue is converted to the residue present in Rap1. Together, these observations suggest a wider role for Rassf adapter protein RAPL and Rap GTPases in cell motility and show that subtle differences between highly similar Rap proteins could be reflected in distinct interactions with common effectors and their cellular function.

The xthA gene product of Archaeoglobus fulgidus is an unspecific DNase.

A thermostable enzyme from the hyperthermophilic sulphate-reducing archaeon, Archaeoglobus fulgidus, was expressed and characterized on the assumption that it is homologous to exonuclease III from Escherichia coli. Sequence similarity database searches were performed based on the amino acid sequence of exonuclease III. The 774 bp long gene was isolated from a culture sample and cloned into different vectors. Expression proved successful by transforming pET28_Af_Exo in Origami B(DE3) containing a tRNA plasmid with extra copies of argU, ileY and leuW tRNA genes as a host strain. The lack of thioredoxin reductase (trxB) and glutathione reductase (gor) in Origami B(DE3) allowed formation of disulfide bridges in the cytosol. Purification was performed by heat treatment of the soluble fraction at 80 degrees C for 30 min followed by a two-step ion exchange chromatography. The activity of the enzyme could be maintained. Optimal activity was achieved at 80 degrees C and at a pH of 7. Within the characterization of the protein we could not find any data verifying exonucleolytic activity in the presence of Mg2+ as described [Ankenbauer, W., Laue, F., Sobek, H., & Greif, M. (2000), patent number WO2001023583]. Instead strong DNA binding properties of the enzyme and nicking activities of double stranded DNA comparable to unspecific DNases could be observed. In contrast to exonuclease III from Escherichia coli, the xthA gene product of Archaeoglobus fulgidus is able to degrade supercoiled plasmids and shows no preferences for blunt or recessed 3'-termini of linear double stranded DNA. The enzyme is inhibited by EDTA and shows only weak activity when replacing Mg2+ with Ca2+ ions.