Structural basis of human PDZD8–Rab7 interaction for the ER-late endosome tethering

AbstractThe membrane contact sites (MCSs) between the ER and late endosomes (LEs) are essential for the regulation of endosomal protein sorting, dynamics, and motility. PDZD8 is an ER transmembrane protein containing a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain. PDZD8 tethers the ER to late endosomes and lysosomes by associating its C-terminal coiled-coil (CC) with the LE Rab7. To identify the structural determinants for the PDZD8–Rab7 interaction, we determined the crystal structure of the human PDZD8 CC domain in complex with the GTP-bound form of Rab7. The PDZD8 CC contains one short helix and the two helices forming an antiparallel coiled-coil. Two Rab7 molecules bind to the opposite sides of the PDZD8 CC in a 2:1 ratio. The switch I/II and interswitch regions of the GTP-loaded Rab7 form the binding interfaces, which correlates with the GTP-dependent interaction of PDZD8 and Rab7. Analysis of the protein interaction by isothermal titration calorimetry confirms that two Rab7 molecules bind the PDZD8 CC in a GTP-dependent manner. The structural model of the PDZD8 CC–Rab7 complex correlates with the recruitment of PDZD8 at the LE–ER interface and its role in lipid transport and regulation.

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Annexin A6 modulates TBC1D15/Rab7/StARD3 axis to control endosomal cholesterol export in NPC1 cells

Cellular and Molecular Life Sciences ◽

10.1007/s00018-019-03330-y ◽

2019 ◽

Vol 77 (14) ◽

pp. 2839-2857 ◽

Cited By ~ 4

Author(s):

Elsa Meneses-Salas ◽

Ana García-Melero ◽

Kristiina Kanerva ◽

Patricia Blanco-Muñoz ◽

Frederic Morales-Paytuvi ◽

...

Keyword(s):

Late Endosome ◽

Dependent Manner ◽

Lipid Transfer ◽

Cholesterol Accumulation ◽

Membrane Contact Sites ◽

Late Endosomes ◽

Membrane Contact ◽

Domain 3 ◽

Niemann Pick ◽

Mutant Cells

Abstract Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.

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Structural Basis for the Interaction between p53 Transactivation Domain and the Mediator Subunit MED25

Molecules ◽

10.3390/molecules23102726 ◽

2018 ◽

Vol 23 (10) ◽

pp. 2726 ◽

Cited By ~ 5

Author(s):

Min-Sung Lee ◽

Kyungeun Lim ◽

Mi-Kyung Lee ◽

Seung-Wook Chi

Keyword(s):

Molecular Interaction ◽

Transcription Initiation ◽

Structural Model ◽

Structural Basis ◽

Titration Calorimetry ◽

Virus Protein ◽

Sequence Motif ◽

Transactivation Domain ◽

Transcriptional Activators ◽

Interaction Domain

Eukaryotic transcription initiation is mediated by interactions between transcriptional activators and the mediator coactivator complex. Molecular interaction of p53 transcription factor with mediator complex subunit 25 (MED25) is essential for its target gene transcription. In this study, we characterized the molecular interaction between p53 transactivation domain (p53TAD) and activator interaction domain (ACID) of MED25 using nuclear magnetic resonance (NMR) spectroscopy. The NMR chemical shift perturbation and isothermal titration calorimetry (ITC) data showed that p53TAD interacted with MED25 ACID mainly through the p53TAD2 sequence motif. Taken together with the mutagenesis data, the refined structural model of MED25 ACID/p53TAD2 peptide complex showed that an amphipathic α-helix of p53TAD2 peptide bound an elongated hydrophobic groove of MED25 ACID. Furthermore, our results revealed the highly conserved mechanism of MED25 interaction with intrinsically unfolded acidic TADs from the transcriptional activators p53, ERM (Ets-related molecule), and herpes simplex virus protein 16 (VP16).

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Biochemical and structural studies of target lectin SapL1 from the emerging opportunistic microfungus Scedosporium apiospermum

Scientific Reports ◽

10.1038/s41598-021-95008-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Dania Martínez-Alarcón ◽

Viviane Balloy ◽

Jean-Philippe Bouchara ◽

Roland J. Pieters ◽

Annabelle Varrot

Keyword(s):

Structural Basis ◽

Soluble Form ◽

Titration Calorimetry ◽

Scedosporium Apiospermum ◽

Dependent Manner ◽

Bronchial Epithelial ◽

Life Threatening ◽

Opportunistic Fungal Pathogen ◽

Conidial Surface

AbstractScedosporium apiospermum is an emerging opportunistic fungal pathogen responsible for life-threatening infections in humans. Host–pathogen interactions often implicate lectins that have become therapeutic targets for the development of carbohydrate mimics for antiadhesive therapy. Here, we present the first report on the identification and characterization of a lectin from S. apiospermum named SapL1. SapL1 was found using bioinformatics as a homolog to the conidial surface lectin FleA from Aspergillus fumigatus known to play a role in the adhesion to host glycoconjugates present in human lung epithelium. In our strategy to obtain recombinant SapL1, we discovered the importance of osmolytes to achieve its expression in soluble form in bacteria. Analysis of glycan arrays indicates specificity for fucosylated oligosaccharides as expected. Submicromolar affinity was measured for fucose using isothermal titration calorimetry. We solved SapL1 crystal structure in complex with α-methyl-L-fucoside and analyzed its structural basis for fucose binding. We finally demonstrated that SapL1 binds to bronchial epithelial cells in a fucose-dependent manner. The information gathered here will contribute to the design and development of glycodrugs targeting SapL1.

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Molecular basis for the interaction between stress-inducible phosphoprotein 1 (STIP1) and S100A1

Biochemical Journal ◽

10.1042/bcj20161055 ◽

2017 ◽

Vol 474 (11) ◽

pp. 1853-1866 ◽

Cited By ~ 4

Author(s):

Andrzej Maciejewski ◽

Vania F. Prado ◽

Marco A.M. Prado ◽

Wing-Yiu Choy

Keyword(s):

Calcium Binding ◽

Molecular Basis ◽

Structural Basis ◽

Titration Calorimetry ◽

Dependent Manner ◽

S100 Family ◽

Calcium Dependent ◽

Binding Interface ◽

Large Conformational Change ◽

Α Helix

Stress-inducible phosphoprotein 1 (STIP1) is a cellular co-chaperone, which regulates heat-shock protein 70 (Hsp70) and Hsp90 activity during client protein folding. Members of the S100 family of dimeric calcium-binding proteins have been found to inhibit Hsp association with STIP1 through binding of STIP1 tetratricopeptide repeat (TPR) domains, possibly regulating the chaperone cycle. Here, we investigated the molecular basis of S100A1 binding to STIP1. We show that three S100A1 dimers associate with one molecule of STIP1 in a calcium-dependent manner. Isothermal titration calorimetry revealed that individual STIP1 TPR domains, TPR1, TPR2A and TPR2B, bind a single S100A1 dimer with significantly different affinities and that the TPR2B domain possesses the highest affinity for S100A1. S100A1 bound each TPR domain through a common binding interface composed of α-helices III and IV of each S100A1 subunit, which is only accessible following a large conformational change in S100A1 upon calcium binding. The TPR2B-binding site for S100A1 was predominately mapped to the C-terminal α-helix of TPR2B, where it is inserted into the hydrophobic cleft of an S100A1 dimer, suggesting a novel binding mechanism. Our data present the structural basis behind STIP1 and S100A1 complex formation, and provide novel insights into TPR module-containing proteins and S100 family member complexes.

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Structural Basis for the C-Terminal Domain of Mycobacterium tuberculosis Ribosome Maturation Factor RimM to Bind Ribosomal Protein S19

Biomolecules ◽

10.3390/biom11040597 ◽

2021 ◽

Vol 11 (4) ◽

pp. 597

Author(s):

Haoran Zhang ◽

Qiuxiang Zhou ◽

Chenyun Guo ◽

Liubin Feng ◽

Huilin Wang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Ribosomal Protein ◽

Solution Structure ◽

Molecular Mechanisms ◽

Structural Model ◽

Ribosomal Subunit ◽

Structural Basis ◽

Hydrophobic Core ◽

Terminal Domain ◽

Ribosomal Protein S19

Multidrug-resistant tuberculosis (TB) is a serious threat to public health, calling for the development of new anti-TB drugs. Chaperon protein RimM, involved in the assembly of ribosomal protein S19 into 30S ribosomal subunit during ribosome maturation, is a potential drug target for TB treatment. The C-terminal domain (CTD) of RimM is primarily responsible for binding S19. However, both the CTD structure of RimM from Mycobacterium tuberculosis (MtbRimMCTD) and the molecular mechanisms underlying MtbRimMCTD binding S19 remain elusive. Here, we report the solution structure, dynamics features of MtbRimMCTD, and its interaction with S19. MtbRimMCTD has a rigid hydrophobic core comprised of a relatively conservative six-strand β-barrel, tailed with a short α-helix and interspersed with flexible loops. Using several biophysical techniques including surface plasmon resonance (SPR) affinity assays, nuclear magnetic resonance (NMR) assays, and molecular docking, we established a structural model of the MtbRimMCTD–S19 complex and indicated that the β4-β5 loop and two nonconserved key residues (D105 and H129) significantly contributed to the unique pattern of MtbRimMCTD binding S19, which might be implicated in a form of orthogonality for species-dependent RimM–S19 interaction. Our study provides the structural basis for MtbRimMCTD binding S19 and is beneficial to the further exploration of MtbRimM as a potential target for the development of new anti-TB drugs.

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Type IIa RPTPs and Glycans: Roles in Axon Regeneration and Synaptogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms22115524 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5524

Author(s):

Kazuma Sakamoto ◽

Tomoya Ozaki ◽

Yuji Suzuki ◽

Kenji Kadomatsu

Keyword(s):

Heparan Sulfate ◽

Network Formation ◽

Synapse Formation ◽

Axon Regeneration ◽

Transmembrane Protein ◽

Inhibitory Synapses ◽

Dependent Manner ◽

Axon Terminals ◽

Axon Extension ◽

Receptor Tyrosine Phosphatases

Type IIa receptor tyrosine phosphatases (RPTPs) play pivotal roles in neuronal network formation. It is emerging that the interactions of RPTPs with glycans, i.e., chondroitin sulfate (CS) and heparan sulfate (HS), are critical for their functions. We highlight here the significance of these interactions in axon regeneration and synaptogenesis. For example, PTPσ, a member of type IIa RPTPs, on axon terminals is monomerized and activated by the extracellular CS deposited in neural injuries, dephosphorylates cortactin, disrupts autophagy flux, and consequently inhibits axon regeneration. In contrast, HS induces PTPσ oligomerization, suppresses PTPσ phosphatase activity, and promotes axon regeneration. PTPσ also serves as an organizer of excitatory synapses. PTPσ and neurexin bind one another on presynapses and further bind to postsynaptic leucine-rich repeat transmembrane protein 4 (LRRTM4). Neurexin is now known as a heparan sulfate proteoglycan (HSPG), and its HS is essential for the binding between these three molecules. Another HSPG, glypican 4, binds to presynaptic PTPσ and postsynaptic LRRTM4 in an HS-dependent manner. Type IIa RPTPs are also involved in the formation of excitatory and inhibitory synapses by heterophilic binding to a variety of postsynaptic partners. We also discuss the important issue of possible mechanisms coordinating axon extension and synapse formation.

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Structural basis for the dominant or recessive character of GLIALCAM mutations found in leukodystrophies

Human Molecular Genetics ◽

10.1093/hmg/ddaa009 ◽

2020 ◽

Vol 29 (7) ◽

pp. 1107-1120 ◽

Cited By ~ 2

Author(s):

Xabier Elorza-Vidal ◽

Efren Xicoy-Espaulella ◽

Adrià Pla-Casillanis ◽

Marta Alonso-Gardón ◽

Héctor Gaitán-Peñas ◽

...

Keyword(s):

Structural Model ◽

Clinical Phenotype ◽

Structural Basis ◽

Recessive Character ◽

Adhesion Protein ◽

Recessive Mutations ◽

Cross Links ◽

Interacting Surfaces ◽

Ig Domain ◽

Subcortical Cysts

Abstract Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a type of leukodystrophy characterized by white matter edema, and it is caused mainly by recessive mutations in MLC1 and GLIALCAM genes. These variants are called MLC1 and MLC2A with both types of patients sharing the same clinical phenotype. In addition, dominant mutations in GLIALCAM have also been identified in a subtype of MLC patients with a remitting phenotype. This variant has been named MLC2B. GLIALCAM encodes for an adhesion protein containing two immunoglobulin (Ig) domains and it is needed for MLC1 targeting to astrocyte–astrocyte junctions. Most mutations identified in GLIALCAM abolish GlialCAM targeting to junctions. However, it is unclear why some mutations behave as recessive or dominant. Here, we used a combination of biochemistry methods with a new developed anti-GlialCAM nanobody, double-mutants and cysteine cross-links experiments, together with computer docking, to create a structural model of GlialCAM homo-interactions. Using this model, we suggest that dominant mutations affect different GlialCAM–GlialCAM interacting surfaces in the first Ig domain, which can occur between GlialCAM molecules present in the same cell (cis) or present in neighbouring cells (trans). Our results provide a framework that can be used to understand the molecular basis of pathogenesis of all identified GLIALCAM mutations.

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TBC-2 Regulates RAB-5/RAB-7-mediated Endosomal Trafficking inCaenorhabditis elegans

Molecular Biology of the Cell ◽

10.1091/mbc.e09-11-0947 ◽

2010 ◽

Vol 21 (13) ◽

pp. 2285-2296 ◽

Cited By ~ 51

Author(s):

Laëtitia Chotard ◽

Ashwini K. Mishra ◽

Marc-André Sylvain ◽

Simon Tuck ◽

David G. Lambright ◽

...

Keyword(s):

Rab Gtpase ◽

Endosomal Trafficking ◽

Dependent Manner ◽

Nucleotide Exchange ◽

Late Endosomes ◽

Arginine Finger ◽

Endosome Maturation ◽

Hops Complex

During endosome maturation the early endosomal Rab5 GTPase is replaced with the late endosomal Rab7 GTPase. It has been proposed that active Rab5 can recruit and activate Rab7, which in turn could inactivate and remove Rab5. However, many of the Rab5 and Rab7 regulators that mediate endosome maturation are not known. Here, we identify Caenorhabditis elegans TBC-2, a conserved putative Rab GTPase-activating protein (GAP), as a regulator of endosome to lysosome trafficking in several tissues. We show that tbc-2 mutant animals accumulate enormous RAB-7–positive late endosomes in the intestine containing refractile material. RAB-5, RAB-7, and components of the homotypic fusion and vacuole protein sorting (HOPS) complex, a RAB-7 effector/putative guanine nucleotide exchange factor (GEF), are required for the tbc-2(−) intestinal phenotype. Expression of activated RAB-5 Q78L in the intestine phenocopies the tbc-2(−) large late endosome phenotype in a RAB-7 and HOPS complex-dependent manner. TBC-2 requires the catalytic arginine-finger for function in vivo and displays the strongest GAP activity on RAB-5 in vitro. However, TBC-2 colocalizes primarily with RAB-7 on late endosomes and requires RAB-7 for membrane localization. Our data suggest that TBC-2 functions on late endosomes to inactivate RAB-5 during endosome maturation.

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The δ subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling

Nature Communications ◽

10.1038/s41467-020-20159-3 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Hao-Hong Pei ◽

Tarek Hilal ◽

Zhuo A. Chen ◽

Yong-Heng Huang ◽

Yuan Gao ◽

...

Keyword(s):

Bacillus Subtilis ◽

Nucleic Acids ◽

Rna Polymerase ◽

Active Site ◽

Genome Stability ◽

Slow Growth ◽

Coiled Coil ◽

Main Channel ◽

Dependent Manner ◽

Secondary Channel

AbstractCellular RNA polymerases (RNAPs) can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis, the RNAP δ subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP-δ-HelD complexes. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNAP secondary channel, dismantling the active site and displacing RNA, while a unique helical protrusion inserts into the main channel, prying the β and β′ subunits apart and, aided by δ, dislodging DNA. RNAP is recycled when, after releasing trapped nucleic acids, HelD dissociates from the enzyme in an ATP-dependent manner. HelD abundance during slow growth and a dimeric (RNAP-δ-HelD)2 structure that resembles hibernating eukaryotic RNAP I suggest that HelD might also modulate active enzyme pools in response to cellular cues.

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