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Human L-asparaginase-like protein 1 (ASRGL1) has hydrolytic activity against L-asparagine and isoaspartyl dipeptides. As an N-terminal nucleophile hydrolase family member, its activation depends on an intramolecular autoprocessing step between G167 and T168. In vitro, autoprocessing reaches only 50% completion, which restrains the activity and hampers the full understanding of the activation process. The ASRGL1 dimer interface plays a critical role in intramolecular processing, and the interactions within oligomers can offer relevant information about autoprocessing. In this work, a fully processed trimeric conformation of ASRGL1 was observed for the first time, and we combined biophysical and structural proteomics assays to characterize trimeric ASRGL1. Our analyses show that oligomerization is critical for autoprocessing, hydrolytic activity and thermal stability. The newest trimeric ASRGL1 conformation enhances protein activity and presents a melting temperature deviation of 4.33 °C in comparison to the monomeric conformation. The interaction of the third monomer in the trimeric conformation is driven by an α-helix comprising residues KVNLARLTLF (227-236)., (Copyright © 2020. Published by Elsevier B.V.)

Vertebrates usually have three class V myosin paralogues (MyoV) to control membrane trafficking in the actin-rich cell cortex, but their functional overlapping or differentiation through cargoes selectivity is yet only partially understood. In this work, we reveal that the globular tail domain of MyoVc binds to the active form of small GTPase Rab3A with nanomolar affinity, a feature shared with MyoVa but not with MyoVb. Using molecular docking analyses guided by chemical cross-linking restraints, we propose a model to explain how Rab3A selectively recognizes MyoVa and MyoVc via a distinct binding site from that used by Rab11A. The MyoVa/c binding interface involves multiple residues from both lobules (I and II) and the short helix at the α2-α3 link region, which is conserved between MyoVa and MyoVc, but not in MyoVb. This motif is also responsible for the selective binding of RILPL2 by MyoVa and potentially MyoVc. Together, these findings support the selective recruitment of MyoVa and MyoVc to exocytic pathways via Rab3A and expand our knowledge about the functional evolution of class V myosins. SIGNIFICANCE: Hormone secretion, neurotransmitter release, and cytoplasm membrane recycling are examples of processes that rely on the interaction of molecular motors and Rab GTPases to regulate the intracellular trafficking and tethering of vesicles. Defects in these proteins may cause neurological impairment, immunodeficiency, and other severe disorders, being fatal in some cases. Despite their crucial roles, little is known about how these molecular motors are selectively recruited by specific members of the large family of Rab GTPases. In this study, we unveil the interaction between the actin-based molecular motor Myosin Vc and the small GTPase Rab3A, a key coordinator of vesicle trafficking and exocytosis in mammalian cells. Moreover, we propose a model for their recognition and demonstrate that Rab3A specifically binds to the globular tail of Myosins Va and Vc, but not of Myosin Vb, advancing our knowledge about the molecular basis for the selective recruitment of class V myosins by Rab GTPases., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bifidobacteria represent one of the first colonizers of human gut microbiota, providing to this ecosystem better health and nutrition. To maintain a mutualistic relationship, they have enzymes to degrade and use complex carbohydrates non-digestible by their hosts. To succeed in the densely populated gut environment, they evolved molecular strategies that remain poorly understood. Herein, we report a novel mechanism found in probiotic Bifidobacteria for the depolymerization of the ubiquitous 2-acetamido-2-deoxy-4-O-(β-d-mannopyranosyl)-d-glucopyranose (Man-β-1,4-GlcNAc), a disaccharide that composes the universal core of eukaryotic N-glycans. In contrast to Bacteroidetes, these Bifidobacteria have a specialist and strain-specific β-mannosidase that contains three distinctive structural elements conferring high selectivity for Man-β-1,4-GlcNAc: a lid that undergoes conformational changes upon substrate binding, a tryptophan residue swapped between the two dimeric subunits to accommodate the GlcNAc moiety, and a Rossmann fold subdomain strategically located near to the active site pocket. These key structural elements for Man-β-1,4-GlcNAc specificity are highly conserved in Bifidobacterium species adapted to the gut of a wide range of social animals, including bee, pig, rabbit, and human. Together, our findings uncover an unprecedented molecular strategy employed by Bifidobacteria to selectively uptake carbohydrates from N-glycans in social hosts., (Copyright © 2019 Elsevier Ltd. All rights reserved.)