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.

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 Phosphoinositide Binding by the Pleckstrin Homology Domains of Ipl and Tih1

2002 ◽  
Vol 277 (51) ◽  
pp. 49935-49944 ◽  
Author(s):  
Anjana Saxena ◽  
Pavel Morozov ◽  
Dale Frank ◽  
Raymond Musalo ◽  
Mark A. Lemmon ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Phosphatidylinositol 3 Kinase ◽  
Sequence Comparisons ◽  
Phosphatidylinositol Phosphate ◽  
Bona Fide ◽  
Epidermal Growth ◽  
Green Fluorescent

The Ipl protein consists of a single pleckstrin homology (PH) domain with short N- and C-terminal extensions. This protein is highly conserved among vertebrates, and it acts to limit placental growth in mice. However, its biochemical function is unknown. The closest paralogue of Ipl is Tih1, another small PH domain protein. By sequence comparisons, Ipl and Tih1 define an outlying branch of the PH domain superfamily. Here we describe phosphatidylinositol phosphate (PIP) binding by these proteins. Ipl and Tih1 bind to immobilized PIPs with moderate affinity, but this binding is weaker and more promiscuous than that of prototypical PH domains from the general receptor for phosphoinositides (GRP1), phospholipase C δ1, and dual adaptor for phosphoinositides and phosphotyrosine 1. In COS7 cells exposed to epidermal growth factor, green fluorescent protein (GFP)-Ipl and GFP-Tih1 accumulate at membrane ruffles without clearing from the cytoplasm, whereas control GFP-GRP1 translocates rapidly to the plasma membrane and clears from the cytoplasm. Ras*-Ipl and Ras*-Tih1 fusion proteins both rescuecdc25ts Saccharomyces cerevisiae, but Ras*-Ipl rescues more efficiently in the presence of phosphatidylinositol 3-kinase (PI3K), whereas PI3K-independent rescue is more efficient with Ras*-Tih1. Site-directed mutagenesis defines amino acids in the β1-loop1-β2 regions of Ipl and Tih1 as essential for growth rescue in this assay. Thus, Ipl and Tih1 arebona fidePH domain proteins, with broad specificity and moderate affinity for PIPs.

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 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 Multiple lipid binding sites determine the affinity of PH domains for phosphoinositide-containing membranes

2020 ◽  
Vol 6 (8) ◽  
pp. eaay5736 ◽  
Author(s):  
Eiji Yamamoto ◽  
Jan Domański ◽  
Fiona B. Naughton ◽  
Robert B. Best ◽  
Antreas C. Kalli ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Bound States ◽  
Lipid Binding ◽  
Dependent Manner ◽  
Ph Domain ◽  
Concentration Dependent Manner ◽  
Phosphatidylinositol Phosphate ◽  
Ph Domains ◽  
Experimental Values ◽  
Dynamics Simulations

Association of peripheral proteins with lipid bilayers regulates membrane signaling and dynamics. Pleckstrin homology (PH) domains bind to phosphatidylinositol phosphate (PIP) molecules in membranes. The effects of local PIP enrichment on the interaction of PH domains with membranes is unclear. Molecular dynamics simulations allow estimation of the binding energy of GRP1 PH domain to PIP3-containing membranes. The free energy of interaction of the PH domain with more than two PIP3 molecules is comparable to experimental values, suggesting that PH domain binding involves local clustering of PIP molecules within membranes. We describe a mechanism of PH binding proceeding via an encounter state to two bound states which differ in the orientation of the protein relative to the membrane, these orientations depending on the local PIP concentration. These results suggest that nanoscale clustering of PIP molecules can control the strength and orientation of PH domain interaction in a concentration-dependent manner.

scholarly journals The Lipid Binding Pleckstrin Homology Domain in UNC-104 Kinesin is Necessary for Synaptic Vesicle Transport in Caenorhabditis elegans

2004 ◽  
Vol 15 (8) ◽  
pp. 3729-3739 ◽  
Author(s):  
Dieter R. Klopfenstein ◽  
Ronald D. Vale
Keyword(s):  
Caenorhabditis Elegans ◽  
Synaptic Vesicles ◽  
Critical Role ◽  
Lipid Binding ◽  
Neuronal Cell Body ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology

UNC-104 (KIF1A) is a kinesin motor that transports synaptic vesicles from the neuronal cell body to the terminal. Previous in vitro studies have shown that a Dictyostelium relative of UNC-104 transports liposomes containing acidic phospholipids, but whether this interaction is needed for the recognition and transport of synaptic vesicles in metazoans remains unexplored. Here, we have introduced mutations in the nonmotor domain of UNC-104 and examined whether these mutant motors can rescue an unc-104 Caenorhabditis elegans strain. We show that a pleckstrin homology (PH) domain in UNC-104 is essential for membrane transport in living C. elegans, that this PH domain binds specifically to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), and that point mutants in the PH domain that interfere with PI(4,5)P2 binding in vitro also interfere with UNC-104 function in vivo. Several other lipid-binding modules could not effectively substitute for the UNC-104 PH domain in this in vivo assay. Real time imaging also revealed that a lipid-binding point mutation in the PH domain reduced movement velocity and processivity of individual UNC-104::GFP punctae in neurites. These results reveal a critical role for PI(4,5)P2 binding in UNC-104–mediated axonal transport and shows that the cargo-binding properties of the distal PH domain can affect motor output.

Stratifin, a keratinocyte specific 14–3-3 protein, harbors a pleckstrin homology (PH) domain and enhances protein kinase C activity

1995 ◽  
Vol 108 (11) ◽  
pp. 3569-3579
Author(s):  
E. Dellambra ◽  
M. Patrone ◽  
B. Sparatore ◽  
A. Negri ◽  
F. Ceciliani ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Terminal Differentiation ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Protein Database ◽  
Kinase C ◽  
Human Keratinocyte ◽  
Protein Kinase C Activity

The intrinsic signal(s) responsible for the onset of human keratinocyte terminal differentiation is not yet fully understood. Evidence has been recently accumulated linking the phospholipase-mediated activation of protein kinase C to the coordinate changes in gene expression occurring during keratinocyte terminal differentiation. Here we report the purification of a keratinocyte-derived protein enhancing protein kinase C enzymatic activity. The stimulator eluted as a peak with estimated molecular mass of approximately 70 kDa, while analysis by SDS-PAGE showed a 30 kDa protein migrating as a distinct doublet, suggesting the formation of a 30 kDa homodimer. The amino acid sequence analysis allowed the unambigous identification of the protein kinase C stimulator as a mixture of the highly homologous sigma (stratifin) and zeta isoforms of 14–3-3 proteins, which are homodimers of identical 30 kDa subunits. Mono Q anion exchange chromatography and immunoblot analysis further confirmed that stratifin enhances protein kinase C activity. Stratifin was originally sequenced from a human keratinocyte protein database, but its function was unknown. The pleckstrin homology domain has been recently related to protein translocation to the cell membrane as well as to functional interactions of intracellular proteins involved in signal transduction. We show here that stratifin (and 14–3-3 zeta) harbors a pleckstrin homology domain, and the consequent functional implications will be discussed.

scholarly journals Plant Actin-binding Protein SCAB1 Is Dimeric Actin Cross-linker with Atypical Pleckstrin Homology Domain

2012 ◽  
Vol 287 (15) ◽  
pp. 11981-11990 ◽  
Author(s):  
Wei Zhang ◽  
Yang Zhao ◽  
Yan Guo ◽  
Keqiong Ye
Keyword(s):  
Functional Organization ◽  
Inositol Phosphates ◽  
Binding Protein ◽  
Actin Binding ◽  
Surface Patch ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Actin Binding Protein ◽  
Cross Linker

SCAB1 is a novel plant-specific actin-binding protein that binds, bundles, and stabilizes actin filaments and regulates stomatal movement. Here, we dissected the structure and function of SCAB1 by structural and biochemical approaches. We show that SCAB1 is composed of an actin-binding domain, two coiled-coil (CC) domains, and a fused immunoglobulin and pleckstrin homology (Ig-PH) domain. We determined crystal structures for the CC1 and Ig-PH domains at 1.9 and 1.7 Å resolution, respectively. The CC1 domain adopts an antiparallel helical hairpin that further dimerizes into a four-helix bundle. The CC2 domain also mediates dimerization. At least one of the coiled coils is required for actin binding, indicating that SCAB1 is a bivalent actin cross-linker. The key residues required for actin binding were identified. The PH domain lacks a canonical basic phosphoinositide-binding pocket but can bind weakly to inositol phosphates via a basic surface patch, implying the involvement of inositol signaling in SCAB1 regulation. Our results provide novel insights into the functional organization of SCAB1.

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