Crystal structure of the phosphatidylinositol 3,4-bisphosphate-binding pleckstrin homology (PH) domain of tandem PH-domain-containing protein 1 (TAPP1): molecular basis of lipid specificity

2001 ◽  
Vol 358 (2) ◽  
pp. 287-294 ◽  
Author(s):  
Christine C. THOMAS ◽  
Simon DOWLER ◽  
Maria DEAK ◽  
Dario R. ALESSI ◽  
Daan M.F. van AALTEN
Keyword(s):  
Crystal Structure ◽  
Binding Sites ◽  
Inositol Phosphate ◽  
Second Messengers ◽  
Breakdown Product ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Phosphate Binding ◽  
Lipid Specificity ◽  
Similar Affinity

Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and its immediate breakdown product PtdIns(3,4)P2 function as second messengers in growth factor- and insulin-induced signalling pathways. One of the ways that these 3-phosphoinositides are known to regulate downstream signalling events is by attracting proteins that possess specific PtdIns-binding pleckstrin homology (PH) domains to the plasma membrane. Many of these proteins, such as protein kinase B, phosphoinositide-dependent kinase 1 and the dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1) interact with both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 with similar affinity. Recently, a new PH-domain-containing protein, termed tandem PH-domain-containing protein (TAPP) 1, was described which is the first protein reported to bind PtdIns(3,4)P2 specifically. Here we describe the crystal structure of the PtdIns(3,4)P2-binding PH domain of TAPP1 at 1.4 Å (1 Å = 0.1 nm) resolution in complex with an ordered citrate molecule. The structure is similar to the known structure of the PH domain of DAPP1 around the D-3 and D-4 inositol-phosphate-binding sites. However, a glycine residue adjacent to the D-5 inositol-phosphate-binding site in DAPP1 is substituted for a larger alanine residue in TAPP1, which also induces a conformational change in the neighbouring residues. We show that mutation of this glycine to alanine in DAPP1 converts DAPP1 into a TAPP1-like PH domain that only interacts with PtdIns(3,4)P2, whereas the alanine to glycine mutation in TAPP1 permits the TAPP1 PH domain to interact with PtdIns(3,4,5)P3.

Molecular modelling and site-directed mutagenesis of the inositol 1,3,4,5-tetrakisphosphate-binding pleckstrin homology domain from the Ras GTPase-activating protein GAP1IP4BP

2000 ◽  
Vol 349 (1) ◽  
pp. 333-342 ◽  
Author(s):  
Gyles COZIER ◽  
Richard SESSIONS ◽  
Joanna R. BOTTOMLEY ◽  
Jon S. REYNOLDS ◽  
Peter J. CULLEN
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Inositol Phosphate ◽  
Site Directed Mutagenesis ◽  
Pleckstrin Homology Domain ◽  
Directed Mutagenesis ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Gtpase Activating Protein ◽  
Ras Gtpase

GAP1IP4BP is a Ras GTPase-activating protein (GAP) that in vitro is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. We have studied Ins(1,3,4,5)P4 binding to GAP1IP4BP, and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)P4 binding to Bruton's tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)P4 binding. The crystal structure of the Btk pleckstrin homology (PH) domain in complex with Ins(1,3,4,5)P4 has shown that the binding site is located in a partially buried pocket between the β1/β2- and β3/β4-loops. Many of the residues involved in the binding are conserved in GAP1IP4BP. Therefore we generated a model of the PH domain of GAP1IP4BP in complex with Ins(1,3,4,5)P4 based on the Btk-Ins(1,3,4,5)P4 complex crystal structure. This model had the typical PH domain fold, with the proposed binding site modelling well on the Btk structure. The model has been verified by site-directed mutagenesis of various residues in and around the proposed binding site. These mutations have markedly reduced affinity for Ins(1,3,4,5)P4, indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding.

Phosphoinositide 3-kinase-dependent regulation of phospholipase Cγ

2007 ◽  
Vol 35 (2) ◽  
pp. 229-230 ◽  
Author(s):  
T. Maffucci ◽  
M. Falasca
Keyword(s):  
Phospholipase C ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Second Messengers ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Functional Roles ◽  
Phosphoinositide 3 Kinase

Activation of the enzyme PLC (phospholipase C) leads to the formation of second messengers Ins(1,4,5)P3 and diacylglycerol. RTKs (receptor tyrosine kinases) activate this reaction through PLCγ isoenzymes. It has been shown that PI3K (phosphoinositide 3-kinase) may regulate PLCγ activity through the interaction of PI3K product PtdIns(3,4,5)P3 and the PLCγ PH domain (pleckstrin homology domain). Here, we analyse the potential functional roles of the PI3K/PLC pathway.

scholarly journals Systematic simulation of the interactions of Pleckstrin homology domains with membranes

2021 ◽  
Author(s):  
Kyle I.P. Le Huray ◽  
He Wang ◽  
Frank Sobott ◽  
Antreas C Kalli
Keyword(s):  
Crystal Structure ◽  
Membrane Association ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Interaction Site ◽  
The Family ◽  
Family Level ◽  
Ph Domains ◽  
Anionic Lipids ◽  
Phosphatidylinositol Phosphates

Pleckstrin homology (PH) domains can recruit proteins to membranes by recognition of phosphatidylinositol phosphates (PIPs). Here we report the systematic simulation of the interactions of 100 mammalian PH domains with PIP containing model membranes. Comparison with crystal structures of PH domains bound to PIP analogues demonstrates that our method correctly identifies interactions at known canonical and non-canonical sites, while revealing additional functionally important sites for interaction not observed in the crystal structure, such as for P-Rex1 and Akt1. At the family level, we find that the β1 and β2 strands and their connecting loop constitute the primary PIP interaction site for the majority of PH domains, but we highlight interesting exceptional cases. Simultaneous interaction with multiple PIPs and clustering of PIPs induced by PH domain binding are also observed. Our findings support a general paradigm for PH domain membrane association involving multivalent interactions with anionic lipids.

scholarly journals Structure of autoinhibited Akt1 reveals mechanism of PIP3-mediated activation

2021 ◽  
Vol 118 (33) ◽  
pp. e2101496118
Author(s):  
Linda Truebestein ◽  
Harald Hornegger ◽  
Dorothea Anrather ◽  
Markus Hartl ◽  
Kaelin D. Fleming ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Growth Factor ◽  
Protein Kinase ◽  
Second Messengers ◽  
Kinase Domain ◽  
Activation Loop ◽  
Growth Factor Signaling ◽  
Ph Domain ◽  
Protein Kinase Akt ◽  
Spatiotemporal Control

The protein kinase Akt is one of the primary effectors of growth factor signaling in the cell. Akt responds specifically to the lipid second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] via its PH domain, leading to phosphorylation of its activation loop and the hydrophobic motif of its kinase domain, which are critical for activity. We have now determined the crystal structure of Akt1, revealing an autoinhibitory interface between the PH and kinase domains that is often mutated in cancer and overgrowth disorders. This interface persists even after stoichiometric phosphorylation, thereby restricting maximum Akt activity to PI(3,4,5)P3- or PI(3,4)P2-containing membranes. Our work helps to resolve the roles of lipids and phosphorylation in the activation of Akt and has wide implications for the spatiotemporal control of Akt and potentially lipid-activated kinase signaling in general.

scholarly journals Localization of a high-affinity inositol 1,4,5-trisphosphate/inositol 1,4,5,6-tetrakisphosphate binding domain to the pleckstrin homology module of a new 130 kDa protein: characterization of the determinants of structural specificity

1996 ◽  
Vol 318 (2) ◽  
pp. 561-568 ◽  
Author(s):  
Hiroshi TAKEUCHI ◽  
Takashi KANEMATSU ◽  
Yoshio MISUMI ◽  
Hassan Bin YAAKOB ◽  
Hitoshi YAGISAWA ◽  
...  
Keyword(s):  
Inositol Phosphate ◽  
Expression System ◽  
Specific Radioactivity ◽  
Binding Activity ◽  
Protein Characterization ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Binding Studies ◽  
High Specific Radioactivity ◽  
Ph Domains

We have previously identified a novel 130 kDa protein (p130) which binds Ins(1,4,5)P3 and shares 38% sequence identity with phospholipase C-Δ1 [Kanematsu, Misumi, Watanabe, Ozaki, Koga, Iwanaga, Ikehara and Hirata (1996) Biochem. J. 313, 319–325]. We have now transfected COS-1 cells with genes encoding the entire length of the molecule or one of several truncated mutants, in order to locate the region for binding of Ins(1,4,5)P3. Deletion of N-terminal residues 116–232, the region which corresponds to the pleckstrin homology (PH) domain of the molecule, completely abolished binding activity. This result was confirmed when the PH domain itself (residues 95–232), isolated from a bacterial expression system, was found to bind [3H]Ins(1,4,5)P3. We also found that Ins(1,4,5,6)P4 was as efficacious as Ins(1,4,5)P3 in displacing [3H]Ins(1,4,5)P3, suggesting that these two polyphosphates bind to p130 with similar affinity. This conclusion was confirmed by direct binding studies using [3H]Ins(1,4,5,6)P4 with high specific radioactivity which we prepared ourselves. Binding specificity was also examined with a variety of inositol phosphate derivatives. As is the case with other PH domains characterized to date, we found that the 4,5-vicinal phosphate pair was an essential determinant of ligand specificity. However, the PH domain of p130 exhibited some novel features. For example, the 3- and/or 6-phosphates could also contribute to overall binding; this contrasts with some other PH domains where these phosphate groups decrease ligand affinity by imposing a steric constraint. Secondly, a free monoester 1-phosphate substantially increased binding affinity, which is a situation so far unique to the PH domain of p130.

New phosphate binding sites in the crystal structure of Escherichia coli purine nucleoside phosphorylase complexed with phosphate and formycin A

FEBS Letters ◽  
2012 ◽  
Vol 586 (7) ◽  
pp. 967-971 ◽  
Author(s):  
Zoran Štefanić ◽  
Marta Narczyk ◽  
Goran Mikleušević ◽  
Beata Wielgus-Kutrowska ◽  
Agnieszka Bzowska ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Binding Sites ◽  
Purine Nucleoside Phosphorylase ◽  
Purine Nucleoside ◽  
Phosphate Binding ◽  
Nucleoside Phosphorylase

scholarly journals Detection of novel intracellular agonist responsive pools of phosphatidylinositol 3,4-bisphosphate using the TAPP1 pleckstrin homology domain in immunoelectron microscopy

2004 ◽  
Vol 377 (3) ◽  
pp. 653-663 ◽  
Author(s):  
Stephen A. WATT ◽  
Wendy A. KIMBER ◽  
Ian N. FLEMING ◽  
Nick R. LESLIE ◽  
C. Peter DOWNES ◽  
...  
Keyword(s):  
Immunoelectron Microscopy ◽  
Lipid Binding ◽  
Differential Sensitivity ◽  
Breakdown Product ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Glutathione S Transferase ◽  
Signalling Molecule

PtdIns(3,4)P2, a breakdown product of the lipid second messenger PtdIns(3,4,5)P3, is a key signalling molecule in pathways controlling various cellular events. Cellular levels of PtdIns(3,4)P2 are elevated upon agonist stimulation, mediating downstream signalling pathways by recruiting proteins containing specialized lipid-binding modules, such as the pleckstrin homology (PH) domain. A recently identified protein, TAPP1 (tandem-PH-domain-containing protein 1), has been shown to interact in vitro with high affinity and specificity with PtdIns(3,4)P2 through its C-terminal PH domain. In the present study, we have utilized this PH domain tagged with glutathione S-transferase (GST–TAPP1-PH) as a probe in an on-section immunoelectron microscopy labelling procedure, mapping the subcellular distribution of PtdIns(3,4)P2. As expected, we found accumulation of PtdIns(3,4)P2 at the plasma membrane in response to the agonists platelet-derived growth factor and hydrogen peroxide. Importantly, however, we also found agonist stimulated PtdIns(3,4)P2 labelling of intracellular organelles, including the endoplasmic reticulum and multivesicular endosomes. Expression of the 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) in PTEN-null U87MG cells revealed differential sensitivity of these lipid pools to the enzyme. These data suggest a role for PtdIns(3,4)P2 in endomembrane function.

scholarly journals Structural basis for the association of PLEKHA7 with membrane-embedded phosphatidylinositol lipids

2020 ◽  
Author(s):  
Alexander E. Aleshin ◽  
Yong Yao ◽  
Amer Iftikhar ◽  
Andrey A. Bobkov ◽  
Jinghua Yu ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Cellular Localization ◽  
Inositol Phosphate ◽  
Phosphatidyl Inositol ◽  
Structural Data ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology

SummaryPLEKHA7 (pleckstrin homology domain containing family A member 7) plays key roles in intracellular signaling, cytoskeletal organization and cell adhesion, and is associated with multiple human cancers. The interactions of its pleckstrin homology (PH) domain with membrane phosphatidyl-inositol-phosphate (PIP) lipids, are critical for proper cellular localization and function, and their inhibition is an attractive target for anti-cancer therapy. While structural data can provide insights in this area, little is known about the way in which PLEKHA7 and other PH domains interact with membrane-embedded PIPs. Here we report atomic-resolution structures of the PLEHA7 PH domain and describe the molecular mechanism for its recognition of membrane-bound PIPs. Using X-ray crystallography, nuclear magnetic resonance (NMR), molecular dynamics (MD) simulations, and isothermal titration calorimetry (ITC), we show – in atomic-level detail – that the interaction of PLEKHA7 with PIPs is multivalent and induces PIP clustering. The PIP binding mechanism is distinct from a discrete one-to-one interaction. Our findings reveal a central role of the membrane assembly in mediating protein-PIP association and provide a roadmap for the design of PLEKHA7-PIP inhibitors.

scholarly journals Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain

2017 ◽  
Vol 474 (4) ◽  
pp. 539-556 ◽  
Author(s):  
Tao Ni ◽  
Antreas C. Kalli ◽  
Fiona B. Naughton ◽  
Luke A. Yates ◽  
Omar Naneh ◽  
...  
Keyword(s):  
Inositol Phosphate ◽  
Lipid Binding ◽  
Site Directed Mutagenesis ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Phosphatidylinositol Phosphate ◽  
Protein Membrane ◽  
Dynamics Simulations ◽  
Lipid Nanodiscs

Kindlins co-activate integrins alongside talin. They possess, like talin, a FERM domain (4.1-erythrin–radixin–moiesin domain) comprising F0–F3 subdomains, but with a pleckstrin homology (PH) domain inserted in the F2 subdomain that enables membrane association. We present the crystal structure of murine kindlin-3 PH domain determined at a resolution of 2.23 Å and characterise its lipid binding using biophysical and computational approaches. Molecular dynamics simulations suggest flexibility in the PH domain loops connecting β-strands forming the putative phosphatidylinositol phosphate (PtdInsP)-binding site. Simulations with PtdInsP-containing bilayers reveal that the PH domain associates with PtdInsP molecules mainly via the positively charged surface presented by the β1–β2 loop and that it binds with somewhat higher affinity to PtdIns(3,4,5)P3 compared with PtdIns(4,5)P2. Surface plasmon resonance (SPR) with lipid headgroups immobilised and the PH domain as an analyte indicate affinities of 300 µM for PtdIns(3,4,5)P3 and 1 mM for PtdIns(4,5)P2. In contrast, SPR studies with an immobilised PH domain and lipid nanodiscs as the analyte show affinities of 0.40 µM for PtdIns(3,4,5)P3 and no affinity for PtdIns(4,5)P2 when the inositol phosphate constitutes 5% of the total lipids (∼5 molecules per nanodisc). Reducing the PtdIns(3,4,5)P3 composition to 1% abolishes nanodisc binding to the PH domain, as does site-directed mutagenesis of two lysines within the β1–β2 loop. Binding of PtdIns(3,4,5)P3 by a canonical PH domain, Grp1, is not similarly influenced by SPR experimental design. These data suggest a role for PtdIns(3,4,5)P3 clustering in the binding of some PH domains and not others, highlighting the importance of lipid mobility and clustering for the biophysical assessment of protein–membrane interactions.

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