Structural Principles in Robo Activation and Auto-inhibition.

Proper brain function requires high-precision neuronal expansion and wiring, processes controlled by the transmembrane Roundabout (Robo) receptor family and their Slit ligands. Despite their great importance, the molecular mechanism by which Robos' switch from "off" to "on" states remains unclear. Here, we report a 3.6 Å crystal structure of the intact human Robo2 ectodomain (domains D1-8). We demonstrate that Robo cis dimerization via D4 is conserved through hRobo1, 2, and 3 and the C. elegans homolog SAX-3 and is essential for SAX-3 function in vivo. The structure reveals two levels of auto-inhibition that prevent premature activation: (1) cis blocking of the D4 dimerization interface and (2) trans interactions between opposing Robo receptors that fasten the D4-blocked conformation. Complementary experiments in mouse primary neurons and C. elegans support the auto-inhibition model. These results suggest that Slit stimulation primarily drives the release of Robo auto-inhibition required for dimerization and activation.

Ubiquitin-independent proteasomal degradation driven by C-degron pathways.

Although most eukaryotic proteins are targeted for proteasomal degradation by ubiquitination, a subset have been demonstrated to undergo ubiquitin-independent proteasomal degradation (UbInPD). However, little is known about the molecular mechanisms driving UbInPD and the degrons involved. Utilizing the GPS-peptidome approach, a systematic method for degron discovery, we found thousands of sequences that promote UbInPD; thus, UbInPD is more prevalent than currently appreciated. Furthermore, mutagenesis experiments revealed specific C-terminal degrons required for UbInPD. Stability profiling of a genome-wide collection of human open reading frames identified 69 full-length proteins subject to UbInPD. These included REC8 and CDCA4, proteins which control proliferation and survival, as well as mislocalized secretory proteins, suggesting that UbInPD performs both regulatory and protein quality control functions. In the context of full-length proteins, C termini also play a role in promoting UbInPD. Finally, we found that Ubiquilin family proteins mediate the proteasomal targeting of a subset of UbInPD substrates.

A duplex structure of SARM1 octamers stabilized by a new inhibitor.

In recent years, there has been growing interest in SARM1 as a potential breakthrough drug target for treating various pathologies of axon degeneration. SARM1-mediated axon degeneration relies on its TIR domain NADase activity, but recent structural data suggest that the non-catalytic ARM domain could also serve as a pharmacological site as it has an allosteric inhibitory function. Here, we screened for synthetic small molecules that inhibit SARM1, and tested a selected set of these compounds in a DRG axon degeneration assay. Using cryo-EM, we found that one of the newly discovered inhibitors, a calmidazolium designated TK106, not only stabilizes the previously reported inhibited conformation of the octamer, but also a meta-stable structure: a duplex of octamers (16 protomers), which we have now determined to 4.0 Å resolution. In the duplex, each ARM domain protomer is engaged in lateral interactions with neighboring protomers, and is further stabilized by contralateral contacts with the opposing octamer ring. Mutagenesis of the duplex contact sites leads to a moderate increase in SARM1 activation in cultured cells. Based on our data we propose that the duplex assembly constitutes an additional auto-inhibition mechanism that tightly prevents pre-mature activation and axon degeneration.

Structural History of Human SRGAP2 Proteins.

In the development of the human brain, human-specific genes are considered to play key roles, conferring its unique advantages and vulnerabilities. At the time of Homo lineage divergence from Australopithecus, SRGAP2C gradually emerged through a process of serial duplications and mutagenesis from ancestral SRGAP2A (3.4-2.4 Ma). Remarkably, ectopic expression of SRGAP2C endows cultured mouse brain cells, with human-like characteristics, specifically, increased dendritic spine length and density. To understand the molecular mechanisms underlying this change in neuronal morphology, we determined the structure of SRGAP2A and studied the interplay between SRGAP2A and SRGAP2C. We found that: 1) SRGAP2A homo-dimerizes through a large interface that includes an F-BAR domain, a newly identified F-BAR extension (Fx), and RhoGAP-SH3 domains. 2) SRGAP2A has an unusual inverse geometry, enabling associations with lamellipodia and dendritic spine heads in vivo, and scaffolding of membrane protrusions in cell culture. 3) As a result of the initial partial duplication event (∼3.4 Ma), SRGAP2C carries a defective Fx-domain that severely compromises its solubility and membrane-scaffolding ability. Consistently, SRGAP2A:SRAGP2C hetero-dimers form, but are insoluble, inhibiting SRGAP2A activity. 4) Inactivation of SRGAP2A is sensitive to the level of hetero-dimerization with SRGAP2C. 5) The primal form of SRGAP2C (P-SRGAP2C, existing between ∼3.4 and 2.4 Ma) is less effective in hetero-dimerizing with SRGAP2A than the modern SRGAP2C, which carries several substitutions (from ∼2.4 Ma). Thus, the genetic mutagenesis phase contributed to modulation of SRGAP2A's inhibition of neuronal expansion, by introducing and improving the formation of inactive SRGAP2A:SRGAP2C hetero-dimers, indicating a stepwise involvement of SRGAP2C in human evolutionary history.

Structure-function analysis of ceTIR-1/hSARM1 explains the lack of Wallerian axonal degeneration in C. elegans.

Wallerian axonal degeneration (WD) does not occur in the nematode C. elegans, in contrast to other model animals. However, WD depends on the NADase activity of SARM1, a protein that is also expressed in C. elegans (ceSARM/ceTIR-1). We hypothesized that differences in SARM between species might exist and account for the divergence in WD. We first show that expression of the human (h)SARM1, but not ceTIR-1, in C. elegans neurons is sufficient to confer axon degeneration after nerve injury. Next, we determined the cryoelectron microscopy structure of ceTIR-1 and found that, unlike hSARM1, which exists as an auto-inhibited ring octamer, ceTIR-1 forms a readily active 9-mer. Enzymatically, the NADase activity of ceTIR-1 is substantially weaker (10-fold higher Km) than that of hSARM1, and even when fully active, it falls short of consuming all cellular NAD+. Our experiments provide insight into the molecular mechanisms and evolution of SARM orthologs and WD across species.

Structural basis for SARM1 inhibition and activation under energetic stress.

SARM1, an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.9 and 2.7 Å resolutions. These indicate that SARM1 homo-octamer avoids premature activation by assuming a packed conformation, with ordered inner and peripheral rings, that prevents dimerization and activation of the catalytic domains. This inactive conformation is stabilized by binding of SARM1's own substrate NAD+ in an allosteric location, away from the catalytic sites. This model was validated by mutagenesis of the allosteric site, which led to constitutively active SARM1. We propose that the reduction of cellular NAD+ concentration contributes to the disassembly of SARM1's peripheral ring, which allows formation of active NADase domain dimers, thereby further depleting NAD+ to cause an energetic catastrophe and cell death.

Structural Evidence for an Octameric Ring Arrangement of SARM1.

SARM1 induces axonal degeneration in response to various insults and is therefore considered an attractive drug target for the treatment of neuro-degenerative diseases as well as for brain and spinal cord injuries. SARM1 activity depends on the integrity of the protein's SAM domains, as well as on the enzymatic conversion of NAD+ to ADPR (ADP Ribose) products by the SARM1's TIR domain. Therefore, inhibition of either SAM or TIR functions may constitute an effective therapeutic strategy. However, there is currently no SARM1-directed therapeutic approach available because of an insufficient structural and mechanistic understanding of this protein. In this study, we found that SARM1 assembles into an octameric ring. This arrangement was not described before in other SAM proteins, but is reminiscent of the apoptosome and inflammasome-well-known apoptotic ring-like oligomers. We show that both SARM1 and the isolated tandem SAM1-2 domains form octamers in solution, and electron microscopy analysis reveals an octameric ring of SARM1. We determined the crystal structure of SAM1-2 and found that it also forms a closed octameric ring in the crystal lattice. The SAM1-2 ring interactions are mediated by complementing "lock and key" hydrophobic grooves and inserts and electrostatic charges between the neighboring protomers. We have mutated several interacting SAM1-2 interfaces and measured how these mutations affect SARM1 apoptotic activity in cultured cells, and in this way identified critical oligomerization sites that facilitate cell death. These results highlight the importance of oligomerization for SARM1 function and reveal critical epitopes for future targeted drug development.

Leukocyte Cytoskeleton Polarization Is Initiated by Plasma Membrane Curvature from Cell Attachment.

Cell polarization is important for various biological processes. However, its regulation, particularly initiation, is incompletely understood. Here, we investigated mechanisms by which neutrophils break their symmetry and initiate their cytoskeleton polarization from an apolar state in circulation for their extravasation during inflammation. We show here that a local increase in plasma membrane (PM) curvature resulting from cell contact to a surface triggers the initial breakage of the symmetry of an apolar neutrophil and is required for subsequent polarization events induced by chemical stimulation. This local increase in PM curvature recruits SRGAP2 via its F-BAR domain, which in turn activates PI4KA and results in PM PtdIns4P polarization. Polarized PM PtdIns4P is targeted by RPH3A, which directs PIP5K1C90 and subsequent phosphorylated myosin light chain polarization, and this polarization signaling axis regulates neutrophil firm attachment to endothelium. Thus, this study reveals a mechanism for the initiation of cell cytoskeleton polarization.

Approaching the Roundabout: cis and trans Robo1 Contacts Revealed.

In this issue of Structure,Aleksandrova et al. (2018) present low- and high-resolution structures of Robo1, a key player in axonal guidance. The structures shed light on the arrangement of Robo1 at the plasma membrane and provide evidence for back-to-back trans Robo1 contacts.

Robo Ig4 Is a Dimerization Domain.

Robo receptors play pivotal roles in axonal guidance as well as in neurogenesis, angiogenesis, cell migration, and cancer progression and invasiveness. They are considered to be attractive drug targets for the treatment of cancer, ocular neovascular disorders, chronic kidney diseases, and more. Despite their great importance, the mechanisms by which Robo receptors switch from their "off" to "on" states remain obscure. One possibility involves a monomer-to-dimer or dimer-to-monomer transition that facilitates the recruitment and activation of enzymatic effectors to instigate intracellular signaling. However, it is not known which domains mediate Robo dimerization, or the structural properties of the dimeric interactions. Here, we identify the extracellular Ig4 (D4) as a Robo dimerization domain. We have determined the crystal structure of the tandem Ig4-5 domains (D4-5) of human Robo2 and found that a hydrophobic surface on D4 mediates close homotypic contacts with a reciprocal D4. Analytical ultracentrifugation measurements of intact and mutated D4-5 shows that dimerization through the D4 interface is specific and has a dimerization dissociation constant of 16.9µM in solution. Direct fluorescence resonance energy transfer dimerization measurements in HEK293 cells corroborate the dimerization of transmembrane hRobo2 through D4, and a functional COS-7 cell collapse assay links D4-mediated dimerization with Robo intracellular signaling. The high level of conservation in the D4 dimerization interface throughout all Robo orthologs and paralogs implies that D4-mediated dimerization is a central hallmark in Robo activation and signaling.

Molecular symmetry-constrained systematic search approach to structure solution of the coiled-coil SRGAP2 F-BARx domain.

SRGAP2 (Slit-Robo GTPase-activating protein 2) is a cytoplasmic protein found to be involved in neuronal branching, restriction of neuronal migration and restriction of the length and density of dendritic postsynaptic spines. The extended F-BAR (F-BARx) domain of SRGAP2 generates membrane protrusions when expressed in COS-7 cells, while most F-BARs induce the opposite effect: membrane invaginations. As a first step to understand this discrepancy, the F-BARx domain of SRGAP2 was isolated and crystallized after co-expression with the carboxy domains of the protein. Diffraction data were collected from two significantly non-isomorphous crystals in the same monoclinic C2 space group. A correct molecular-replacment solution was obtained by applying a molecular symmetry-constrained systematic search approach that took advantage of the conserved biological symmetry of the F-BAR domains. It is shown that similar approaches can solve other F-BAR structures that were previously determined by experimental phasing. Diffraction data were reprocessed with a high-resolution cutoff of 2.2 Å, chosen using less strict statistical criteria. This has improved the outcome of multi-crystal averaging and other density-modification procedures.

The Neuronal Migration Factor srGAP2 Achieves Specificity in Ligand Binding through a Two-Component Molecular Mechanism.

srGAP proteins regulate cell migration and morphogenesis by shaping the structure and dynamics of the cytoskeleton and membranes. First discovered as intracellular effectors for the Robo1 axon-guidance receptor, srGAPs were later identified as interacting with several other nuclear and cytoplasmic proteins. In all these cases, the srGAP SH3 domain mediates protein-protein interactions by recognizing a short proline-rich segment on the cognate-binding partner. However, as interactions between the isolated SH3 domain and a selected set of ligands show weak affinity and low specificity, it is not clear how srGAPs are precisely recruited to their signaling sites. Here, we report a two-component molecular mechanism that regulates ligand binding to srGAP2 by on the one hand dramatically tightening their association and on the other, moderately autoinhibiting and restricting binding. Our results allow the design of point mutations for better probing of srGAP2 activities, and may facilitate the identification of new srGAP2 ligands.