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.     

Pleckstrin homology (PH) like domains - versatile modules in protein-protein interaction platforms

The initial reports on pleckstrin homology (PH) domains almost 20years ago described them as sequence feature of proteins involved in signal transduction processes. Investigated at first along the phospholipid binding properties of a small subset of PH representatives, the PH fold turned out to appear as mediator of phosphotyrosine and polyproline peptide binding to other signaling proteins. While phospholipid binding now seems rather the exception among PH-like domains, protein-protein interactions established as more and more important feature of these modules. In this review we focus on the PH superfold as a versatile protein-protein interaction platform and its three-dimensional integration in an increasing number of available multidomain structures.     

Pleckstrin homology (PH) domains and phosphoinositides

PH (pleckstrin homology) domains represent the 11th most common domain in the human proteome. They are best known for their ability to bind phosphoinositides with high affinity and specificity, although it is now clear that less than 10% of all PH domains share this property. Cases in which PH domains bind specific phosphoinositides with high affinity are restricted to those phosphoinositides that have a pair of adjacent phosphates in their inositol headgroup. Those that do not [PtdIns3P, PtdIns5P and PtdIns(3,5)P2] are instead recognized by distinct classes of domains including FYVE domains, PX (phox homology) domains, PHD (plant homeodomain) fingers and the recently identified PROPPINs (b-propellers that bind polyphosphoinositides). Of the 90% of PH domains that do not bind strongly and specifically to phosphoinositides, few are well understood. One group of PH domains appears to bind both phosphoinositides (with little specificity) and Arf (ADP-ribosylation factor) family small G-proteins, and are targeted to the Golgi apparatus where both phosphoinositides and the relevant Arfs are both present. Here, the PH domains may function as coincidence detectors. A central challenge in understanding the majority of PH domains is to establish whether the very low affinity phosphoinositide binding reported in many cases has any functional relevance. For PH domains from dynamin and from Dbl family proteins, this weak binding does appear to be functionally important, although its precise mechanistic role is unclear. In many other cases, it is quite likely that alternative binding partners are more relevant, and that the observed PH domain homology represents conservation of structural fold rather than function.     

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.     

Crystal structure of the PH-BEACH domains of human LRBA/BGL

The beige and Chediak-Higashi syndrome (BEACH) domain defines a large family of eukaryotic proteins that have diverse cellular functions in vesicle trafficking, membrane dynamics, and receptor signaling. The domain is the only module that is highly conserved among all of these proteins, but the exact functions of this domain and the molecular basis for its actions are currently unknown. Our previous studies showed that the BEACH domain is preceded by a novel, weakly conserved pleckstrin homology (PH) domain. We report here the crystal structure at 2.4 A resolution of the PH-BEACH domain of human LRBA/BGL. The PH domain has the same backbone fold as canonical PH domains, despite sharing no sequence homology with them. However, our binding assays demonstrate that the PH domain in the BEACH proteins cannot bind phospholipids. The BEACH domain contains a core of several partially extended peptide segments that is flanked by helices on both sides. The structure suggests intimate association between the PH and the BEACH domains, and surface plasmon resonance studies confirm that the two domains of the protein FAN have high affinity for each other, with a K(d) of 120 nM.     

Pleckstrin homology domains and the cytoskeleton

Pleckstrin homology (PH) domains are 100-120 amino acid protein modules best known for their ability to bind phosphoinositides. All possess an identical core beta-sandwich fold and display marked electrostatic sidedness. The binding site for phosphoinositides lies in the center of the positively charged face. In some cases this binding site is well defined, allowing highly specific and strong ligand binding. In several of these cases the PH domains specifically recognize 3-phosphorylated phosphoinositides, allowing them to drive membrane recruitment in response to phosphatidylinositol 3-kinase activation. Examples of these PH domain-containing proteins include certain Dbl family guanine nucleotide exchange factors, protein kinase B, PhdA, and pleckstrin-2. PH domain-mediated membrane recruitment of these proteins contributes to regulated actin assembly and cell polarization. Many other PH domain-containing cytoskeletal proteins, such as spectrin, have PH domains that bind weakly, and to all phosphoinositides. In these cases, the individual phosphoinositide interactions may not be sufficient for membrane association, but appear to require self-assembly of their host protein and/or cooperation with other anchoring motifs within the same molecule to drive membrane attachment.     

Molecular modeling of the membrane targeting of phospholipase C pleckstrin homology domains

Phospholipases C (PLCs) reversibly associate with membranes to hydrolyze phosphatidylinositol-4, 5-bisphosphate (PI[4,5]P(2)) and comprise four main classes: beta, gamma, delta, and epsilon. Most eukaryotic PLCs contain a single, N-terminal pleckstrin homology (PH) domain, which is thought to play an important role in membrane targeting. The structure of a single PLC PH domain, that from PLCdelta1, has been determined; this PH domain binds PI(4,5)P(2) with high affinity and stereospecificity and has served as a paradigm for PH domain functionality. However, experimental studies demonstrate that PH domains from different PLC classes exhibit diverse modes of membrane interaction, reflecting the dissimilarity in their amino acid sequences. To elucidate the structural basis for their differential membrane-binding specificities, we modeled the three-dimensional structures of all mammalian PLC PH domains by using bioinformatic tools and calculated their biophysical properties by using continuum electrostatic approaches. Our computational analysis accounts for a large body of experimental data, provides predictions for those PH domains with unknown functions, and indicates functional roles for regions other than the canonical lipid-binding site identified in the PLCdelta1-PH structure. In particular, our calculations predict that (1). members from each of the four PLC classes exhibit strikingly different electrostatic profiles than those ordinarily observed for PH domains in general, (2). nonspecific electrostatic interactions contribute to the membrane localization of PLCdelta-, PLCgamma-, and PLCbeta-PH domains, and (3). phosphorylation regulates the interaction of PLCbeta-PH with its effectors through electrostatic repulsion. Our molecular models for PH domains from all of the PLC classes clearly demonstrate how a common structural fold can serve as a scaffold for a wide range of surface features and biophysical properties that support distinctive functional roles.     

Structure and dynamics of the human pleckstrin DEP domain: distinct molecular features of a novel DEP domain subfamily

Pleckstrin1 is a major substrate for protein kinase C in platelets and leukocytes, and comprises a central DEP (disheveled, Egl-10, pleckstrin) domain, which is flanked by two PH (pleckstrin homology) domains. DEP domains display a unique alpha/beta fold and have been implicated in membrane binding utilizing different mechanisms. Using multiple sequence alignments and phylogenetic tree reconstructions, we find that 6 subfamilies of the DEP domain exist, of which pleckstrin represents a novel and distinct subfamily. To clarify structural determinants of the DEP fold and to gain further insight into the role of the DEP domain, we determined the three-dimensional structure of the pleckstrin DEP domain using heteronuclear NMR spectroscopy. Pleckstrin DEP shares main structural features with the DEP domains of disheveled and Epac, which belong to different DEP subfamilies. However, the pleckstrin DEP fold is distinct from these structures and contains an additional, short helix alpha4 inserted in the beta4-beta5 loop that exhibits increased backbone mobility as judged by NMR relaxation measurements. Based on sequence conservation, the helix alpha4 may also be present in the DEP domains of regulator of G-protein signaling (RGS) proteins, which are members of the same DEP subfamily. In pleckstrin, the DEP domain is surrounded by two PH domains. Structural analysis and charge complementarity suggest that the DEP domain may interact with the N-terminal PH domain in pleckstrin. Phosphorylation of the PH-DEP linker, which is required for pleckstrin function, could regulate such an intramolecular interaction. This suggests a role of the pleckstrin DEP domain in intramolecular domain interactions, which is distinct from the functions of other DEP domain subfamilies found so far.     

Crystal structure of the BEACH domain reveals an unusual fold and extensive association with a novel PH domain

The BEACH domain is highly conserved in a large family of eukaryotic proteins, and is crucial for their functions in vesicle trafficking, membrane dynamics and receptor signaling. However, it does not share any sequence homology with other proteins. Here we report the crystal structure at 2.9 A resolution of the BEACH domain of human neurobeachin. It shows that the BEACH domain has a new and unusual polypeptide backbone fold, as the peptide segments in its core do not assume regular secondary structures. Unexpectedly, the structure also reveals that the BEACH domain is in extensive association with a novel, weakly conserved pleckstrin-homology (PH) domain. Consistent with the structural analysis, biochemical studies show that the PH and BEACH domains have strong interactions, suggesting they may function as a single unit. Functional studies in intact cells demonstrate the requirement of both the PH and the BEACH domains for activity. A prominent groove at the interface between the two domains may be used to recruit their binding partners.     

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.     

Crystal structures of the BAR-PH and PTB domains of human APPL1

APPL1 interacts with adiponectin receptors and other important signaling molecules. It contains a BAR and a PH domain near its N terminus, and the two domains may function as a unit (BAR-PH domain). We report here the crystal structures of the BAR-PH and PTB domains of human APPL1. The structures reveal novel features for BAR domain dimerization and for the interactions between the BAR and PH domains. The BAR domain dimer of APPL1 contains two four-helical bundles, whereas other BAR domain dimers have only three helices in each bundle. The PH domain is located at the opposite ends of the BAR domain dimer. Yeast two-hybrid assays confirm the interactions between the BAR and PH domains. Lipid binding assays show that the BAR, PH, and PTB domains can bind phospholipids. The ability of APPL1 to interact with multiple signaling molecules and phospholipids supports an important role for this adaptor in cell signaling.     

Comprehensive identification of PIP3-regulated PH domains from C. elegans to H. sapiens by model prediction and live imaging

Phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol(3,4,5)-trisphosphate (PIP3) control cell growth, migration, and other processes by recruiting proteins with pleckstrin homology (PH) domains and possibly other domains to the plasma membrane (PM). However, previous experimental and structural work with PH domains left conflicting evidence about which ones are PIP3 regulated. Here we used live-cell confocal imaging of 130 YFP-conjugated mouse PH domains and found that 20% translocated to the PM in response to receptor-generated PIP3 production. We developed a recursive-learning algorithm to predict PIP3 regulation of 1200 PH domains from different eukaryotes and validated that it accurately predicts PIP3 regulation. Strikingly, this algorithm showed that PIP3 regulation is specified by amino acids across the PH domain, not just the PIP3-binding pocket, and must have evolved several times independently from PIP3-insensitive ancestral PH domains. Finally, our algorithm and live-cell experiments provide a functional survey of PH domains in different species, showing that PI3K regulation increased from approximately two C. elegans and four Drosophila to 40 vertebrate proteins.     

The PH Domain and the Polybasic c Domain of Cytohesin-1 Cooperate specifically in Plasma Membrane Association and Cellular Function

Recruitment of intracellular proteins to the plasma membrane is a commonly found requirement for the initiation of signal transduction events. The recently discovered pleckstrin homology (PH) domain, a structurally conserved element found in ~100 signaling proteins, has been implicated in this function, because some PH domains have been described to be involved in plasma membrane association. Furthermore, several PH domains bind to the phosphoinositides phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate in vitro, however, mostly with low affinity. It is unclear how such weak interactions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the structural and functional requirements for membrane association of cytohesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-terminal polybasic sequence of cytohesin-1 (c domain) are necessary for plasma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosensor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosphate binds to the PH domain and c domain together with high affinity (100 nM), whereas the isolated PH domain has a substantially lower affinity (2–3 μM). The cooperativity of both elements appears specific, because a chimeric protein, consisting of the c domain of cytohesin-1 and the PH domain of the β-adrenergic receptor kinase does not associate with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation–isoprenylation motif partially restored the biological function, but the specific targeting to the plasma membrane was not retained. Thus we conclude that two elements of cytohesin-1, the PH domain and the c domain, are required and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we observed a similar functional cooperativity of the PH domain of Bruton’s tyrosine kinase with the adjacent Bruton’s tyrosine kinase motif, a novel zinc-containing fold.     

Specificity of the myotubularin family of phosphatidylinositol-3-phosphatase is determined by the PH/GRAM domain

Myotubularins (MTM) are a large subfamily of lipid phosphatases that specifically dephosphorylate at the D3 position of phosphatidylinositol 3-phosphate (PI(3)P) in PI(3)P and PI(3,5)P2. We recently found that MTMR6 specifically inhibits the Ca2+-activated K+ channel, KCa3.1, by dephosphorylating PI(3)P. We now show that inhibition is specific for MTMR6 and other MTMs do not inhibit KCa3.1. By replacing either or both of the coiled-coil (CC) and pleckstrin homology/GRAM (PH/G) domains of MTMs that failed to inhibit KCa3.1 with the CC and PH/G domains of MTMR6, we found that chimeric MTMs containing both the MTMR6 CC and PH/G domains functioned like MTMR6 to inhibit KCa3.1 channel activity, whereas chimeric MTMs containing either domain alone did not. Immunofluorescent microscopy demonstrated that both the MTMR6 CC and PH/G domains are required to co-localize MTMR6 to the plasma membrane with KCa3.1. These findings support a model in which two specific low affinity interactions are required to co-localize MTMR6 with KCa3.1: 1) between the CC domains on MTMR6 and KCa3.1 and (2) between the PH/G domain and a component of the plasma membrane. Our inability to detect significant interaction of the MTMR6 G/PH domain with phosphoinositides suggests that this domain may bind a protein. Identifying the specific binding partners of the CC and PH/G domains on other MTMs will provide important clues to the specific functions regulated by other MTMs as well as the mechanism(s) whereby loss of some MTMs lead to disease.     

ARAP3 binding to phosphatidylinositol-(3,4,5)-trisphosphate depends on N-terminal tandem PH domains and adjacent sequences

Pleckstrin homology (PH) domains are modules characterised by a conserved three-dimensional protein fold. Several PH domains bind phosphoinositides with high affinity and specificity whilst most others do not. ARAP3 is a dual GTPase activating protein for Arf6 and RhoA which was identified in a screen for phosphatidylinositol-(3,4,5)-trisphophate (PtdIns(3,4,5)P₃) binding proteins. It is a regulator of cell shape and adhesion, and is itself regulated by PtdIns(3,4,5)P_3, which acts to recruit ARAP3 to the plasma membrane and to catalytically activate it. We show here that ARAP3 binds to PtdIns(3,4,5)P₃ in an unusual, PH domain-dependent manner. None of the five PH domains are sufficient to bind PtdIns(3,4,5)P₃ in isolation. Instead, the minimal PtdIns(3,4,5)P₃ binding fragment comprises ARAP3's N-terminal tandem PH domains, and an N-terminal linker region. For substantial binding, the N-terminal sterile alpha motif (SAM) domain is also required. Site-directed mutagenesis of either of the two N-terminal PH domains within the fragment greatly reduces binding to PtdIns(3,4,5)P₃, however, in the context of the full-length protein, point mutations in the second PH domain have a lesser effect on binding, whilst deletion of any one of the five PH domains abolishes PtdIns(3,4,5)P₃ binding. We propose a mechanism by which basic residues from the N-terminal tandem PH domains, and from elsewhere in the protein synergise to mediate strong, specific PtdIns(3,4,5)P₃ binding.     

Using HHsearch to tackle proteins of unknown function: A pilot study with PH domains

Advances in membrane cell biology are hampered by the relatively high proportion of proteins with no known function. Such proteins are largely or entirely devoid of structurally significant domain annotations. Structural bioinformaticians have developed profile‐profile tools such as HHsearch (online version called HHpred), which can detect remote homologies that are missed by tools used to annotate databases. Here we have applied HHsearch to study a single structural fold in a single model organism as proof of principle. In the entire clan of protein domains sharing the pleckstrin homology domain fold in yeast, systematic application of HHsearch accurately identified known PH‐like domains. It also predicted 16 new domains in 13 yeast proteins many of which are implicated in intracellular traffic. One of these was Vps13p, where we confirmed the functional importance of the predicted PH‐like domain. Even though such predictions require considerable work to be corroborated, they are useful first steps. HHsearch should be applied more widely, particularly across entire proteomes of model organisms, to significantly improve database annotations.      

Multiple roles of pleckstrin homology domains in phospholipase Cbeta function

Since their discovery almost 10 years ago pleckstrin homology (PH) domains have been identified in a wide variety of proteins. Here, we focus on two proteins whose PH domains play a defined functional role, phospholipase C (PLC)-beta(2) and PLCdelta(1). While the PH domains of both proteins are responsible for membrane targeting, their specificity of membrane binding drastically differs. However, in both these proteins the PH domains work to modulate the activity of their catalytic core upon interaction with either phosphoinositol lipids or G protein activators. These observations show that these PH domains are not simply binding sites tethered onto their host enzyme but are intimately associated with their catalytic core. This property may be true for other PH domains.     

Sequence-specific RNA binding mediated by the RNase PH domain of components of the exosome

We have previously demonstrated that PM-Scl-75, a component of the human exosome complex involved in RNA maturation and mRNA decay, can specifically interact with RNAs containing an AU-rich instability element. Through the analysis of a series of deletion mutants, we have now shown that a 266 amino acid fragment representing the RNase PH domain is responsible for the sequence-specific binding to AU-rich elements. Furthermore, we found that the RNase PH domains from two other exosomal components, OIP2 and RRP41, as well as from Escherichia coli polynucleotide phosphorylase, are all capable of specifically interacting with RNAs containing an AU-rich element with similar affinities. Finally, we demonstrate that the interaction of the RNase PH domain of PM-Scl-75 is readily competed by poly(U), but only inefficiently using other homopolymeric RNAs. These data demonstrate that RNase PH domains in general have an affinity for U- and AU-rich sequences, and broaden the potential role in RNA biology of proteins containing these domains.     

A phosphoinositide-binding sequence is shared by PH domain target molecules—a model for the binding of PH domains to proteins

Pleckstrin homology (PH) domains have been proven to bind phosphoinositides (PI) and inositolphosphates (IP). On the other hand, a binding of PH domains to proteins is still a matter of debate. The goal of this work was to identify potential PH domain protein target sites and to build a model for PH domain–protein binding. A candidate sequence, called HIKE, was identified by sequence homology analysis of the proteins that are considered the strongest PH binding candidates, i.e., G_β, PKC, and Akt. HIKE contains a PI binding sequence and fulfills several criteria for a potential PH-binding site, i.e., it is present in other PH-binding candidates, lies in regulatory regions independently predicted to bind PH domains, and is conserved in 3-D structure among different molecules. These findings and the similarities with the mode of binding of PTB and PDZ domains suggest a β strand–β strand coordination model for PH–protein binding. The HIKE model predicts that membrane anchoring of PH domains and their targets could be a critical step in their interaction, which would consistently explain why PH–protein binding has only been detected in the presence of PI. Proteins 31:1–9, 1998. © 1998 Wiley-Liss, Inc.