An Actin Network Dispatches Ciliary GPCRs into Extracellular Vesicles to Modulate Signaling.

Signaling receptors dynamically exit cilia upon activation of signaling pathways such as Hedgehog. Here, we find that when activated G protein-coupled receptors (GPCRs) fail to undergo BBSome-mediated retrieval from cilia back into the cell, these GPCRs concentrate into membranous buds at the tips of cilia before release into extracellular vesicles named ectosomes. Unexpectedly, actin and the actin regulators drebrin and myosin 6 mediate ectosome release from the tip of cilia. Mirroring signal-dependent retrieval, signal-dependent ectocytosis is a selective and effective process that removes activated signaling molecules from cilia. Congruently, ectocytosis compensates for BBSome defects as ectocytic removal of GPR161, a negative regulator of Hedgehog signaling, permits the appropriate transduction of Hedgehog signals in Bbs mutants. Finally, ciliary receptors that lack retrieval determinants such as the anorexigenic GPCR NPY2R undergo signal-dependent ectocytosis in wild-type cells. Our data show that signal-dependent ectocytosis regulates ciliary signaling in physiological and pathological contexts.

Nucleotide binding and conformational switching in the hexameric ring of a AAA+ machine.

ClpX, a AAA+ ring homohexamer, uses the energy of ATP binding and hydrolysis to power conformational changes that unfold and translocate target proteins into the ClpP peptidase for degradation. In multiple crystal structures, some ClpX subunits adopt nucleotide-loadable conformations, others adopt unloadable conformations, and each conformational class exhibits substantial variability. Using mutagenesis of individual subunits in covalently tethered hexamers together with fluorescence methods to assay the conformations and nucleotide-binding properties of these subunits, we demonstrate that dynamic interconversion between loadable and unloadable conformations is required to couple ATP hydrolysis by ClpX to mechanical work. ATP binding to different classes of subunits initially drives staged allosteric changes, which set the conformation of the ring to allow hydrolysis and linked mechanical steps. Subunit switching between loadable and unloadable conformations subsequently isomerizes or resets the configuration of the nucleotide-loaded ring and is required for mechanical function.

Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine.

ClpX is a AAA+ machine that uses the energy of ATP binding and hydrolysis to unfold native proteins and translocate unfolded polypeptides into the ClpP peptidase. The crystal structures presented here reveal striking asymmetry in ring hexamers of nucleotide-free and nucleotide-bound ClpX. Asymmetry arises from large changes in rotation between the large and small AAA+ domains of individual subunits. These differences prevent nucleotide binding to two subunits, generate a staggered arrangement of ClpX subunits and pore loops around the hexameric ring, and provide a mechanism for coupling conformational changes caused by ATP binding or hydrolysis in one subunit to flexing motions of the entire ring. Our structures explain numerous solution studies of ClpX function, predict mechanisms for pore elasticity during translocation of irregular polypeptides, and suggest how repetitive conformational changes might be coupled to mechanical work during the ATPase cycle of ClpX and related molecular machines.

Loss of the BBSome perturbs endocytic trafficking and disrupts virulence of Trypanosoma brucei.

Cilia (eukaryotic flagella) are present in diverse eukaryotic lineages and have essential motility and sensory functions. The cilium's capacity to sense and transduce extracellular signals depends on dynamic trafficking of ciliary membrane proteins. This trafficking is often mediated by the Bardet-Biedl Syndrome complex (BBSome), a protein complex for which the precise subcellular distribution and mechanisms of action are unclear. In humans, BBSome defects perturb ciliary membrane protein distribution and manifest clinically as Bardet-Biedl Syndrome. Cilia are also important in several parasites that cause tremendous human suffering worldwide, yet biology of the parasite BBSome remains largely unexplored. We examined BBSome functions in Trypanosoma brucei, a flagellated protozoan parasite that causes African sleeping sickness in humans. We report that T. brucei BBS proteins assemble into a BBSome that interacts with clathrin and is localized to membranes of the flagellar pocket and adjacent cytoplasmic vesicles. Using BBS gene knockouts and a mouse infection model, we show the T. brucei BBSome is dispensable for flagellar assembly, motility, bulk endocytosis, and cell viability but required for parasite virulence. Quantitative proteomics reveal alterations in the parasite surface proteome of BBSome mutants, suggesting that virulence defects are caused by failure to maintain fidelity of the host-parasite interface. Interestingly, among proteins altered are those with ubiquitination-dependent localization, and we find that the BBSome interacts with ubiquitin. Collectively, our data indicate that the BBSome facilitates endocytic sorting of select membrane proteins at the base of the cilium, illuminating BBSome roles at a critical host-pathogen interface and offering insights into BBSome molecular mechanisms.

Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation.

The HslUV proteolytic machine consists of HslV, a double-ring self-compartmentalized peptidase, and one or two AAA+ HslU ring hexamers that hydrolyze ATP to power the unfolding of protein substrates and their translocation into the proteolytic chamber of HslV. Here, we use genetic tethering and disulfide bonding strategies to construct HslU pseudohexamers containing mixtures of ATPase active and inactive subunits at defined positions in the hexameric ring. Genetic tethering impairs HslV binding and degradation, even for pseudohexamers with six active subunits, but disulfide-linked pseudohexamers do not have these defects, indicating that the peptide tether interferes with HslV interactions. Importantly, pseudohexamers containing different patterns of hydrolytically active and inactive subunits retain the ability to unfold protein substrates and/or collaborate with HslV in their degradation, supporting a model in which ATP hydrolysis and linked mechanical function in the HslU ring operate by a probabilistic mechanism.

Coordinated gripping of substrate by subunits of a AAA+ proteolytic machine.

Hexameric ATP-dependent proteases and protein remodeling machines use conserved loops that line the axial pore to apply force to substrates during the mechanical processes of protein unfolding and translocation. Whether loops from multiple subunits act independently or coordinately in these processes is a critical aspect of the mechanism but is currently unknown for any AAA+ machine. By studying covalently linked hexamers of the Escherichia coli ClpX unfoldase bearing different numbers and configurations of wild-type and mutant pore loops, we show that loops function synergistically, and the number of wild-type loops required for efficient degradation is dependent on the stability of the protein substrate. Our results support a mechanism in which a power stroke initiated in one subunit of the ClpX hexamer results in the concurrent movement of all six pore loops, which coordinately grip and apply force to the substrate.

Expanding the ligandable proteome by paralog hopping with covalent probes.

More than half of the ~20,000 protein-encoding human genes have at least one paralog. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to a subset of paralogous proteins. Here, we explore whether such covalent compound-cysteine interactions can be used to discover ligandable pockets in paralogs that lack the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we mutated the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling (ABPP) that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide-N112C-CCNE1 interaction into a NanoBRET-ABPP assay capable of identifying compounds that reversibly inhibit both N112C- and WT-CCNE1:CDK2 complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings thus provide a roadmap for leveraging electrophile-cysteine interactions to extend the ligandability of the proteome beyond covalent chemistry.

Glycine dimerization motif in the N-terminal transmembrane domain of the high density lipoprotein receptor SR-BI required for normal receptor oligomerization and lipid transport.

Scavenger receptor class B, type I (SR-BI), a CD36 superfamily member, is an oligomeric high density lipoprotein (HDL) receptor that mediates negatively cooperative HDL binding and selective lipid uptake. We identified in the N-terminal transmembrane (N-TM) domain of SR-BI a conserved glycine dimerization motif, G(15)X(2)G(18)X(3)AX(2)G(25), of which the submotif G(18)X(3)AX(2)G(25) significantly contributes to homodimerization and lipid uptake activity. SR-BI variants were generated by mutations (single or multiple Gly → Leu substitutions) or by replacing the N-TM domain with those from other CD36 superfamily members containing (croquemort) or lacking (lysosomal integral membrane protein (LIMP) II) this glycine motif (chimeras). None of the SR-BI variants exhibited altered surface expression (based on antibody binding) or HDL binding. However, the G15L/G18L/G25L triple mutant exhibited reductions in cell surface homo-oligomerization (>10-fold) and the rate of selective lipid uptake (∼ 2-fold). Gly(18) and Gly(25) were necessary for normal lipid uptake activity of SR-BI and the SR-BI/croquemort chimera. The lipid uptake activity of the glycine motif-deficient SR-BI/LIMP II chimera was low but could be increased by introducing glycines at positions 18 and 25. The rate of lipid uptake mediated by SR-BI/LIMP II chimeras was proportional to the extent of receptor oligomerization. Thus, the glycine dimerization motif G(18)X(3)AX(2)G(25) in the N-TM domain of SR-BI contributes substantially to the homo-oligomerization and lipid transport activity of SR-BI but does not influence the negative cooperativity of HDL binding. Oligomerization-independent binding cooperativity suggests that classic allostery is not involved and that the negative cooperativity is probably the consequence of a "lattice effect" (interligand steric interference accompanying binding to adjacent receptors).

Design and Validation of Inducible TurboCARs with Tunable Induction and Combinatorial Cytokine Signaling.

Although cytokine support can enhance CAR T-cell function, coadministering cytokines or engineering CAR T cells to secrete cytokines can result in toxicities. To mitigate these safety risks, we engineered iTurboCAR T cells that coexpress a novel inducible Turbo (iTurbo) cytokine signaling domain. iTurbo domains consist of modular components that are customizable to a variety of activating inputs, as well as cytokine signaling outputs multiplexable for combinatorial signaling outcomes. Unlike most canonical cytokine receptors that are heterodimeric, iTurbo domains leverage a compact, homodimeric design that minimizes viral vector cargo. Using an iTurbo domain activated by the clinically validated dimerizer, AP1903, homodimeric iTurbo domains instigated signaling that mimicked the endogenous heterodimeric cytokine receptor. Different iTurbo domains programmed iTurboCAR T cells toward divergent phenotypes and resulted in improved antitumor efficacy. iTurbo domains, therefore, offer the flexibility for user-programmable signaling outputs, permitting control over cellular phenotype and function while minimizing viral cargo footprint.

Ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia.

Regulated trafficking of G protein-coupled receptors (GPCRs) controls cilium-based signaling pathways. ß-Arrestin, a molecular sensor of activated GPCRs, and the BBSome, a complex of Bardet-Biedl syndrome (BBS) proteins, are required for the signal-dependent exit of ciliary GPCRs, but the functional interplay between ß-arrestin and the BBSome remains elusive. Here we find that, upon activation, ciliary GPCRs become tagged with ubiquitin chains comprising K63 linkages (UbK63) in a ß-arrestin-dependent manner before BBSome-mediated exit. Removal of ubiquitin acceptor residues from the somatostatin receptor 3 (SSTR3) and from the orphan GPCR GPR161 demonstrates that ubiquitination of ciliary GPCRs is required for their regulated exit from cilia. Furthermore, targeting a UbK63-specific deubiquitinase to cilia blocks the exit of GPR161, SSTR3, and Smoothened (SMO) from cilia. Finally, ubiquitinated proteins accumulate in cilia of mammalian photoreceptors and Chlamydomonas cells when BBSome function is compromised. We conclude that Ub chains mark GPCRs and other unwanted ciliary proteins for recognition by the ciliary exit machinery.

The proteasome 19S cap and its ubiquitin receptors provide a versatile recognition platform for substrates.

Proteins are targeted to the proteasome by the attachment of ubiquitin chains, which are markedly varied in structure. Three proteasome subunits-Rpn10, Rpn13, and Rpn1-can recognize ubiquitin chains. Here we report that proteins with single chains of K48-linked ubiquitin are targeted for degradation almost exclusively through binding to Rpn10. Rpn1 can act as a co-receptor with Rpn10 for K63 chains and for certain other chain types. Differences in targeting do not correlate with chain affinity to receptors. Surprisingly, in steady-state assays Rpn13 retarded degradation of various single-chain substrates. Substrates with multiple short ubiquitin chains can be presented for degradation by any of the known receptors, whereas those targeted to the proteasome through a ubiquitin-like domain are degraded most efficiently when bound by Rpn13 or Rpn1. Thus, the proteasome provides an unexpectedly versatile binding platform that can recognize substrates targeted for degradation by ubiquitin chains differing greatly in length and topology.

The Molecular Architecture of Native BBSome Obtained by an Integrated Structural Approach.

The unique membrane composition of cilia is maintained by a diffusion barrier at the transition zone that is breached when the BBSome escorts signaling receptors out of cilia. Understanding how the BBSome removes proteins from cilia has been hampered by a lack of structural information. Here, we present a nearly complete Cα model of BBSome purified from cow retina. The model is based on a single-particle cryo-electron microscopy density map at 4.9-Å resolution that was interpreted with the help of comprehensive Rosetta-based structural modeling constrained by crosslinking mass spectrometry data. We find that BBSome subunits have a very high degree of interconnectivity, explaining the obligate nature of the complex. Furthermore, like other coat adaptors, the BBSome exists in an autoinhibited state in solution and must thus undergo a conformational change upon recruitment to membranes by the small GTPase ARL6/BBS3. Our model provides the first detailed view of the machinery enabling ciliary exit.

A dynamic model for replication protein A (RPA) function in DNA processing pathways.

Processing of DNA in replication, repair and recombination pathways in cells of all organisms requires the participation of at least one major single-stranded DNA (ssDNA)-binding protein. This protein protects ssDNA from nucleolytic damage, prevents hairpin formation and blocks DNA reannealing until the processing pathway is successfully completed. Many ssDNA-binding proteins interact physically and functionally with a variety of other DNA processing proteins. These interactions are thought to temporally order and guide the parade of proteins that 'trade places' on the ssDNA, a model known as 'hand-off', as the processing pathway progresses. How this hand-off mechanism works remains poorly understood. Recent studies of the conserved eukaryotic ssDNA-binding protein replication protein A (RPA) suggest a novel mechanism by which proteins may trade places on ssDNA by binding to RPA and mediating conformation changes that alter the ssDNA-binding properties of RPA. This article reviews the structure and function of RPA, summarizes recent studies of RPA in DNA replication and other DNA processing pathways, and proposes a general model for the role of RPA in protein-mediated hand-off.

BBSome trains remove activated GPCRs from cilia by enabling passage through the transition zone.

A diffusion barrier at the transition zone enables the compartmentalization of signaling molecules by cilia. The BBSome and the small guanosine triphosphatase Arl6, which triggers BBSome coat polymerization, are required for the exit of activated signaling receptors from cilia, but how diffusion barriers are crossed when membrane proteins exit cilia remains to be determined. In this study, we found that activation of the ciliary G protein-coupled receptors (GPCRs) Smoothened and SSTR3 drove the Arl6-dependent assembly of large, highly processive, and cargo-laden retrograde BBSome trains. Single-molecule imaging revealed that the assembly of BBSome trains enables the lateral transport of ciliary GPCRs across the transition zone. However, the removal of activated GPCRs from cilia was inefficient because a second periciliary diffusion barrier was infrequently crossed. We conclude that exit from cilia is a two-step process in which BBSome/Arl6 trains first move activated GPCRs through the transition zone before a periciliary barrier can be crossed.

Structure of the 4-1BB/4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab.

4-1BB (CD137, TNFRSF9) is an inducible costimulatory receptor expressed on activated T cells. Clinical trials of two agonist antibodies, utomilumab (PF-05082566) and urelumab (BMS-663513), are ongoing in multiple cancer indications, and both antibodies demonstrate distinct activities in the clinic. To understand these differences, we solved structures of the human 4-1BB/4-1BBL complex, the 4-1BBL trimer alone, and 4-1BB bound to utomilumab or urelumab. The 4-1BB/4-1BBL complex displays a unique interaction between receptor and ligand when compared with other TNF family members. Furthermore, our ligand-only structure differs from previously published data. Utomilumab, a ligand-blocking antibody, binds 4-1BB between CRDs 3 and 4. In contrast, urelumab binds 4-1BB CRD-1, away from the ligand binding site. Finally, cell-based assays demonstrate utomilumab is a milder agonist than urelumab. Collectively, our data provide a deeper understanding of the 4-1BB signaling complex, providing a template for future development of next generation 4-1BB targeted biologics.

Structural basis for membrane targeting of the BBSome by ARL6.

The BBSome is a coat-like ciliary trafficking complex composed of proteins mutated in Bardet-Biedl syndrome (BBS). A critical step in BBSome-mediated sorting is recruitment of the BBSome to membranes by the GTP-bound Arf-like GTPase ARL6. We have determined crystal structures of Chlamydomonas reinhardtii ARL6-GDP, ARL6-GTP and the ARL6-GTP-BBS1 complex. The structures demonstrate how ARL6-GTP binds the BBS1 ß-propeller at blades 1 and 7 and explain why GTP- but not GDP-bound ARL6 can recruit the BBSome to membranes. Single point mutations in the ARL6-GTP-BBS1 interface abolish the interaction of ARL6 with the BBSome and prevent the import of BBSomes into cilia. Furthermore, we show that BBS1 with the M390R mutation, responsible for 30% of all reported BBS disease cases, fails to interact with ARL6-GTP, thus providing a molecular rationale for patient pathologies.

Mechanochemical basis of protein degradation by a double-ring AAA+ machine.

Molecular machines containing double or single AAA+ rings power energy-dependent protein degradation and other critical cellular processes, including disaggregation and remodeling of macromolecular complexes. How the mechanical activities of double-ring and single-ring AAA+ enzymes differ is unknown. Using single-molecule optical trapping, we determine how the double-ring ClpA enzyme from Escherichia coli, in complex with the ClpP peptidase, mechanically degrades proteins. We demonstrate that ClpA unfolds some protein substrates substantially faster than does the single-ring ClpX enzyme, which also degrades substrates in collaboration with ClpP. We find that ClpA is a slower polypeptide translocase and that it moves in physical steps that are smaller and more regular than steps taken by ClpX. These direct measurements of protein unfolding and translocation define the core mechanochemical behavior of a double-ring AAA+ machine and provide insight into the degradation of proteins that unfold via metastable intermediates.

The intraflagellar transport protein IFT27 promotes BBSome exit from cilia through the GTPase ARL6/BBS3.

The sorting of signaling receptors into and out of cilia relies on the BBSome, a complex of Bardet-Biedl syndrome (BBS) proteins, and on the intraflagellar transport (IFT) machinery. GTP loading onto the Arf-like GTPase ARL6/BBS3 drives assembly of a membrane-apposed BBSome coat that promotes cargo entry into cilia, yet how and where ARL6 is activated remains elusive. Here, we show that the Rab-like GTPase IFT27/RABL4, a known component of IFT complex B, promotes the exit of BBSome and associated cargoes from cilia. Unbiased proteomics and biochemical reconstitution assays show that, upon disengagement from the rest of IFT-B, IFT27 directly interacts with the nucleotide-free form of ARL6. Furthermore, IFT27 prevents aggregation of nucleotide-free ARL6 in solution. Thus, we propose that IFT27 separates from IFT-B inside cilia to promote ARL6 activation, BBSome coat assembly, and subsequent ciliary exit, mirroring the process by which BBSome mediates cargo entry into cilia.

Engineering fluorescent protein substrates for the AAA+ Lon protease.

AAA+ proteases, such as Escherichia coli Lon, recognize protein substrates by binding to specific peptide degrons and then unfold and translocate the protein into an internal degradation chamber for proteolysis. For some AAA+ proteases, attaching specific degrons to the N- or C-terminus of green fluorescent protein (GFP) generates useful substrates, whose unfolding and degradation can be monitored by loss of fluorescence, but Lon fails to degrade appropriately tagged GFP variants at a significant rate. Here, we demonstrate that Lon catalyzes robust unfolding and degradation of circularly permuted variants of GFP with a ß20 degron appended to the N terminus or a sul20 degron appended to the C terminus. Lon degradation of non-permuted GFP-sul20 is very slow, in part because the enzyme cannot efficiently extract the degron-proximal C-terminal ß-strand to initiate denaturation. The circularly permuted GFP substrates described here allow convenient high-throughput assays of the kinetics of Lon degradation in vitro and also permit assays of Lon proteolysis in vivo.