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.

scholarly journals Phosphotyrosine protein of molecular mass 30 kDa binds specifically to the positively charged region of the pleckstrin N-terminal pleckstrin homology domain

1999 ◽  
Vol 342 (2) ◽  
pp. 423-430
Author(s):  
Limin LIU ◽  
Mary MAKOWSKE
Keyword(s):  
Binding Site ◽  
Inositol Phosphates ◽  
Site Directed Mutagenesis ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Glutathione S Transferase ◽  
Protein Protein Interaction ◽  
Ph Domains ◽  
Positively Charged

It has been proposed that phosphoinositides and inositol phosphates serve as general ligands for members of the structurally related pleckstrin homology (PH) domain family. The N-terminal PH domain of pleckstrin (N-PH), in contrast with other PH domains, does not bind to any of these ligands with the high affinity expected for a physiological interaction. To examine whether N-PH might instead mediate protein-protein interaction, a fusion protein with glutathione S-transferase (GST) expressing N-PH (GST-N-PH) was used to screen [35S]methionine metabolically labelled HL-60 and Bac1.2F5 cell lysates for potential binding partners. A 30 kDa binding protein was identified in both cell lines. Binding to N-PH demonstrated specificity, because binding was approx. 10-fold higher than when an equimolar amount of pleckstrin C-terminal PH domain (GST-C-PH) was used as probe. The 30 kDa protein could also be metabolically labelled with [32P]Pi and proved to be a tyrosine-phosphorylated protein. Binding to N-PH could be specifically inhibited with phosphotyrosine but not with phosphothreonine; the inhibition was concentration-dependent. Site-directed mutagenesis indicated that a positively charged region previously identified as the phosphoinositide-binding site in N-PH and other PH domains, rather than a putative phosphotyrosine-binding region previously identified in structurally similar phosphotyrosine-binding (PTB) domains, served as the binding site. These results suggest that the positively charged region of N-PH has the potential to interact with a protein ligand that contains phosphotyrosine.

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.

scholarly journals Myo1c Binds Phosphoinositides through a Putative Pleckstrin Homology Domain

2006 ◽  
Vol 17 (11) ◽  
pp. 4856-4865 ◽  
Author(s):  
David E. Hokanson ◽  
Joseph M. Laakso ◽  
Tianming Lin ◽  
David Sept ◽  
E. Michael Ostap
Keyword(s):  
Binding Site ◽  
Membrane Trafficking ◽  
Cellular Localization ◽  
Membrane Binding ◽  
Pleckstrin Homology Domain ◽  
Dependent Manner ◽  
Ph Domain ◽  
Pleckstrin Homology

Myo1c is a member of the myosin superfamily that binds phosphatidylinositol-4,5-bisphosphate (PIP2), links the actin cytoskeleton to cellular membranes and plays roles in mechano-signal transduction and membrane trafficking. We located and characterized two distinct membrane binding sites within the regulatory and tail domains of this myosin. By sequence, secondary structure, and ab initio computational analyses, we identified a phosphoinositide binding site in the tail to be a putative pleckstrin homology (PH) domain. Point mutations of residues known to be essential for polyphosphoinositide binding in previously characterized PH domains inhibit myo1c binding to PIP2 in vitro, disrupt in vivo membrane binding, and disrupt cellular localization. The extended sequence of this binding site is conserved within other myosin-I isoforms, suggesting they contain this putative PH domain. We also characterized a previously identified membrane binding site within the IQ motifs in the regulatory domain. This region is not phosphoinositide specific, but it binds anionic phospholipids in a calcium-dependent manner. However, this site is not essential for in vivo membrane binding.

scholarly journals Binding of phosphatidylinositol 3,4,5-trisphosphate to the pleckstrin homology domain of protein kinase B induces a conformational change

2003 ◽  
Vol 375 (3) ◽  
pp. 531-538 ◽  
Author(s):  
Christine C. MILBURN ◽  
Maria DEAK ◽  
Sharon M. KELLY ◽  
Nick C. PRICE ◽  
Dario R. ALESSI ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Binding Site ◽  
Conformational Change ◽  
Conformational Changes ◽  
Protein Kinase B ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Variable Loop ◽  
Solution Studies

Protein kinase B (PKB/Akt) is a key regulator of cell growth, proliferation and metabolism. It possesses an N-terminal pleckstrin homology (PH) domain that interacts with equal affinity with the second messengers PtdIns(3,4,5)P3 and PtdIns(3,4)P2, generated through insulin and growth factor-mediated activation of phosphoinositide 3-kinase (PI3K). The binding of PKB to PtdIns(3,4,5)P3/PtdIns(3,4)P2 recruits PKB from the cytosol to the plasma membrane and is also thought to induce a conformational change that converts PKB into a substrate that can be activated by the phosphoinositide-dependent kinase 1 (PDK1). In this study we describe two high-resolution crystal structures of the PH domain of PKBα in a noncomplexed form and compare this to a new atomic resolution (0.98 Å, where 1 Å=0.1 nm) structure of the PH domain of PKBα complexed to Ins(1,3,4,5)P4, the head group of PtdIns(3,4,5)P3. Remarkably, in contrast to all other PH domains crystallized so far, our data suggest that binding of Ins(1,3,4,5)P4 to the PH domain of PKB, induces a large conformational change. This is characterized by marked changes in certain residues making up the phosphoinositide-binding site, formation of a short α-helix in variable loop 2, and a movement of variable loop 3 away from the lipid-binding site. Solution studies with CD also provided evidence of conformational changes taking place upon binding of Ins(1,3,4,5)P4 to the PH domain of PKB. Our data provides the first structural insight into the mechanism by which the interaction of PKB with PtdIns(3,4,5)P3/PtdIns(3,4)P2 induces conformational changes that could enable PKB to be activated by PDK1.

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.

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 Membrane activity of the phospholipase C-δ1 pleckstrin homology (PH) domain

2005 ◽  
Vol 389 (2) ◽  
pp. 435-441 ◽  
Author(s):  
Frits M. Flesch ◽  
Jong W. Yu ◽  
Mark A. Lemmon ◽  
Koert N. J. Burger
Keyword(s):  
Binding Site ◽  
Phospholipase C ◽  
Membrane Surface ◽  
Pleckstrin Homology Domain ◽  
Membrane Insertion ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Lipid Mixture ◽  
Membrane Activity ◽  
Binding Domains

PH-PLCδ1 [the PH domain (pleckstrin homology domain) of PLCδ1 (phospholipase C-δ1)] is among the best-characterized phosphoinositide-binding domains. PH-PLCδ1 binds with high specificity to the headgroup of PtdIns(4,5)P2, but little is known about its interfacial properties. In the present study, we show that PH-PLCδ1 is also membrane-active and can insert significantly into PtdIns(4,5)P2-containing monolayers at physiological (bilayer-equivalent) surface pressures. However, this membrane activity appears to involve interactions distinct from those that target PH-PLCδ1 to the PtdIns(4,5)P2 headgroup. Whereas the majority of PtdIns(4,5)P2-bound PH-PLCδ1 can be displaced by adding excess of soluble headgroup [Ins(1,4,5)P3], membrane activity of PH-PLCδ1 cannot. PH-PLCδ1 differs from other phosphoinositide-binding domains in that its membrane insertion does not require that the phosphoinositide-binding site be occupied. Significant monolayer insertion remains when the phosphoinositide-binding site is mutated, and PH-PLCδ1 can insert into monolayers that contain no PtdIns(4,5)P2 at all. Our results suggest a model in which reversible membrane binding of PH-PLCδ1, mediated by PtdIns(4,5)P2 or other acidic phospholipids, occurs without membrane insertion. Accumulation of the PH domain at the membrane surface enhances the efficiency of insertion, but does not significantly affect its extent, whereas the presence of phosphatidylethanolamine and cholesterol in the lipid mixture promotes the extent of insertion. This is the first report of membrane activity in an isolated PH domain and has implications for understanding the membrane targeting by this common type of domain.

scholarly journals Crystal structure of CotA laccase complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel binding site

2016 ◽  
Vol 72 (4) ◽  
pp. 328-335 ◽  
Author(s):  
Zhongchuan Liu ◽  
Tian Xie ◽  
Qiuping Zhong ◽  
Ganggang Wang
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
The Novel ◽  
Wild Type ◽  
Outer Coat ◽  
Cota Laccase ◽  
Key Residues

The CotA laccase fromBacillus subtilisis an abundant component of the spore outer coat and has been characterized as a typical laccase. The crystal structure of CotA complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in a hole motif has been solved. The novel binding site was about 26 Å away from the T1 binding pocket. Comparison with known structures of other laccases revealed that the hole is a specific feature of CotA. The key residues Arg476 and Ser360 were directly bound to ABTS. Site-directed mutagenesis studies revealed that the residues Arg146, Arg429 and Arg476, which are located at the bottom of the novel binding site, are essential for the oxidation of ABTS and syringaldazine. Specially, a Thr480Phe variant was identified to be almost 3.5 times more specific for ABTS than for syringaldazine compared with the wild type. These results suggest this novel binding site for ABTS could be a potential target for protein engineering of CotA laccases.

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