Structure and phosphatidylinositol-(3,4)-bisphosphate binding of the C-terminal PH domain of human pleckstrin

Pleckstrin is the major target of protein kinase C (PKC) in blood platelets. Its phosphorylation triggers responses that ultimately lead to platelet activation and blood clot formation. Pleckstrin consists of three domains: a pleckstrin homology (PH) domain at both termini and a central DEP (Dishevelled, Egl-1, Pleckstrin) domain. Here, we report the solution nuclear magnetic resonance (NMR) structure of the C-terminal PH domain (C-PH) of human pleckstrin-1. We show that this PH domain binds phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) with high specificity in protein lipid overlay assays. Using NMR titration experiments and mutational analysis, residues involved in binding to PtdIns(3,4)P2 are identified. The binding site is formed by a patch of basic residues from the beta1 and beta2 strands and the beta1-beta2 loop. Since PtdIns(3,4)P2 is an important signaling molecule in platelets, our data suggest a C-PH dependent regulation of pleckstrin function in response to PtdIns(3,4)P2.     

Structure of the split PH domain and distinct lipid-binding properties of the PH-PDZ supramodule of alpha-syntrophin

Pleckstrin homology (PH) domains play diverse roles in cytoskeletal dynamics and signal transduction. Split PH domains represent a unique subclass of PH domains that have been implicated in interactions with complementary partial PH domains 'hidden' in many proteins. Whether partial PH domains exist as independent structural units alone and whether two halves of a split PH domain can fold together to form an intact PH domain are not known. Here, we solved the structure of the PH(N)-PDZ-PH(C) tandem of alpha-syntrophin. The split PH domain of alpha-syntrophin adopts a canonical PH domain fold. The isolated partial PH domains of alpha-syntrophin, although completely unfolded, remain soluble in solution. Mixing of the two isolated domains induces de novo folding and yields a stable PH domain. Our results demonstrate that two complementary partial PH domains are capable of binding to each other to form an intact PH domain. We further showed that the PH(N)-PDZ-PH(C) tandem forms a functionally distinct supramodule, in which the split PH domain and the PDZ domain function synergistically in binding to inositol phospholipids.     

Crystal structure of the human Fe65-PTB1 domain

The neuronal adaptor protein Fe65 is involved in brain development, Alzheimer disease amyloid precursor protein (APP) signaling, and proteolytic processing of APP. It contains three protein-protein interaction domains, one WW domain, and a unique tandem array of phosphotyrosine-binding (PTB) domains. The N-terminal PTB domain (Fe65-PTB1) was shown to interact with a variety of proteins, including the low density lipoprotein receptor-related protein (LRP-1), the ApoEr2 receptor, and the histone acetyltransferase Tip60. We have determined the crystal structures of human Fe65-PTB1 in its apo- and in a phosphate-bound form at 2.2 and 2.7A resolution, respectively. The overall fold shows a PTB-typical pleckstrin homology domain superfold. Although Fe65-PTB1 has been classified on an evolutionary basis as a Dab-like PTB domain, it contains attributes of other PTB domain subfamilies. The phosphotyrosine-binding pocket resembles IRS-like PTB domains, and the bound phosphate occupies the binding site of the phosphotyrosine (Tyr(P)) within the canonical NPXpY recognition motif. In addition Fe65-PTB1 contains a loop insertion between helix alpha2 and strand beta2(alpha2/beta2 loop) similar to members of the Shc-like PTB domain subfamily. The structural comparison with the Dab1-PTB domain reveals a putative phospholipid-binding site opposite the peptide binding pocket. We suggest Fe65-PTB1 to interact with its target proteins involved in translocation and signaling of APP in a phosphorylation-dependent manner.     

Solution structure of the SEA domain from the murine homologue of ovarian cancer antigen CA125 (MUC16)

Human CA125, encoded by the MUC16 gene, is an ovarian cancer antigen widely used for a serum assay. Its extracellular region consists of tandem repeats of SEA domains. In this study we determined the three-dimensional structure of the SEA domain from the murine MUC16 homologue using multidimensional NMR spectroscopy. The domain forms a unique alpha/beta sandwich fold composed of two alpha helices and four antiparallel beta strands and has a characteristic turn named the TY-turn between alpha1 and alpha2. The internal mobility of the main chain is low throughout the domain. The residues that form the hydrophobic core and the TY-turn are fully conserved in all SEA domain sequences, indicating that the fold is common in the family. Interestingly, no other residues are conserved throughout the family. Thus, the sequence alignment of the SEA domain family was refined on the basis of the three-dimensional structure, which allowed us to classify the SEA domains into several subfamilies. The residues on the surface differ between these subfamilies, suggesting that each subfamily has a different function. In the MUC16 SEA domains, the conserved surface residues, Asn-10, Thr-12, Arg-63, Asp-75, Asp-112, Ser-115, and Phe-117, are clustered on the beta sheet surface, which may be functionally important. The putative epitope (residues 58-77) for anti-MUC16 antibodies is located around the beta2 and beta3 strands. On the other hand the tissue tumor marker MUC1 has a SEA domain belonging to another subfamily, and its GSVVV motif for proteolytic cleavage is located in the short loop connecting beta2 and beta3.     

Analysis of GEF activity of Bcr protein DH domain

Dbl protein and DH (Dbl homology) domains are key regulators of RhoGTPases and promote GDP release from the complex with GTPase. About 70 DH-containing proteins are found. DH domain is localized in tandem with PH (pleckstrin homology) domain in many proteins. Bcr protein is a partner of Abl in reciprocal translocation t(9;22) which leads to Philadelphia chromosome formation. In the present study we have cloned Bcr DH and PH domains into the vector for mammalian expression. GEF activity of Bcr DH domain was studied alone and together with PH domain. Our data suggest, that Bcr DH domains does not reveal GEF activity against RhoGTPases RhoA, Cdc42 and Racl subfamilies in vivo.     

Structural basis for dimerization of the Grb10 Src homology 2 domain. Implications for ligand specificity

Grb7, Grb10, and Grb14 are members of a distinct family of adapter proteins that interact with various receptor tyrosine kinases upon receptor activation. Proteins in this family contain several modular signaling domains including a pleckstrin homology (PH) domain, a BPS (between PH and SH2) domain, and a C-terminal Src homology 2 (SH2) domain. Although SH2 domains are typically monomeric, we show that the Grb10 SH2 domain and also full-length Grb10 gamma are dimeric in solution under physiologic conditions. The crystal structure of the Grb10 SH2 domain at 1.65-A resolution reveals a non-covalent dimer whose interface comprises residues within and flanking the C-terminal alpha helix, which are conserved in the Grb7/Grb10/Grb14 family but not in other SH2 domains. Val-522 in the BG loop (BG3) and Asp-500 in the EF loop (EF1) are positioned to interfere with the binding of the P+3 residue of a phosphopeptide ligand. These structural features of the Grb10 SH2 domain will favor binding of dimeric, turn-containing phosphotyrosine sequences, such as the phosphorylated activation loops in the two beta subunits of the insulin and insulin-like growth factor-1 receptors. Moreover, the structure suggests the mechanism by which the Grb7 SH2 domain binds selectively to pTyr-1139 (pYVNQ) in Her2, which along with Grb7 is co-amplified in human breast cancers.     

Structural characterization of the split pleckstrin homology domain in phospholipase C-gamma1 and its interaction with TRPC3

Phospholipase C (PLC)-gamma is unique among the PLC enzymes because each PLC-gamma isozyme contains a split pleckstrin homology (PH) domain with an SH2SH2SH3 tandem repeat insertion (where SH indicates Src homology domain) in the middle of its sequence. Split PH domains exist in a number of other proteins that play crucial signaling roles. However, little is known about the structure and function of split PH domains. The C-terminal half of the PLC-gamma split PH domain has been implicated to interact directly with the TRPC3 calcium channel, thereby providing a direct coupling mechanism between PLC-gamma and agonist-induced calcium entry. However, this interaction has not been proved by direct biochemical or structural studies. Here we determined the three-dimensional structure of the split PH domain of PLC-gamma1, and we found that the split PH domain of the enzyme folds into a canonical PH domain fold with high thermostability. The SH2SH2SH3 insertion between the beta3 and beta4 strands does not change the structure of the split PH domain. In contrast to the majority of phospholipid-binding PH domains, the PLC-gamma1 split PH domain lacks the signature lipid-binding motif located between the beta1 and beta2 strands. Consistent with this structural feature, the split PH domain of PLC-gamma1 does not bind to phospholipids. Multiple biochemical and biophysical experiments have argued against a direct interaction between TRPC3 and the C-terminal half of the PLC-gamma1 split PH domain. Our data pointed to the existence of a yet to be elucidated interaction mechanism between TRPC3 and PLC-gamma1.     

Substrate specificity and recognition is conferred by the pleckstrin homology domain of the Dbl family guanine nucleotide exchange factor P-Rex2

Dbl family guanine nucleotide exchange factors (GEFs) are characterized by the presence of a catalytic Dbl homology domain followed invariably by a lipid-binding pleckstrin homology (PH) domain. To date, substrate recognition and specificity of this family of GEFs has been reported to be mediated exclusively via the Dbl homology domain. Here we report the novel and unexpected finding that, in the Dbl family Rac-specific GEF P-Rex2, it is the PH domain that confers substrate specificity and recognition. Moreover, the beta3beta4 loop of the PH domain of P-Rex2 is the determinant for Rac1 recognition, as substitution of the beta3beta4 loop of the PH domain of Dbs (a RhoA- and Cdc42-specific GEF) with that of P-Rex2 confers Rac1-specific binding capability to the PH domain of Dbs. The contact interface between the PH domain of P-Rex2 and Rac1 involves the switch loop and helix 3 of Rac1. Moreover, substitution of helix 3 of Cdc42 with that of Rac1 now enables the PH domain of P-Rex2 to bind this Cdc42 chimera. Despite having the ability to recognize this chimeric Cdc42, P-Rex2 is unable to catalyze nucleotide exchange on Cdc42, suggesting that recognition of substrate and catalysis are two distinct events. Thus substrate recognition can now be added to the growing list of functions that are being attributed to the PH domain of Dbl family GEFs.     

Phosphorylated pleckstrin induces cell spreading via an integrin-dependent pathway

Pleckstrin is a 40-kD phosphoprotein containing NH(2)- and COOH-terminal pleckstrin homology (PH) domains separated by a disheveled-egl 10-pleckstrin (DEP) domain. After platelet activation, pleckstrin is rapidly phosphorylated by protein kinase C. We reported previously that expressed phosphorylated pleckstrin induces cytoskeletal reorganization and localizes in microvilli along with glycoproteins, such as integrins. Given the role of integrins in cytoskeletal organization and cell spreading, we investigated whether signaling from pleckstrin cooperated with signaling pathways involving the platelet integrin, alphaIIbbeta3. Pleckstrin induced cell spreading in both transformed (COS-1 & CHO) and nontransformed (REF52) cell lines, and this spreading was regulated by pleckstrin phosphorylation. In REF52 cells, pleckstrin-induced spreading was matrix dependent, as evidenced by spreading of these cells on fibrinogen but not on fibronectin. Coexpression with alphaIIbbeta3 did not enhance pleckstrin-mediated cell spreading in either REF52 or CHO cells. However, coexpression of the inactive variant alphaIIbbeta3 Ser753Pro, or beta3 Ser753Pro alone, completely blocked pleckstrin-induced spreading. This implies that alphaIIbbeta3 Ser753Pro functions as a competitive inhibitor by blocking the effects of an endogenous receptor that is used in the signaling pathway involved in pleckstrin-induced cell spreading. Expression of a chimeric protein composed of the extracellular and transmembrane portion of Tac fused to the cytoplasmic tail of beta3 completely blocked pleckstrin-mediated spreading, whereas chimeras containing the cytoplasmic tail of beta3 Ser753Pro or alphaIIb had no effect. This suggests that the association of an unknown signaling protein with the cytoplasmic tail of an endogenous integrin beta-chain is also required for pleckstrin-induced spreading. Thus, expressed phosphorylated pleckstrin promotes cell spreading that is both matrix and integrin dependent. To our knowledge, this is the first example of a mutated integrin functioning as a dominant negative inhibitor.     

Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity

The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-gamma1 with translational elongation factor (EF)-1alpha, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-gamma1 followed by peptide sequence analysis, we identified EF-1alpha as a binding partner of a split PH domain of PLC-gamma1. Analysis by site-directed mutagenesis of the PH domain revealed that the beta2-sheet of a split PH domain is critical for the interaction with EF-1alpha. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)). So the PH domain of PLC-gamma1 binds to both EF-1alpha and PIP(2). The binding affinity of EF-1alpha to the GST.PH domain fusion protein increased in the presence of PIP(2), although PIP(2) does not bind to EF-1alpha directly. This suggests that EF-1alpha may control the binding affinity between the PH domain and PIP(2). PLC-gamma1 is substantially activated in the presence of EF-1alpha with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-gamma1 (Y509A/F510A) that lost its binding affinity to EF-1alpha shows basal level activity. Taken together, our data show that EF-1alpha plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-gamma1 activity via a split PH domain.     

Pleckstrin Associates with Plasma Membranes and Induces the Formation of Membrane Projections: Requirements for Phosphorylation and the NH2-terminal PH Domain

Pleckstrin homology (PH) domains are sequences of ~100 amino acids that form “modules” that have been proposed to facilitate protein/protein or protein/lipid interactions. Pleckstrin, first described as a substrate for protein kinase C in platelets and leukocytes, is composed of two PH domains, one at each end of the molecule, flanking an intervening sequence of 147 residues. Evidence is accumulating to support the hypothesis that PH domains are structural motifs that target molecules to membranes, perhaps through interactions with Gβγ or phosphatidylinositol 4,5-bisphosphate (PIP₂), two putative PH domain ligands. In the present studies, we show that pleckstrin associates with membranes in human platelets. We further demonstrate that, in transfected Cos-1 cells, pleckstrin associates with peripheral membrane ruffles and dorsal membrane projections. This association depends on phosphorylation of pleckstrin and requires the presence of its NH₂-terminal, but not its COOH-terminal, PH domain. Moreover, PH domains from other molecules cannot effectively substitute for pleckstrin's NH₂terminal PH domain in directing membrane localization. Lastly, we show that wild-type pleckstrin actually promotes the formation of membrane projections from the dorsal surface of transfected cells, and that this morphologic change is similarly PH domain dependent. Since we have shown previously that pleckstrin-mediated inhibition of PIP₂ metabolism by phospholipase C or phosphatidylinositol 3-kinase also requires pleckstrin phosphorylation and an intact NH₂-terminal PH domain, these results suggest that: (a) pleckstrin's NH₂terminal PH domain may regulate pleckstrin's activity by targeting it to specific areas within the cell membrane; and (b) pleckstrin may affect membrane structure, perhaps via interactions with PIP₂ and/or other membrane-bound ligands.     

Characterization of RhoA-binding kinase ROKalpha implication of the pleckstrin homology domain in ROKalpha function using region-specific antibodies

Rho-binding kinase alpha (ROKalpha) is a serine/threonine kinase with multiple functional domains involved in actomyosin assembly. It has previously been documented that the C terminus part of ROKalpha interacts with the N-terminal kinase domain and thereby regulates its catalytic activity. Here we used antibodies against different domains of ROKalpha and were able to reveal some structural aspects that are essential for the specific functions of ROKalpha. Antibodies against the kinase domain revealed that this part of the protein is highly complex and inaccessible. Further experiments confirmed that this domain could undergo inter- and intramolecular interactions in a complex manner, which regulates the kinase catalytic activity. Other antibodies that raised against the coiled-coil domain, Rho binding domain, and the pleckstrin homology (PH) domain were all effective in recognizing the native proteins in an immunoprecipitation assay. Only the anti-Rho binding domain antibodies could activate the kinase independent of RhoA. The PH antibodies had no apparent effects on the catalytic activity but were effective in blocking actomyosin assembly and cell contractility. Likewise, mutations of the PH domains can abrogate its dominant negative effects on actin morphology. The subsequent disruption of endogenous ROK localization to the actomyosin network by overexpressing the PH domain is supportive of a role of the PH domain of ROK in targeting the kinase to these structures.     

Structure of a PH domain from the C. elegans muscle protein UNC-89 suggests a novel function

BACKGROUND: Pleckstrin homology (PH) domains constitute a structurally conserved family present in many signaling and regulatory proteins. PH domains have been shown to bind to phospholipids, and many function in membrane targeting. They generally have a strong electrostatic polarization and interact with negatively charged phospholipids via the positive pole. On the basis of electrostatic modeling, however, we have previously identified a class of PH domains with a predominantly negative charge and predicted that these domains recognize other targets. Here, we report the first experimental structure of such a PH domain. RESULTS: The structure of the PH domain from Caenorhabditis elegans muscle protein UNC-89 has been determined by heteronuclear NMR. The domain adopts the classic PH fold, but has an unusual closed conformation of the "inositol binding loops. This creates a small opening to a deep hydrophobic pocket lined with negative charges on one side, and provides a molecular explanation for the lack of association with inositol-1,4,5-triphosphate. As predicted, the PH domain of UNC-89 has a strongly negative overall electrostatic potential. Modeling the Dbl homology (DH)-linked PH domains from the C. elegans genome shows that a large proportion of these modules are negatively charged. CONCLUSIONS: We present the first structure of a PH domain with a strong negative overall electrostatic potential. The presence of a deep pocket lined with negative charges suggests that the domain binds to ligands other than acidic phospholipids. The abundance of this class of PH domain in the C. elegans genome suggests a prominent role in mediating protein-protein interactions.     

Pleckstrin homology domain 1 of mouse alpha 1-syntrophin binds phosphatidylinositol 4,5-bisphosphate

Mouse alpha 1-syntrophin sequences were produced as chimeric fusion proteins in bacteria and found to bind phosphatidylinositol 4, 5-bisphosphate (PtdIns4,5P2). Half-maximal binding occurred at 1.9 microM PtdIns4,5P2 and when 1.2 PtdIns4,5P2 were added per syntrophin. Binding was specific for PtdIns4,5P2 and did not occur with six other tested lipids including the similar phosphatidylinositol 4-phosphate. Binding was localized to the N-terminal pleckstrin homology domain (PH1); the second, C-terminal PH2 domain did not bind lipids. Key residues in PtdIns4,5P2 binding to a PH domain were found to be conserved in alpha-syntrophins' PH1 domains and absent in PH2 domains, suggesting a molecular basis for binding.     

NMR structure of the apoptosis- and inflammation-related NALP1 pyrin domain

Signaling in apoptosis and inflammation is often mediated by proteins of the death domain superfamily in the Fas/FADD/Caspase-8 or the Apaf-1/Caspase-9 pathways. This superfamily currently comprises the death domain (DD), death effector domain (DED), caspase recruitment domain (CARD), and pyrin domain (PYD) subfamilies. The PYD subfamily is most abundant, but three-dimensional structures are only available for the subfamilies DD, DED, and CARD, which have an antiparallel arrangement of six alpha helices as common fold. This paper presents the NMR structure of PYD of NALP1, a protein that is involved in the innate immune response and is a component of the inflammasome. The structure of NALP1 PYD differs from all other known death domain superfamily structures in that the third alpha helix is replaced by a flexibly disordered loop. This unique feature appears to relate to the molecular basis of familial Mediterranean fever (FMF), a genetic disease caused by single-point mutations.     

Structural Basis of DEPTOR to Recognize Phosphatidic Acid Using its Tandem DEP Domains

DEP domain containing mTOR-interacting protein (DEPTOR) plays pivotal roles in regulating metabolism, growth, autophagy and apoptosis by functions as an endogenous inhibitor of mTOR signaling pathway. Activated by phosphatidic acid, a second messenger in mTOR signaling, DEPTOR dissociates from mTORC1 complex with unknown mechanism. Here, we present a 1.5 Å resolution crystal structure, which shows that the N-terminal two tandem DEP domains of hDEPTOR fold into a dumbbell-shaped structure, protruding the characteristic β-hairpin arms of DEP domains on each side. An 18 amino acids DDEX motif at the end of DEP2 interacts with DEP1 and stabilizes the structure. Biochemical studies showed that the tandem DEP domains directly interact with phosphatidic acid using two distinct positively charged patches. These results provide insights into mTOR activation upon phosphatidic acid stimulation.     

Structural and functional differences of SWIRM domain subtypes

SWIRM is a conserved domain found in several chromatin-associated proteins. Based on their sequences, the SWIRM family members can be classified into three subfamilies, which are represented by Swi3, LSD1, and Ada2. Here we report the SWIRM structure of human MYb-like, Swirm and Mpn domain-containing protein-1 (MYSM1). The MYSM1 SWIRM structure forms a compact HTH-related fold comprising five alpha-helices, which best resembles the Swi3 SWIRM structure, among the known SWIRM structures. The MYSM1 and Swi3 SWIRM structures are more similar to the LSD1 structure than the Ada2alpha structure. The SWIRM domains of MYSM1 and LSD1 lacked DNA binding activity, while those of Ada2alpha and the human Swi3 counterpart, SMARCC2, bound DNA. The dissimilarity in the DNA-binding ability of the MYSM1 and SMARCC2 SWIRM domains might be due to a couple of amino acid differences in the last helix. These results indicate that the SWIRM family has indeed diverged into three structural subfamilies (Swi3/MYSM1, LSD1, and Ada2 types), and that the Swi3/MYSM1-type subfamily has further diverged into two functionally distinct groups. We also solved the structure of the SANT domain of MYSM1, and demonstrated that it bound DNA with a similar mode to that of the c-Myb DNA-binding domain.     

The Ras/p120 GTPase-activating protein (GAP) interaction is regulated by the p120 GAP pleckstrin homology domain

Pleckstrin homology domains are structurally conserved functional domains that can undergo both protein/protein and protein/lipid interactions. Pleckstrin homology domains can mediate inter- and intra-molecular binding events to regulate enzyme activity. They occur in numerous proteins including many that interact with Ras superfamily members, such as p120 GAP. The pleckstrin homology domain of p120 GAP is located in the NH(2)-terminal, noncatalytic region of p120 GAP. Overexpression of the noncatalytic domains of p120 GAP may modulate Ras signal transduction pathways. Here, we demonstrate that expression of the isolated pleckstrin homology domain of p120 GAP specifically inhibits Ras-mediated signaling and transformation but not normal cellular growth. Furthermore, we show that the pleckstrin homology domain binds the catalytic domain of p120 GAP and interferes with the Ras/GAP interaction. Thus, we suggest that the pleckstrin homology domain of p120 GAP may specifically regulate the interaction of Ras with p120 GAP via competitive intra-molecular binding.     

Structural basis of phosphoinositide binding to kindlin-2 protein pleckstrin homology domain in regulating integrin activation

Kindlins are a subclass of FERM-containing proteins that have recently emerged as key regulators of integrin receptor activation and signaling. As compared with the conventional FERM domain, the kindlin FERM domain contains an inserted pleckstrin homology (PH) domain that recognizes membrane phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). Using NMR spectroscopy, we show that PIP3 site-specifically binds to kindlin-2 PH with substantial chemical shift changes that are much larger than PIP2. This suggests an enhanced association of kindlin-2 with membrane as mediated by PIP3 upon its conversion from PIP2 by phosphoinositide-3 kinase, a known regulator of integrin activation. We determined the NMR structure of the kindlin-2 PH domain bound to the head group of PIP3, inositol 1,3,4,5-tetraphosphate (IP4). The structure reveals a canonical PH domain fold, yet with a distinct IP4 binding pocket that appears highly conserved for the kindlin family members. Functional experiments demonstrate that although wild type kindlin-2 is capable of cooperating with integrin activator talin to induce synergistic integrin α(IIb)β(3) activation, this ability is significantly impaired for a phosphoinositide binding-defective kindlin-2 mutant. These results define a specific PIP3 recognition mode for the kindlin PH domain. Moreover, they shed light upon a mechanism as to how the PH domain mediates membrane engagement of kindlin-2 to promote its binding to integrin and cooperation with talin for regulation of integrin activation.     

Phosphatidylserine induces functional and structural alterations of the membrane-associated pleckstrin homology domain of phospholipase C-delta1

The membrane binding affinity of the pleckstrin homology (PH) domain of phospholipase C (PLC)-delta1 was investigated using a vesicle coprecipitation assay and the structure of the membrane-associated PH domain was probed using solid-state (13)C NMR spectroscopy. Twenty per cent phosphatidylserine (PS) in the membrane caused a moderate but significant reduction of the membrane binding affinity of the PH domain despite the predicted electrostatic attraction between the PH domain and the head groups of PS. Solid-state NMR spectra of the PH domain bound to the phosphatidylcholine (PC)/PS/phosphatidylinositol 4,5-bisphosphate (PIP(2)) (75 : 20 : 5) vesicle indicated loss of the interaction between the amphipathic alpha2-helix of the PH domain and the interface region of the membrane which was previously reported for the PH domain bound to PC/PIP(2) (95 : 5) vesicles. Characteristic local conformations in the vicinity of Ala88 and Ala112 induced by the hydrophobic interaction between the alpha2-helix and the membrane interface were lost in the structure of the PH domain at the surface of the PC/PS/PIP(2) vesicle, and consequently the structure becomes identical to the solution structure of the PH domain bound to d-myo-inositol 1,4,5-trisphosphate. These local structural changes reduce the membrane binding affinity of the PH domain. The effects of PS on the PH domain were reversed by NaCl and MgCl(2), suggesting that the effects are caused by electrostatic interaction between the protein and PS. These results generally suggest that the structure and function relationships among PLCs and other peripheral membrane proteins that have similar PH domains would be affected by the local lipid composition of membranes.