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 domains of tec family protein kinases.

Pleckstrin homology (PH) domains have been shown to be involved in different interactions, including binding to inositol compounds, protein kinase C isoforms, and heterotrimeric G proteins. In some cases, the most important function of PH domains is transient localisation of proteins to membranes, where they can interact with their partners. Tec family protein tyrosine kinases contain a PH domain. In Btk, also PH domain mutations lead into an immunodeficiency, X-linked agammaglobulinemia (XLA). A new disease-causing mutation was identified in the PH domain. The structures for the PH domains of Bmx, Itk, and Tec were modelled based on Btk structure. The domains seem to have similar scaffolding and electrostatic polarisation but to have some differences in the binding regions. The models provide new insight into the specificity, function, and regulation of Tec family kinases.     

Genome-wide analysis of membrane targeting by S. cerevisiae pleckstrin homology domains

Pleckstrin homology (PH) domains are small protein modules known for their ability to bind phosphoinositides and to drive membrane recruitment of their host proteins. We investigated phosphoinositide binding (in vitro and in vivo) and subcellular localization, and we modeled the electrostatic properties for all 33 PH domains encoded in the S. cerevisiae genome. Only one PH domain (from Num1p) binds phosphoinositides with high affinity and specificity. Six bind phosphoinositides with moderate affinity and little specificity and are membrane targeted in a phosphoinositide-dependent manner. Although all of the remaining 26 yeast PH domains bind phosphoinositides very weakly or not at all, three were nonetheless efficiently membrane targeted. Our proteome-wide analysis argues that membrane targeting is important for only approximately 30% of yeast PH domains and is defined by binding to both phosphoinositides and other targets. These findings have significant implications for understanding the function of proteins that contain this common domain.     

Enigmatic relationship of Pleckstrin homology domain with phospholipid breakdown mediated signal transduction.

Research into phospholipid signaling continues to flourish, as more and more bioactive lipids and proteins are being identified and their actions characterised. The Pleckstrin homology (PH) domain is one such newly recognized protein module thought to play an important role in intracellular signal transduction. The tertiary structures of several PH domains have been determined, some of them complexed with ligands and on the basis of structural similarities between PH domains and lipid binding proteins it has been suggested that PH domains may be binding to lipophilic molecules. In fact many of the proteins that contain this domain can interfere with the membrane association. This review examines the specificity of this binding and illustrates the importance of charge-charge interactions in PIP2-PH domain complex formation. The precise physiological functions of PH domain in vivo remains to be explored therefore this review examines the biochemical aspects of the interaction of PH domains with phospholipid breakdown mediated products and proto-oncogenic serine-threonine kinase (Akt), protein tyrosine kinases, which have been found to be a target of phospholipid second messengers. Thus, number of cellular processes mediated by this way, ranging from insulin signaling and protein synthesis to differentiation and cell survival are regulated by this intracellular signaling protein module.     

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.     

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.     

ICln159 folds into a pleckstrin homology domain-like structure. Interaction with kinases and the splicing factor LSm4

ICln is a multifunctional protein involved in regulatory mechanisms as different as membrane ion transport and RNA splicing. The protein is water-soluble, and during regulatory volume decrease after cell swelling, it is able to migrate from the cytosol to the cell membrane. Purified, water-soluble ICln is able to insert into lipid bilayers to form ion channels. Here, we show that ICln159, a truncated ICln mutant, which is also able to form ion channels in lipid bilayers, belongs to the pleckstrin homology (PH) domain superfold family of proteins. The ICln PH domain shows unusual properties as it lacks the electrostatic surface polarization seen in classical PH domains. However, similar to many classical PH domain-containing proteins, ICln interacts with protein kinase C, and in addition, interacts with cAMP-dependent protein kinase and cGMP-dependent protein kinase type II but not cGMP-dependent protein kinase type Ibeta. A major phosphorylation site for all three kinases is Ser-45 within the ICln PH domain. Furthermore, ICln159 interacts with LSm4, a protein involved in splicing and mRNA degradation, suggesting that the ICln159 PH domain may serve as a protein-protein interaction platform.     

Structure basis and unconventional lipid membrane binding properties of the PH-C1 tandem of rho kinases

Rho kinase (ROCK), a downstream effector of Rho GTPase, is a serine/threonine protein kinase that regulates many crucial cellular processes via control of cytoskeletal structures. The C-terminal PH-C1 tandem of ROCKs has been implicated to play an autoinhibitory role by sequestering the N-terminal kinase domain and reducing its kinase activity. The binding of lipids to the pleckstrin homology (PH) domain not only regulates the localization of the protein but also releases the kinase domain from the close conformation and thereby activates its kinase activity. However, the molecular mechanism governing the ROCK PH-C1 tandem-mediated lipid membrane interaction is not known. In this study, we demonstrate that ROCK is a new member of the split PH domain family of proteins. The ROCK split PH domain folds into a canonical PH domain structure. The insertion of the atypical C1 domain in the middle does not alter the structure of the PH domain. We further show that the C1 domain of ROCK lacks the diacylglycerol/phorbol ester binding pocket seen in other canonical C1 domains. Instead, the inserted C1 domain and the PH domain function cooperatively in binding to membrane bilayers via the unconventional positively charged surfaces on each domain. Finally, the analysis of all split PH domains with known structures indicates that split PH domains represent a unique class of tandem protein modules, each possessing distinct structural and functional features.     

Critical role of the pleckstrin homology and cysteine-rich domains in Vav signaling and transforming activity

Vav family proteins are members of the Dbl family of guanine nucleotide exchange factors and activators of Rho family small GTPases. In addition to the Dbl homology (DH) domain important for guanine nucleotide exchange factor catalytic function, all Dbl family proteins contain an adjacent pleckstrin homology (PH) domain that serves to regulate DH domain activity. Although the role of the PH domain in Vav function has been evaluated extensively, its precise role and whether it serves a distinct role in different Vav proteins remain unresolved. Additionally, the precise role of an adjacent cysteine-rich domain (CRD) in regulating DH domain function is also unclear. In this study, we evaluated the contribution of these putative protein-protein or protein-lipid interaction domains to Vav signaling and transforming activity. In contrast to previous observations, we found that the PH domain is critical for Vav transforming activity. Similarly, the CRD was also essential and served a function distinct from that of the PH domain. Although mutation of either domain reduced Vav membrane association, addition of plasma membrane targeting sequences to either the CRD or PH domain mutant proteins did not restore Vav transforming activity. This result contrasts with other Dbl family proteins, where a membrane targeting sequence alone was sufficient to restore the loss of function caused by mutation of the PH domain. Furthermore, green fluorescent protein fusion proteins containing the PH domain or CRD, or both, failed to target to the plasma membrane, suggesting that these two domains also serve regulatory functions independent of promoting membrane localization. Finally, we found that phosphatidylinositol 3-kinase activation may promote Vav membrane association via phosphatidylinositol 3,4,5-triphosphate binding to the PH domain.     

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.     

Loss of phosphatidylinositol 3-phosphate binding by the C-terminal Tiam-1 pleckstrin homology domain prevents in vivo Rac1 activation without affecting membrane targeting

Dbl family guanine nucleotide exchange factors (GEFs) for Rho family small GTPases invariably contain a pleckstrin homology (PH) domain that immediately follows their Dbl homology (DH) domain. Although the DH domain is responsible for GEF activity, the role of the PH domain is less clear. We previously reported that PH domains from several Dbl family members bind phosphoinositides with very low affinity (K(d) values in the 10 microM range). This suggests that, unlike several other PH domains, those from Dbl proteins will not function as independent membrane-targeting modules. To determine the functional relevance of low affinity phosphoinositide binding, we mutated the corresponding PH domain from Tiam-1 to abolish its weak, specific binding to phosphatidylinositol 3-phosphate. We first confirmed in vitro that phosphoinositide binding by the isolated DH/PH domain was impaired by the mutations but that intrinsic GEF activity was unaffected. We then introduced the PH domain mutations into full-length Tiam-1 and found that its ability to activate Rac1 or serum response factor in vivo was abolished. Immunofluorescence studies showed that membrane targeting of Tiam-1 was essentially unaffected by mutations in the C-terminal PH domain. Our studies therefore indicate that low affinity phosphatidylinositol 3-phosphate binding by the C-terminal PH domain may be critical for in vivo regulation and activity of Tiam-1 but that the PH domain exerts its regulatory effects without altering membrane targeting. We suggest instead that ligand binding to the PH domain induces conformational and/or orientational changes at the membrane surface that are required for maximum exchange activity of its adjacent DH domain.     

Crystal structure of the DH/PH fragment of Dbs without bound GTPase

Dbl proteins are guanine nucleotide exchange factors for Rho GTPases, containing adjacent Dbl homology (DH) and pleckstrin homology (PH) domains. This domain architecture is virtually invariant and typically required for full exchange potential. Several structures of DH/PH fragments bound to GTPases implicate the PH domain in nucleotide exchange. To more fully understand the functional linkage between DH and PH domains, we have determined the crystal structure of the DH/PH fragment of Dbs without bound GTPase. This structure is generally similar to previously determined structures of Dbs bound to GTPases albeit with greater apparent mobility between the DH and PH domains. These comparisons suggest that the DH and PH domains of Dbs are spatially primed for binding GTPases and small alterations in intradomain conformations that may be elicited by subtle biological responses, such as altered phosphoinositide levels, are sufficient to enhance exchange by facilitating interactions between the PH domain and GTPases.     

The Dbs PH domain contributes independently to membrane targeting and regulation of guanine nucleotide-exchange activity

Dbl family GEFs (guanine nucleotide-exchange factors) for the Rho GTPases almost invariably contain a PH (pleckstrin homology) domain adjacent to their DH (Dbl homology) domain. The DH domain is responsible for GEF activity, and the PH domain plays a regulatory role that remains poorly understood. We demonstrated previously that Dbl family PH domains bind phosphoinositides with low affinity and cannot function as independent membrane targeting modules. In the present study, we show that dimerization of a Dbs (Dbl's big sister) DH/PH domain fragment is sufficient to drive it to the plasma membrane through a mechanism involving PH domain-phosphoinositide interactions. Thus, the Dbs PH domain could play a significant role in membrane targeting if it co-operates with other domains in the protein. We also show that mutations that prevent phosphoinositide binding by the Dbs PH domain significantly impair cellular GEF activity even in chimaeric proteins that are robustly membrane targeted by farnesylation or by the PH domain of phospholipase C-delta1. This finding argues that the Dbs PH domain plays a regulatory role that is independent of its ability to aid membrane targeting. Thus, we suggest that the PH domain plays dual roles, contributing independently to membrane localization of Dbs (as part of a multi-domain interaction) and allosteric regulation of the DH domain.     

Comparison of sequence-based and structure-based phylogenetic trees of homologous proteins: Inferences on protein evolution

Several studies based on the known three-dimensional (3-D) structures of proteins show that two homologous proteins with insignificant sequence similarity could adopt a common fold and may perform same or similar biochemical functions. Hence, it is appropriate to use similarities in 3-D structure of proteins rather than the amino acid sequence similarities in modelling evolution of distantly related proteins. Here we present an assessment of using 3-D structures in modelling evolution of homologous proteins. Using a dataset of 108 protein domain families of known structures with at least 10 members per family we present a comparison of extent of structural and sequence dissimilarities among pairs of proteins which are inputs into the construction of phylogenetic trees. We find that correlation between the structure-based dissimilarity measures and the sequence-based dissimilarity measures is usually good if the sequence similarity among the homologues is about 30% or more. For protein families with low sequence similarity among the members, the correlation coefficient between the sequence-based and the structure-based dissimilarities are poor. In these cases the structure-based dendrogram clusters proteins with most similar biochemical functional properties better than the sequence-similarity based dendrogram. In multi-domain protein families and disulphide-rich protein families the correlation coefficient for the match of sequence-based and structure-based dissimilarity (SDM) measures can be poor though the sequence identity could be higher than 30%. Hence it is suggested that protein evolution is best modelled using 3-D structures if the sequence similarities (SSM) of the homologues are very low.     

The pleckstrin homology domain of phospholipase C-beta(2) links the binding of gbetagamma to activation of the catalytic core

Pleckstrin homology (PH) domains are membrane tethering devices found in many signal transducing proteins. These domains also couple to the betagamma subunits of GTP binding proteins (G proteins), but whether this association transmits allosteric information to the catalytic core is unclear. To address this question, we constructed protein chimeras in which the PH domain of phospholipase C-beta(2) (PLC-beta(2)), which is regulated by Gbetagamma, replaces the PH domain of PLC-delta(1) which binds to, but is not regulated by, Gbetagamma. We found that attachment of the PH domain of PLC-beta(2) onto PLC-delta(1) not only causes the membrane-binding properties of PLC-delta(1) to become similar to those of PLC-beta(2), but also results in a Gbetagamma-regulated enzyme. Thus, PH domains are more than simple tethering devices and mediate regulatory signals to the host protein.     

Serum and glucocorticoid-regulated protein kinases: variations on a theme

The phosphatidylinositol 3' kinase (PI3K)-signaling pathway plays a critical role in a variety of cellular responses such as modulation of cell survival, glucose homeostasis, cell division, and cell growth. PI3K generates important lipid second messengers-phosphatidylinositides that are phosphorylated at the 3' position of their inositol ring head-group. These membrane restricted lipids act by binding with high affinity to specific protein domains such as the pleckstrin homology (PH) domain. Effectors of PI3K include molecules that harbor such domains such as phosphoinositide-dependent kinase (PDK1) and protein kinase B (PKB), also termed Akt. The mammalian genome encodes three different PKB genes (alpha, beta, and gamma; Akt1, 2, and 3, respectively) and each is an attractive target for therapeutic intervention in diseases such as glioblastoma and breast cancer. A second family of three protein kinases, termed serum and glucocorticoid-regulated protein kinases (SGKs), is structurally related to the PKB family including regulation by PI3K but lack a PH domain. However, in addition to PH domains, a second class of 3' phosphorylated inositol phospholipid-binding domains exists that is termed Phox homology (PX) domain: this domain is found in one of the SGKs (SGK3). Here, we summarize knowledge of the three SGK isoforms and compare and contrast them to PKB with respect to their possible importance in cellular regulation and potential as therapeutic targets.     

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.     

Rational design and purification of human Bruton's tyrosine kinase SH3-SH2 protein for structure-function studies

Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase consisting of N-terminal pleckstrin homology (PH) domain followed by Tec homology (TH) domain, Src homology 3 and 2 (SH3 and SH2) domains, and a C-terminal kinase domain. Mutations in the human BTK gene cause the severe immunodeficiency disease X-linked agammaglobulinemia (XLA). The structural and functional basis of several XLA-causing mutations remains unknown, since only the structures of the PH and SH3 domains of human Btk are currently available. In this study, we overexpressed and purified a protein consisting of the SH3 and SH2 domains of human Btk for biochemical and structural analysis. The purified protein was only partially soluble and had a tendency to dimerize, which made it unsuitable for further studies. To overcome the problems of low solubility and dimerization, subdomain interactions were engineered without altering the function of the protein.     

Recruitment of Dok-R to the EGF receptor through its PTB domain is required for attenuation of Erk MAP kinase activation.

Dok (for downstream of tyrosine kinases) proteins are a newly identified family of docking molecules that are characterized by the presence of an amino-terminal pleckstrin homology (PH) domain, a central putative phosphotyrosine-binding (PTB) domain and numerous potential sites of tyrosine phosphorylation [1] [2] [3] [4] [5] [6]. Here, we explore the potential role of the Dok family member Dok-R (also known as p56(Dok2) or FRIP) in signaling pathways mediated by the epidermal growth factor (EGF) receptor. An intact PTB domain in Dok-R was critical for its association with two PTB-binding consensus sites on the EGF receptor and the PH domain further contributed to stable in vivo binding and tyrosine phosphorylation of Dok-R. Multiple sites on Dok-R were tyrosine-phosphorylated following EGF stimulation; phosphorylated Tyr276 and Tyr304 are proposed to dock the tandem Src homology 2 (SH2) domains of the p21(Ras) GTPase-activating protein rasGAP and Tyr351 mediates an association with the SH2 domain of the adapter protein Nck. Interestingly, we have found that Dok-R could attenuate EGF-stimulated mitogen-activated protein (MAP) kinase activation independently of its association with rasGAP. Together, these results suggest that Dok-R has an important role downstream of growth factor receptors as a potential negative regulator of signal transduction.