Structural and Evolutionary Division of Phosphotyrosine Binding (PTB) Domains

131

151

Transit of proteins through the endosomal organelle following endocytosis is critical for regulating the homeostasis of cell-surface proteins and controlling signal transduction pathways. However, the mechanisms that control these membrane-transport processes are poorly understood. The Phox-homology (PX) domain-containing proteins sorting nexin (SNX) 17, SNX27, and SNX31 have emerged recently as key regulators of endosomal recycling and bind conserved Asn-Pro-Xaa-Tyr-sorting signals in transmembrane cargos via an atypical band, 4.1/ezrin/radixin/moesin (FERM) domain. Here we present the crystal structure of the SNX17 FERM domain bound to the sorting motif of the P-selectin adhesion protein, revealing both the architecture of the atypical FERM domain and the molecular basis for recognition of these essential sorting sequences. We further show that the PX-FERM proteins share a promiscuous ability to bind a wide array of putative cargo molecules, including receptor tyrosine kinases, and propose a model for their coordinated molecular interactions with membrane, cargo, and regulatory proteins.endosome | protein crystallography | X-ray scattering | membrane trafficking T he cell-surface levels of signaling and adhesion receptors, nutrient transporters, ion channels, and many other proteins are tightly regulated by opposing endocytic, exocytic, and endosomal recycling transport pathways. The selective sorting of these transmembrane proteins is the consequence of their interaction with essential adaptor proteins via conserved motifs present in their cytosolic tails, generally based on short linear amino acid sequences such as the Yxxϕ, DxxLL, and [DE]xxxL [LI] motifs (where ϕ is any bulky hydrophobic side-chain, and x is any residue) recognized by clathrin adaptors (1-3). The first identified sorting signal was the Asn-Pro-Xaa-Tyr (NPxY) motif, initially described in the LDL receptor (LDLR) isolated from patients suffering from familial hypercholesterolemia, where mutation of the sequence results in defective internalization and cholesterol uptake (4). The recent structure of the autosomal recessive hypercholesterolemia protein (ARH) phosphotyrosinebinding (PTB) domain in complex with the LDLR intracellular domain (ICD) provides the molecular basis of LDLR recognition and internalization within clathrin-coated pits by endocytic adaptor proteins (5).Although little is known about the sorting sequences and mechanisms required for competing endosome-to-cell surface recycling pathways, emerging evidence shows that the NPxY motif not only is vital to internalization but also aids cargo recycling via organelle-specific recognition by the endosomal protein, sorting nexin 17 (SNX17). SNX17 is a member of the Phox-homology (PX) domain-containing protein family and has been shown to be a critical regulator of endosomal sorting and cell-surface recycling of several essential cargo molecules. These include P-selectin (6-8), amyloid precursor protein (APP) (9, 10), integrins (11,12), and members of the LDL receptor family (13-17) ...

Section: Resultsmentioning

confidence: 99%

Structural basis for endosomal trafficking of diverse transmembrane cargos by PX-FERM proteins

Ghai

Bugarčić

Liu

et al. 2013

131

151

“…The PH fold consists of a β-sandwich structure that contains seven antiparallel β-strands forming two orthogonal β-sheets, and is capped by a C-terminal helix (denoted as α2). Although most PTB domains that have been solved share low levels of sequence identity, structure-based alignments reveal that the core topological structure is evolutionary conserved (19). Like the PTB domains of Shc, disabled, and numb, the Mint1 PTB domain contains an N-terminal helix (α1) inserted between strands β1 and β2 (17,(20)(21)(22).…”

Section: Resultsmentioning

confidence: 99%

“…Thus, the APP peptide binds at a groove formed by strand β5 and helix α2 of the Mint1 PTB domain (Fig. 3B), a binding mode that is shared among PTB do- mains (17,19). However, in the Mint1 PTB* domain structure, the helix α3 formed by the C-terminal extension (termed the "autoinhibitory helix") packs against strand β2 and helices α1 and α2, thereby blocking the center of the APP binding site.…”

Section: Resultsmentioning

confidence: 99%

Autoinhibition of Mint1 adaptor protein regulates amyloid precursor protein binding and processing

Matos

Xu²,

Dulubova

et al. 2012

Mint adaptor proteins bind to the amyloid precursor protein (APP) and regulate APP processing associated with Alzheimer's disease; however, the molecular mechanisms underlying Mint regulation in APP binding and processing remain unclear. Biochemical, biophysical, and cellular experiments now show that the Mint1 phosphotyrosine binding (PTB) domain that binds to APP is intramolecularly inhibited by the adjacent C-terminal linker region. The crystal structure of a C-terminally extended Mint1 PTB fragment reveals that the linker region forms a short α-helix that folds back onto the PTB domain and sterically hinders APP binding. This intramolecular interaction is disrupted by mutation of Tyr633 within the Mint1 autoinhibitory helix leading to enhanced APP binding and β-amyloid production. Our findings suggest that an autoinhibitory mechanism in Mint1 is important for regulating APP processing and may provide novel therapies for Alzheimer's disease.M ints [also known as X11s or amyloid precursor protein (APP) binding, family A] are a family of adaptor proteins encoded by three genes: neuron-specific Mint1 and Mint2, and the ubiquitously expressed Mint3 (1, 2). Mints consist of a divergent N-terminal region and conserved C-terminal sequences composed of a phosphotyrosine binding (PTB) domain and two tandem PDZ (postsynaptic density-95/discs large/zona occludens-1) domains. The PTB domain of all Mints interacts directly with a conserved YENPTY cytoplasmic motif of the APP that is essential for regulating APP trafficking and processing (3-6). APP is a type I transmembrane protein that is sequentially cleaved by site-specific proteases (7-10). Abnormal processing of APP generates Aβ peptides leading to the accumulation of extracellular plaques that are characteristic of Alzheimer's disease (AD). β-Secretase is the first amyloidogenic cleavage, generating a soluble APP ectodomain (sAPPβ) and a C-terminal fragment (CTFβ). CTFβ is subsequently cleaved by γ-secretase (a protein complex of anterior pharyx defective 1, nicastrin, presenilin, and presenilin enhancer 2) to release the small peptides Aβ40 and Aβ42, the latter having an increased propensity to form amyloid aggregates found in the brains of AD patients (11)(12)(13)(14). Alternatively, APP is sequentially cleaved by α-secretase within the Aβ region then γ-secretase, which initiates the nonamyloidogenic or nontoxic pathway (9, 10).Considerable evidence indicates that the APP-Mint interaction is biologically significant because loss of each individual Mint protein delays age-dependent production of amyloid plaques in transgenic AD mouse models (15). Past studies have indicated that the N-and C-terminal regions of Mints play an important role in regulating APP binding and cleavage events based on deletion analysis of Mints in a heterologous expression system (6, 16). The Mint N terminus was shown to promote APP binding and Aβ production, whereas the C-terminal region disrupted PTB-mediated binding to APP and decreased Aβ peptides (6, 16). However, the structural basis ...

“…SH2 and PTB domains are docking sites for protein-protein interaction (19,20). Although PTB stands for phosphotyrosine (pY) binding, many PTB domains can bind their ligands in a pY-independent manner, and the PTB domain in tensin is phylogenetically grouped with this latter class of PTB domains (20).…”

Section: Dlc1 and Dlc3 Bind To Both The Tensin-src Homology 2 (Sh2) Andmentioning

confidence: 99%

Oncogenic inhibition by a deleted in liver cancer gene requires cooperation between tensin binding and Rho-specific GTPase-activating protein activities

Qian¹,

Li²,

Asmussen³

et al. 2007

184

243

The three deleted in liver cancer genes (DLC1-3) encode RhoGTPase-activating proteins (RhoGAPs) whose expression is frequently down-regulated or silenced in a variety of human malignancies. The RhoGAP activity is required for full DLC-dependent tumor suppressor activity. Here we report that DLC1 and DLC3 bind to human tensin1 and its chicken homolog. The binding has been mapped to the tensin Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains at the C terminus of tensin proteins. Distinct DLC1 sequences are required for SH2 and PTB binding. DCL binding to both domains is constitutive under basal conditions. The SH2 binding depends on a tyrosine in DCL1 (Y442) but is phosphotyrosine-independent, a highly unusual feature for SH2 binding. DLC1 competed with the binding of other proteins to the tensin C terminus, including ␤3-integrin binding to the PTB domain. Point mutation of a critical tyrosine residue (Y442F) in DLC1 rendered the protein deficient for binding the tensin SH2 domain and binding full-length tensin. The Y442F protein was diffusely cytoplasmic, in contrast to the localization of wild-type DLC1 to focal adhesions, but it retained the ability to reduce the intracellular levels of Rho-GTP. The Y442F mutant displayed markedly reduced biological activity, as did a mutant that was RhoGAP-deficient. The results suggest that DLC1 is a multifunctional protein whose biological activity depends on cooperation between its tensin binding and RhoGAP activities, although neither activity depends on the other. DLC1 ͉ Src homology 2 and phosphotyrosine binding domains ͉ tumor suppressor gene