Alfred Wittinghofer

The BBSome is a heterooctameric protein complex that plays a central role in primary cilia homeostasis. Its malfunction causes the severe ciliopathy Bardet-Biedl syndrome (BBS). The complex acts as a cargo adapter that recognizes signaling proteins such as GPCRs and links them to the intraflagellar transport machinery. The underlying mechanism is poorly understood. Here we present a high-resolution cryo-EM structure of a human heterohexameric core subcomplex of the BBSome. The structure reveals the architecture of the complex in atomic detail. It explains how the subunits interact with each other and how disease-causing mutations hamper this interaction. The complex adopts a conformation that is open for binding to membrane-associated GTPase Arl6 and a large positively charged patch likely strengthens the interaction with the membrane. A prominent negatively charged cleft at the center of the complex is likely involved in binding of positively charged signaling sequences of cargo proteins.

The BBSome is a heterooctameric protein complex that plays a central role in primary cilia homeostasis. Its malfunction causes the severe ciliopathy Bardet-Biedl syndrome (BBS). The complex acts as a cargo adapter that recognizes signaling proteins such as GPCRs and links them to the intraflagellar transport machinery. The underlying mechanism is poorly understood. Here we present a high-resolution cryo-EM structure of a human heterohexameric core subcomplex of the BBSome. The structure reveals the architecture of the complex in atomic detail. It explains how the subunits interact with each other and how disease-causing mutations hamper this interaction. The complex adopts a conformation that is open for binding to membrane-associated GTPase Arl6 and a large positively charged patch likely strengthens the interaction with the membrane. A prominent negatively charged cleft at the center of the complex is likely involved in binding of positively charged signaling sequences of cargo proteins.

Retinitis pigmentosa 2 (RP2) is the causative gene for a form of X-linked retinal degeneration. RP2 was previously shown to have GTPase-activating protein (GAP) activity towards the small GTPase ARL3 via its N-terminus, but the function of the C-terminus remains elusive. Here, we report a novel interaction between RP2 and osteoclast-stimulating factor 1 (OSTF1), an intracellular protein that indirectly enhances osteoclast formation and activity and is a negative regulator of cell motility. Moreover, this interaction is abolished by a human pathogenic mutation in RP2. We utilized a structure-based approach to pinpoint the binding interface to a strictly conserved cluster of residues on the surface of RP2 that spans both the C- and N-terminal domains of the protein, and which is structurally distinct from the ARL3-binding site. In addition, we show that RP2 is a positive regulator of cell motility in vitro, recruiting OSTF1 to the cell membrane and preventing its interaction with the migration regulator Myo1E.

Cilia are small, antenna-like structures on the surface of eukaryotic cells that harbor a unique set of sensory proteins, including GPCRs and other membrane proteins. The transport of these proteins involves the BBSome, an eight-membered protein complex that is recruited to ciliary membranes by the G-protein Arl6. BBSome malfunction leads to Bardet-Biedl syndrome, a ciliopathy with severe consequences. Short ciliary targeting sequences (CTS) have been identified that trigger the transport of ciliary proteins. However, mechanistic studies that relate ciliary targeting to BBSome binding are missing. Here we used heterologously expressed BBSome subcomplexes to analyze the complex architecture and to investigate the binding of GPCRs and other receptors to the BBSome. A stable heterohexameric complex was identified that binds to GPCRs with interactions that only partially overlap with previously described CTS, indicating a more complex recognition than anticipated. Arl6•GTP does not affect these interactions, suggesting no direct involvement in cargo loading/unloading.

The inositol polyphosphate 5′-phosphatase E (INPP5E) localizes to cilia. We showed that the carrier protein phosphodiesterase 6 delta subunit (PDE6δ) mediates the sorting of farnesylated INPP5E into cilia due to high affinity binding and release by the ADP-ribosylation factor (Arf)-like protein Arl3·GTP. However, the dynamics of INPP5E transport into and inside the ciliary compartment are not fully understood. Here, we investigate the movement of INPP5E using live cell fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) analysis. We show that PDE6δ and the dynein transport system are essential for ciliary sorting and entry of INPP5E. However, its innerciliary transport is regulated solely by the intraflagellar transport (IFT) system, independent from PDE6δ activity and INPP5E farnesylation. By contrast, movement of Arl3 into and within cilia occurs freely by diffusion and IFT-independently. The farnesylation defective INPP5E CaaX box mutant loses the exclusive ciliary localization. The accumulation of this mutant at centrioles after photobleaching suggests an affinity trap mechanism for ciliary entry, that in case of the wild type is overcome by the interaction with PDE6δ. Collectively, we postulate a three-step mechanism regulating ciliary localization of INPP5E, consisting of farnesylation- and PDE6δ-mediated targeting, INPP5E-PDE6δ complex diffusion into the cilium with transfer to the IFT system, and retention inside cilia.

Arl2 and Arl3 are Arf-like small GTP-binding proteins of the Arf subfamily of the Ras superfamily. Despite their structural similarity and sharing of many interacting partners, Arl2 and Arl3 have different biochemical properties and biological functions. Growing evidence suggest that Arl2 and Arl3 play a fundamental role as regulators of trafficking of lipid modified proteins between different compartments. Here we highlight the similarities and differences between these 2 homologous proteins and discuss the sorting mechanism of lipidated cargo into the ciliary compartment through the carriers PDE6δ and Unc119 and the release factors Arl2 and Arl3.

The controlled derivatization of natural products is of great importance for their use in drug discovery. The ideally rapid generation of compound libraries for structure–activity relationship studies is of particular concern. We here use modified biosynthesis for the generation of such a library of reduced polyketides to interfere with the oncogenic KRas pathway. The polyketide is derivatized via side chain alteration, and variations in its redox pattern and in its backbone chain length through manipulation in the corresponding polyketide synthase. Structural and biophysical analyses revealed the nature of the interaction between the polyketides and KRas-interacting protein PDE6δ. Non-natural polyketides with low nanomolar affinity to PDE6δ were identified.

The phosphodiesterase 6 delta subunit (PDE6δ) shuttles several farnesylated cargos between membranes. The cargo sorting mechanism between cilia and other compartments is not understood. Here we show using the inositol polyphosphate 5′-phosphatase E (INPP5E) and the GTP-binding protein (Rheb) that cargo sorting depends on the affinity towards PDE6δ and the specificity of cargo release. High-affinity cargo is exclusively released by the ciliary transport regulator Arl3, while low-affinity cargo is released by Arl3 and its non-ciliary homologue Arl2. Structures of PDE6δ/cargo complexes reveal the molecular basis of the sorting signal which depends on the residues at the −1 and −3 positions relative to farnesylated cysteine. Structure-guided mutation allows the generation of a low-affinity INPP5E mutant which loses exclusive ciliary localization. We postulate that the affinity to PDE6δ and the release by Arl2/3 in addition to a retention signal are the determinants for cargo sorting and enrichment at its destination.

This study shows that the prenylated C‐terminus of RPGR can bind to PDE6δ with high affinity, suggesting two distinct binding sites of the RPGR/PDE6δ complex. The serine residue at the −3 position relative to the prenylated cysteine seems to play a key role in defining the selectivity of PDE6δ towards ciliary prenylated cargo.