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Metazoans require the sorting nexin (SNX) protein, SNX27, to recycle hundreds of important transmembrane protein receptors from endosomes to the plasma membrane. Cargo recycling by SNX27 requires its interaction with retromer, a heterotrimer known to assemble on membranes with multiple sorting nexins, including SNX-BAR proteins and SNX3. SNX27 has also been functionally linked to SNX-BARs, but the molecular basis of this interaction has been unknown. We identify a direct biochemical interaction between the conserved and flexible SNX1/SNX2 N-terminus and full-length SNX27 using purified proteins in pulldown experiments. Sequence alignments indicate both SNX1 and SNX2 contain two short and conserved stretches of acidic residues bearing a DxF motif in their flexible N-terminal regions. Biochemical pulldown and mapping experiments reveal forty residues in the N-terminus of either SNX1 or SNX2 can mediate binding to SNX27. SNX27 truncation analysis demonstrates the SNX27 FERM domain binds the SNX1 N-terminus. Calorimetry experiments quantified binding between the SNX1 N-terminus and SNX27 in the low micromolar affinity range (KD ∼10 μM) and suggest the second DxF motif may play a more prominent role in binding. Mutation of either DxF sequence in SNX1 abrogates measurable binding to SNX27 in the calorimeter. Modelling from both predicted and experimentally determined structures suggests the SNX27 FERM domain could accommodate both DxF motifs simultaneously. Together, these data suggest SNX27 is directly linked to specific SNX-BAR proteins through binding acidic motifs in the SNX1 or SNX2 N-terminus.

SUMMARY NMDA receptor (NMDAR)-dependent Ca2+ influx underpins multiple forms of synaptic plasticity. Most synaptic NMDAR currents in the adult forebrain are mediated by GluN2A-containing receptors, which are rapidly inserted into synapses during long-term potentiation (LTP); however, the underlying molecular mechanisms remain poorly understood. In this study, we show that GluN2A is phosphorylated at Ser-1459 by Ca2+/calmodulin-dependent kinase IIα (CaMKIIα) in response to glycine stimulation that mimics LTP in primary neurons. Phosphorylation of Ser-1459 promotes GluN2A interaction with the sorting nexin 27 (SNX27)-retromer complex, thereby enhancing the endosomal recycling of NMDARs. Loss of SNX27 or CaMKIIα function blocks the glycine-induced increase in GluN2A-NMDARs on the neuronal membrane. Interestingly, mutations of Ser-1459, including the rare S1459G human epilepsy variant, prolong the decay times of NMDAR-mediated synaptic currents in heterosynapses by increasing the duration of channel opening. These findings not only identify a critical role of Ser-1459 phosphorylation in regulating the function of NMDARs, but they also explain how the S1459G variant dysregulates NMDAR function., Graphical Abstract, In brief Yong et al. identify that activity-dependent phosphorylation of Ser-1459 in the GluN2A C-terminal domain by CaMKIIα promotes its interaction with the SNX27-retromer complex, thereby enhancing the surface expression of NMDARs during synaptic potentiation. Mutations of Ser-1459 prolong the decay times of NMDAR-mediated synaptic currents by increasing the duration of channel opening.

Many eukaryotes assemble an actin- and myosin-based cytokinetic ring (CR) on the plasma membrane (PM) for cell division, but how it is anchored there remains unclear. In Schizosaccharomyces pombe, the F-BAR protein Cdc15 links the PM via its F-BAR domain to proteins in the CR's interior via its SH3 domain. However, Cdc15's F-BAR domain also directly binds formin Cdc12, suggesting that Cdc15 may polymerize a protein network directly adjacent to the membrane. Here, we determine that the F-BAR domain binds Cdc12 using residues on the face opposite its membrane-binding surface. These residues also bind paxillin-like Pxl1, promoting its recruitment with calcineurin to the CR. Mutation of these F-BAR domain residues results in a shallower CR, with components localizing ∼35% closer to the PM than in wild type, and aberrant CR constriction. Thus, F-BAR domains serve as oligomeric membrane-bound platforms that can modulate the architecture of an entire actin structure.

The enteric protozoan parasite, Entamoeba histolytica, an etiological agent of amebiasis, is involved in the adhesion and destruction of human tissues. Worldwide, the parasite causes about 50 million cases of amebiasis and 100,000 deaths annually. EhRabX3, a unique amoebic Rab GTPase with tandem G-domains, possesses an unusually large number of cysteine residues in its N-terminal domain. Crystal structure of EhRabX3 revealed an intra-molecular disulfide bond between C39 and C163 which is critical for maintaining the 3-dimensional architecture and biochemical function of this protein. The remaining six cysteine residues were found to be surface exposed and predicted to be involved in inter-molecular disulfide bonds. In the current study, using biophysical and mutational approaches, we have investigated the role of the cysteine residues in the assembly of EhRabX3 oligomer. The self-association of EhRabX3 is found to be redox sensitive, in vitro. Furthermore, the oligomeric conformation of EhRabX3 failed to bind and exchange the guanine nucleotide, indicating structural re-organization of the active site. Altogether, our results provide valuable insights into the redox-dependent oligomerization of EhRabX3 and its implication on nucleotide binding.

Autophagy is a lysosomal degradation pathway that degrades cytosolic constituents, including whole organelles and intracellular pathogens. Previous studies on various autophagy related genes revealed the importance of the Atg12-Atg5-Atg16 complex in autophagy. Atg16L1 is an effector of Golgi-resident Rab33B and the molecular mechanism of the interaction of Rab33B with either Atg16L1 or in complex with Atg5 is still elusive. In the current study, using the pull down and calorimetric approaches, we have dissected the molecular insights into the interaction of Rab33B with different regions of mouse Atg16L1 as well as with the dimeric complex, Atg5-mAtg16L1. Our in vitro observation suggests that Atg5 is pre-requisite for the augmented nucleotide dependent interaction of Rab33B with the dimeric complex, Atg5-Atg16L1. Moreover, the results reported here suggest that Arg-24 of Atg16L1 is crucial for its interaction with Atg5 which will have further implication in the binding of the dimeric complex to Rab33B.

The enteric protozoan parasite, Entamoeba histolytica, is the causative agent of amoebic dysentery, liver abscess and colitis in human. Vesicular trafficking plays a key role in the survival and virulence of the protozoan and is regulated by various Rab GTPases. EhRabX3 is a catalytically inefficient amoebic Rab protein, which is unique among the eukaryotic Ras superfamily by virtue of its tandem domain organization. Here, we report the crystal structures of GDP-bound fast hydrolyzing mutant (V71A/K73Q) and GTP-bound wild type EhRabX3 at 3.1 and 2.8Å resolutions, respectively. Though both G-domains possess "phosphate binding loop containing nucleoside triphosphate hydrolases fold", only the N-terminal domain binds to guanine nucleotide. The relative orientation of the N-terminal domain and C-terminal domain is stabilized by numerous inter-domain interactions. Compared to other Ras superfamily members, both the GTPase domains displayed large deviation in switch II perhaps due to non-conservative substitutions in this region. As a result, entire switch II is restructured and moved away from the nucleotide binding pocket, providing a rationale for the diminished GTPase activity of EhRabX3. The N-terminal GTPase domain possesses unusually large number of cysteine residues. X-ray crystal structure of the fast hydrolyzing mutant of EhRabX3 revealed that C39 and C163 formed an intra-molecular disulfide bond. Subsequent mutational and biochemical studies suggest that C39 and C163 are critical for maintaining the structural integrity and function of EhRabX3. Structure-guided functional investigation of cysteine mutants could provide the physiological implications of the disulfide bond and could allow us to design potential inhibitors for the better treatment of intestinal amebiasis.

Phosphorylation of phosphoinositides by the class II phosphatidylinositol 3-kinase (PI3K) PI3K-C2α is essential for many processes, including neuroexocytosis and formation of clathrin-coated vesicles. A defining feature of the class II PI3Ks is a C-terminal module composed of phox-homology (PX) and C2 membrane interacting domains; however, the mechanisms that control their specific cellular localization remain poorly understood. Here we report the crystal structure of the C2 domain of PI3K-C2α in complex with the phosphoinositide head-group mimic inositol hexaphosphate, revealing two distinct pockets for membrane binding. The C2 domain preferentially binds to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate, and low-resolution structures of the combined PX-C2 module by small-angle X-ray scattering reveal a compact conformation in which cooperative lipid binding by each domain binding can occur. Finally, we demonstrate an unexpected role for calcium in perturbing the membrane interactions of the PX-C2 module, which we speculate may be important for regulating the activity of PI3K-C2α.