Structures and interaction analyses of integrin ?M?2 cytoplasmic tails.
ABSTRACT: Integrins are heterodimeric (? and ? subunits) signal transducer proteins involved in cell adhesions and migrations. The cytosolic tails of integrins are essential for transmitting bidirectional signaling and also implicated in maintaining the resting states of the receptors. In addition, cytosolic tails of integrins often undergo post-translation modifications like phosphorylation. However, the consequences of phosphorylation on the structures and interactions are not clear. The leukocyte-specific integrin ?M?2 is essential for myeloid cell adhesion, phagocytosis, and degranulation. In this work, we determined solution structures of the myristoylated cytosolic tail of ?M and a Ser phosphorylated variant in dodecylphosphocholine micelles by NMR spectroscopy. Furthermore, the interactions between non-phosphorylated and phosphorylated ?M tails with ?2 tail were investigated by NMR and fluorescence resonance energy transfer (FRET). The three-dimensional structures of the 24-residue cytosolic tail of ?M or phosphorylated ?M are characterized by an N-terminal amphipathic helix and a loop at the C terminus. The residues at the loop are involved in packing interactions with the hydrophobic face of the helix. 15N-1H heteronuclear single quantum coherence experiments identified residues of ?M and ?2 tails that may be involved in the formation of a tail-tail heterocomplex. We further examined interactions between myristoylated ?2 tail in dodecylphosphocholine micelles with dansylated ?M tail peptides by FRET. These studies revealed enhanced interactions between ?M or phosphorylated ?M tails with ?2 tail with Kd values ?5.2±0.6 and ?4.4±0.7 ?m, respectively. Docked structures of tail-tail complexes delineated that the ?M/?2 interface at the cytosolic region could be sustained by a network of polar interactions, ionic interactions, and/or hydrogen bonds.
Project description:<h4>Background</h4>Integrins are signal transducer proteins involved in a number of vital physiological processes including cell adhesion, proliferation and migration. Integrin molecules are hetero-dimers composed of two distinct subunits, ? and ?. In humans, 18 ? and 8 ? subunits are combined into 24 different integrin molecules. Each of the subunit comprises a large extracellular domain, a single pass transmembrane segment and a cytosolic tail (CT). The CTs of integrins are vital for bidirectional signal transduction and in maintaining the resting state of the receptors. A large number of intracellular proteins have been found to interact with the CTs of integrins linking integrins to the cytoskeleton.<h4>Methodology/principal findings</h4>In this work, we have investigated structure and interactions of CTs of the leukocyte specific integrin ?X?2. We determined the atomic resolution structure of a myristoylated CT of ?X in perdeuterated dodecylphosphocholine (DPC) by NMR spectroscopy. Our results reveal that the 35-residue long CT of ?X adopts an ?-helical conformation for residues F4-N17 at the N-terminal region. The remaining residues located at the C-terminal segment of ?X delineate a long loop of irregular conformations. A segment of the loop maintains packing interactions with the helical structure by an extended non-polar surface of the ?X CT. Interactions between ?X and ?2 CTs are demonstrated by (15)N-(1)H HSQC NMR experiments. We find that residues constituting the polar face of the helical conformation of ?X are involved in interactions with the N-terminal residues of ?2 CT. A docked structure of the CT complex indicates that a network of polar and/or salt-bridge interactions may sustain the heteromeric interactions.<h4>Conclusions/significance</h4>The current study provides important insights into the conservation of interactions and structures among different CTs of integrins.
Project description:The Shc adaptor protein, particularly its p52 isoform, has been identified as a primary signaling partner for the tyrosine(s)-phosphorylated cytoplasmic tails of activated β3 integrins. Inspired by our recent structure of the Shc PTB domain in complex with a bi-phosphorylated peptide derived from β3 cytoplasmic tail, we have initiated the investigation of Shc interaction with phospholipids of the membrane. We are particularly focused on PtdIns and their effects on Shc mediated integrin signaling in vitro. Here we present thermodynamic profiles and molecular details of the interactions between Shc, integrin, and PtdIns, all of which have been studied by ITC and solution NMR methods. A model of p52 Shc interaction with phosphorylated β3 integrin cytoplasmic tail at the cytosolic face of the plasma membrane is proposed based on these data.
Project description:Integrins are involved in cell migration and adhesion. A large number of proteins interact with the cytoplasmic tails of integrins. Dok1 is a negative regulator of integrin activation and it binds to the phosphorylated membrane proximal NxxY motif in a number of integrin ? tails. The ? tail of the ?2 integrins contains a non-phosphorylatable NxxF motif. Hence it is unclear how Dok1 associates with the ?2 integrins. We showed in this study using NMR and cell based analyses that residues Ser745 and Ser756 in the integrin ?2 tail, which are adjacent to the NxxF motif, are required for Dok1 interaction. NMR analyses detected significant chemical shift changes and higher affinity interactions between Dok1 phospho-tyrosine binding (PTB) domain and integrin ?2 tail peptide containing pSer756 compared to pSer745. The phosphorylated ?2 peptide occupies the canonical ligand binding pocket of Dok1 based on the docked structure of the ?2 tail-Dok1 PTB complex. Taken together, our data suggest an alternate phosphorylation switch in ?2 integrins that regulates Dok1 binding. This could be important for cells of the immune system and their functions.
Project description:Integrins are a family of heterodimeric (?+?) adhesion receptors that play key roles in many cellular processes. Integrins are unusual in that their functions can be modulated from both outside and inside the cell. Inside-out signaling is mediated by binding adaptor proteins to the flexible cytoplasmic tails of the ?- and ?-integrin subunits. Talin is one well-known intracellular activator, but various other adaptors bind to integrin tails, including 14-3-3-?, ?a member of the 14-3-3 family of dimeric proteins that have a preference for binding phosphorylated sequence motifs. Phosphorylation of a threonine in the ?2 integrin tail has been shown to modulate ?2/14-3-3-? interactions, and recently, the ?4 integrin tail was reported to bind to 14-3-3-? and associate with paxillin in a ternary complex that is regulated by serine phosphorylation. Here, we use a range of biophysical techniques to characterize interactions between 14-3-3-? and the cytoplasmic tails of ?4, ?1, ?2 and ?3 integrins. The X-ray structure of the 14-3-3-?/?4 complex indicates a canonical binding mode for the ?4 phospho-peptide, but unexpected features are also observed: residues outside the consensus 14-3-3-? binding motif are shown to be essential for an efficient interaction; in contrast, a short ?2 phospho-peptide is sufficient for high-affinity binding to 14-3-3-?. In addition, we report novel 14-3-3-?/integrin tail interactions that are independent of phosphorylation. Of the integrin tails studied, the strongest interaction with 14-3-3-? is observed for the ?1A variant. In summary, new insights about 14-3-3-?/integrin tail interactions that have implications for the role of these molecular associations in cells are described.
Project description:Integrins are large membrane-spanning receptors fundamental to cell adhesion and migration. Integrin adhesiveness for the extracellular matrix is activated by the cytoskeletal protein talin via direct binding of its phosphotyrosine-binding-like F3 domain to the cytoplasmic tail of the beta integrin subunit. The phosphotyrosine-binding domain of the signaling protein Dok1, on the other hand, has an inactivating effect on integrins, a phenomenon that is modulated by integrin tyrosine phosphorylation. Using full-length tyrosine-phosphorylated (15)N-labeled beta3, beta1A, and beta7 integrin tails and an NMR-based protein-protein interaction assay, we show that talin1 binds to the NPXY motif and the membrane-proximal portion of beta3, beta1A, and beta7 tails, and that the affinity of this interaction is decreased by integrin tyrosine phosphorylation. Dok1 only interacts weakly with unphosphorylated tails, but its affinity is greatly increased by integrin tyrosine phosphorylation. The Dok1 interaction remains restricted to the integrin NPXY region, thus phosphorylation inhibits integrin activation by increasing the affinity of beta integrin tails for a talin competitor that does not form activating membrane-proximal interactions with the integrin. Key residues governing these specificities were identified by detailed structural analysis, and talin1 was engineered to bind preferentially to phosphorylated integrins by introducing the mutation D372R. As predicted, this mutation affects talin1 localization in live cells in an integrin phosphorylation-specific manner. Together, these results indicate that tyrosine phosphorylation is a common mechanism for regulating integrin activation, despite subtle differences in how these integrins interact with their binding proteins.
Project description:Integrins require an activation step before ligand binding and signaling that is mediated by talin and kindlin binding to the β integrin cytosolic domain (β-tail). Conflicting reports exist about the contribution of phosphorylation of a conserved threonine motif in the β1-tail (β1-pT788/pT789) to integrin activation. We show that widely used and commercially available antibodies against β1-pT788/pT789 integrin do not detect specific β1-pT788/pT789 integrin signals in immunoblots of several human and mouse cell lysates but bind bi-phosphorylated threonine residues in numerous proteins, which were identified by mass spectrometry experiments. Furthermore, we found that fibroblasts and epithelial cells expressing the phospho-mimicking β1-TT788/789DD integrin failed to activate β1 integrins and displayed reduced integrin ligand binding, adhesion initiation and cell spreading. These cellular defects are specifically caused by the inability of kindlin to bind β1-tail polypeptides carrying a phosphorylated threonine motif or phospho-mimicking TT788/789DD substitutions. Our findings indicate that the double-threonine motif in β1-class integrins is not a major phosphorylation site but if phosphorylated would curb integrin function.
Project description:Integrins are a family of alpha/beta heterodimeric membrane proteins, which mediate cell-cell and cell-matrix interactions. The molecular mechanisms by which integrins are activated and cluster are currently poorly understood. One hypothesis posits that the cytoplasmic tails of the alpha and beta subunits interact strongly with one another in a 1:1 interaction, and that this interaction is modulated in the course of the activation of alphaIIbbeta3 [Hughes, P. E., et al. (1996) J. Biol. Chem. 271, 6571-6574]. To examine the structural basis for this interaction, protein fragments encompassing the transmembrane helix plus cytoplasmic tails of the alpha and beta subunits of alphaIIbbeta3 were expressed and studied in phospholipid micelles at physiological salt concentrations. Analyses of these fragments by analytical ultracentrifugation, NMR, circular dichroism, and electrophoresis indicated that they had very little or no tendency to interact with one another. Instead, they formed homomeric interactions, with the alpha- and beta-fragments forming dimers and trimers, respectively. Thus, these regions of the protein structure may contribute to the clustering of integrins that accompanies cellular adhesion.
Project description:The first reported receptor for SARS-CoV-2 on host cells was the angiotensin-converting enzyme 2 (ACE2). However, the viral spike protein also has an RGD motif, suggesting that cell surface integrins may be co-receptors. We examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif (ELM) resource and identified candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton, and cell signaling. These SLiM candidates are highly conserved in vertebrates and may interact with the μ2 subunit of the endocytosis-associated AP2 adaptor complex, as well as with various protein domains (namely, I-BAR, LC3, PDZ, PTB, and SH2) found in human signaling and regulatory proteins. Several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, such as in response to tyrosine phosphorylation status. Candidate LC3-interacting region (LIR) motifs are present in the tails of integrin β<sub>3</sub> and ACE2, suggesting that these proteins could directly recruit autophagy components. Our findings identify several molecular links and testable hypotheses that could uncover mechanisms of SARS-CoV-2 attachment, entry, and replication against which it may be possible to develop host-directed therapies that dampen viral infection and disease progression. Several of these SLiMs have now been validated to mediate the predicted peptide interactions.
Project description:Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation.
Project description:The adhesion of integrins to the extracellular matrix is regulated by binding of the cytoskeletal protein talin to the cytoplasmic tail of the ?-integrin subunit. Structural studies of this interaction have hitherto largely focused on the ?3-integrin, one member of the large and diverse integrin family. Here, we employ NMR to probe interactions and dynamics, revealing marked structural diversity in the contacts between ?1A, ?1D, and ?3 tails and the Talin1 and Talin2 isoforms. Coupled with analysis of recent structures of talin/? tail complexes, these studies elucidate the thermodynamic determinants of this heterogeneity and explain why the Talin2/?1D isoforms, which are co-localized in striated muscle, form an unusually tight interaction. We also show that talin/integrin affinity can be enhanced 1000-fold by deleting two residues in the ? tail. Together, these studies illustrate how the integrin/talin interaction has been fine-tuned to meet varying biological requirements.