Mapping Critical Residues in ATG11’s Coiled-Coil 2 Domain that Block Multiple Interactions and Disrupt Selective Autophagy
Ontology highlight
ABSTRACT:
SUBMITTER: Meyer M
PROVIDER: S-EPMC8805157 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Selective autophagy is the capture of specific cytosolic contents in double-membrane vesicles that subsequently fuse with the vacuole or lysosome, thereby delivering cargo for degradation. Selective autophagy receptors (SARs) mark the cargo for degradation and, in yeast, recruit Atg11, the scaffolding protein for selective autophagy initiation. The mitochondrial protein Atg32 is the yeast SAR that mediates mitophagy, the selective autophagic capture of mitochondria. Atg11-Atg32 interactions concentrate Atg32 into puncta that are thought to represent sites of mitophagy initiation. However, it is unclear how Atg11 concentrates Atg32 to generate mitophagy initiation sites. We show here that the coiled coil 3 (CC3) domain of Atg11 is required for concentrating Atg32 into puncta. We determined the structure of the majority of the CC3, demonstrating that the CC3 forms a parallel homodimer whose dimer interface is formed by a small number of hydrophobic residues. We further show that the CC3 interface is not required for Atg11 dimerization but is required for shaping Atg32 into functional mitophagy initiation sites and for delivery of mitochondria to the vacuole. Our findings suggest that Atg11 self-interactions help concentrate SARs as a necessary precondition for cargo capture.
| S-EPMC7603899 | biostudies-literature
Project description:Autophagy recycles cytoplasmic components by sequestering them in double membrane-surrounded autophagosomes. The two proteins Atg11 and Atg17 are scaffolding components of the Atg1 kinase complex. Atg17 recruits and tethers Atg9-donor vesicles, and the corresponding Atg1 kinase complex induces the formation of nonselective autophagosomes. Atg11 initiates selective autophagy and coordinates the switch to nonselective autophagy by recruiting Atg17. The molecular function of Atg11 remained, however, less well understood. Here, we demonstrate that Atg11 is activated by cargo through a direct interaction with autophagy receptors. Activated Atg11 dimerizes and tethers Atg9 vesicles, which leads to the nucleation of phagophores in direct vicinity of cargo. Starvation reciprocally regulates the activity of both tethering factors by initiating the degradation of Atg11 while Atg17 is activated. This allows Atg17 to sequester and tether Atg9 vesicles independent of cargo to nucleate nonselective phagophores. Our data reveal insights into the molecular mechanisms governing cargo selection and specificity in autophagy.
| S-EPMC6687201 | biostudies-literature
Project description:Autophagy, an essential eukaryotic homeostasis pathway, allows the sequestration of unwanted, damaged, or harmful cytoplasmic components in vesicles called autophagosomes, permitting subsequent lysosomal degradation and nutrient recycling. Autophagosome nucleation is mediated by class III phosphatidylinositol-3-kinase complexes that include two key autophagy proteins, BECN1/Beclin 1 and ATG14/BARKOR, which form parallel heterodimers via their coiled-coil domains (CCDs). Here we present the 1.46 Å X-ray crystal structure of the antiparallel, human BECN1 CCD homodimer, which represents BECN1 oligomerization outside the autophagosome nucleation complex. We use circular dichroism and small-angle X-ray scattering (SAXS) to show that the ATG14 CCD is significantly disordered but becomes more helical in the BECN1:ATG14 heterodimer, although it is less well-folded than the BECN1 CCD homodimer. SAXS also indicates that the BECN1:ATG14 heterodimer is more curved than other BECN1-containing CCD dimers, which has important implications for the structure of the autophagosome nucleation complex. A model of the BECN1:ATG14 CCD heterodimer that agrees well with the SAXS data shows that BECN1 residues at the homodimer interface are also responsible for heterodimerization, allowing us to identify ATG14 interface residues. Finally, we verify the role of BECN1 and ATG14 interface residues in binding by assessing the impact of point mutations of these residues on co-immunoprecipitation of the partner and demonstrate that these mutations abrogate starvation-induced upregulation of autophagy but do not impact basal autophagy. Thus, this research provides insights into structures of the BECN1 CCD homodimer and the BECN1:ATG14 CCD heterodimer and identifies interface residues that are important for BECN1:ATG14 heterodimerization and for autophagy.
| S-EPMC5116031 | biostudies-literature
Project description:Polar residues lying between adjacent ?-helical chains of coiled-coils often contribute to coiled-coil curvature and flexibility, while more typical core hydrophobic residues anneal the chains together. In tropomyosins, ranging from smooth and skeletal muscle to cytoplasmic isoforms, a highly conserved Asp at residue 137 places negative charges within the tropomyosin coiled-coil core in a position which may affect the conformation needed for tropomyosin binding and regulatory movements on actin. Proteolytic susceptibility suggested that substituting a canonical Leu for the naturally occurring Asp at residue 137 increases inter-chain rigidity by stabilizing the tropomyosin coiled-coil. Using molecular dynamics, we now directly assess changes in coiled-coil curvature and flexibility caused by such mutants. Although the coiled-coil flexibility is modestly diminished near the residue 137 mutation site, as expected, a delocalized increase in flexibility along the overall coiled-coil is observed. Even though the average shape of the D137L tropomyosin is straighter than that of wild-type tropomyosin, it is still capable of binding actin due to this increase in flexibility. We conclude that the conserved, non-canonical Asp-137 destabilizes the local structure resulting in a local flexible region in the middle of tropomyosin that normally is important for tropomyosin steady-state equilibrium position on actin.
| S-EPMC3384579 | biostudies-literature
Project description:Coiled-coil regions were among the first protein motifs described structurally and theoretically. The simplicity of the motif promises that coiled-coil regions can be detected with reasonable accuracy and precision in any protein sequence. Here, we re-evaluated the most commonly used coiled-coil prediction tools with respect to the most comprehensive reference data set available, the entire Protein Data Bank, down to each amino acid and its secondary structure. Apart from the 30-fold difference in minimum and maximum number of coiled coils predicted the tools strongly vary in where they predict coiled-coil regions. Accordingly, there is a high number of false predictions and missed, true coiled-coil regions. The evaluation of the binary classification metrics in comparison with naïve coin-flip models and the calculation of the Matthews correlation coefficient, the most reliable performance metric for imbalanced data sets, suggests that the tested tools' performance is close to random. This implicates that the tools' predictions have only limited informative value. Coiled-coil predictions are often used to interpret biochemical data and are part of in-silico functional genome annotation. Our results indicate that these predictions should be treated very cautiously and need to be supported and validated by experimental evidence.
| S-EPMC8203680 | biostudies-literature
Project description:Autophagy is an intracellular degradation system in which the formation of an autophagosome is a key event. In budding yeast, autophagosomes are generated from the preautophagosomal structure (PAS), in which Atg11 and Atg17 function as scaffolds essential for selective and nonselective types of autophagy, respectively. Structural studies have been extensively performed on Atg17, but not on Atg11, preventing us from understanding the selective type of the PAS. Here, we purified and characterized Atg11. Biophysical analyses, including analytical ultracentrifugation and CD, showed that Atg11 behaves as an elongated homodimer abundant in ?-helices in solution. Moreover, truncation analyses suggested that Atg11 has a parallel coiled-coil architecture, in contrast to the antiparallel dimeric architecture of Atg17.
| S-EPMC5757174 | biostudies-literature
Project description:ATG14 binding to BECN/Beclin homologs is essential for autophagy, a critical catabolic homeostasis pathway. Here, we show that the ?-helical, coiled-coil domain (CCD) of BECN2, a recently identified mammalian BECN1 paralog, forms an antiparallel, curved homodimer with seven pairs of nonideal packing interactions, while the BECN2 CCD and ATG14 CCD form a parallel, curved heterodimer stabilized by multiple, conserved polar interactions. Compared to BECN1, the BECN2 CCD forms a weaker homodimer, but binds more tightly to the ATG14 CCD. Mutation of nonideal BECN2 interface residues to more ideal pairs improves homodimer self-association and thermal stability. Unlike BECN1, all BECN2 CCD mutants bind ATG14, although more weakly than wild type. Thus, polar BECN2 CCD interface residues result in a metastable homodimer, facilitating dissociation, but enable better interactions with polar ATG14 residues stabilizing the BECN2:ATG14 heterodimer. These structure-based mechanistic differences in BECN1 and BECN2 homodimerization and heterodimerization likely dictate competitive ATG14 recruitment.
| S-EPMC5405433 | biostudies-literature
Project description:Delineating functionally normal variants from functionally abnormal variants in tumor suppressor proteins is critical for cancer surveillance, prognosis, and treatment options. BRCA1 is a protein that has many variants of uncertain significance which are not yet classified as functionally normal or abnormal. In vitro functional assays can be used to identify the functional impact of a variant when the variant has not yet been categorized through clinical observation. Here we employ a homology-directed repair (HDR) reporter assay to evaluate over 300 missense and nonsense BRCA1 variants between amino acid residues 1280 and 1576, which encompasses the coiled-coil and serine cluster domains. Functionally abnormal variants tended to cluster in residues known to interact with PALB2, which is critical for homology-directed repair. Multiplexed results were confirmed by singleton assay and by ClinVar database variant interpretations. Comparison of multiplexed results to designated benign or likely benign or pathogenic or likely pathogenic variants in the ClinVar database yielded 100% specificity and 100% sensitivity of the multiplexed assay. Clinicians can reference the results of this functional assay for help in guiding cancer treatment and surveillance options. These results are the first to evaluate this domain of BRCA1 using a multiplexed approach and indicate the importance of this domain in the DNA repair process.
2023-10-13 | GSE237142 | GEO
Project description:As the cellular power plant, mitochondria play a significant role in homeostasis. To maintain the proper quality and quantity of mitochondria requires both mitochondrial degradation and division. A selective type of autophagy, mitophagy, drives the degradation of excess or damaged mitochondria, whereas division is controlled by a specific fission complex; however, the relationship between these two processes, especially the role of mitochondrial fission during mitophagy, remains unclear. In this study, we report that mitochondrial fission is important for the progression of mitophagy. When mitophagy is induced, the fission complex is recruited to the degrading mitochondria through an interaction between Atg11 and Dnm1; interfering with this interaction severely blocks mitophagy. These data establish a paradigm for selective organelle degradation.
| S-EPMC3720741 | biostudies-literature
Project description:Thanatos-associated [THAP] domain-containing apoptosis-associated protein 1 (THAP1) is a DNA-binding protein that has been recently associated with DYT6 dystonia, a hereditary movement disorder involving sustained, involuntary muscle contractions. A large number of dystonia-related mutations have been identified in THAP1 in diverse patient populations worldwide. Previous reports have suggested that THAP1 oligomerizes with itself via a C-terminal coiled-coil domain, raising the possibility that DYT6 mutations in this region might affect this interaction. In this study, we examined the ability of wild-type THAP1 to bind itself and the effects on this interaction of the following disease mutations: C54Y, F81L, ?F132, T142A, I149T, Q154fs180X, and A166T. The results confirmed that wild-type THAP1 associated with itself and most of the DYT6 mutants tested, except for the Q154fs180X variant, which loses most of the coiled-coil domain because of a frameshift at position 154. However, deletion of C-terminal residues after position 166 produced a truncated variant of THAP1 that was able to bind the wild-type protein. The interaction of THAP1 with itself therefore required residues within a 13-amino acid region (aa 154-166) of the coiled-coil domain. Further inspection of this sequence revealed elements highly consistent with previous descriptions of leucine zippers, which serve as dimerization domains in other transcription factor families. Based on this similarity, a structural model was generated to predict how hydrophobic residues in this region may mediate dimerization. These observations offer additional insight into the role of the coiled-coil domain in THAP1, which may facilitate future analyses of DYT6 mutations in this region.
| S-EPMC3166381 | biostudies-literature