Contrasting membrane interaction mechanisms of AP180 N-terminal homology (ANTH) and epsin N-terminal homology (ENTH) domains

Epsin and AP180/CALM are endocytotic accessory proteins that have been implicated in the formation of clathrin-coated pits. Both proteins have phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding domains in their N termini, but these domains are structurally and functionally different. To understand the basis of their distinct properties, we measured the PtdIns(4,5)P2-dependent membrane binding of the epsin N-terminal homology (ENTH) domain and the AP180 N-terminal homology (ANTH) domain by means of surface plasmon resonance and monolayer penetration techniques and also calculated the effect of PtdIns(4,5)P2 on the electrostatic potential of these domains. PtdIns(4,5)P2 enhances the electrostatic membrane association of both domains; however, PtdIns(4,5)P2 binding exerts distinct effects on their membrane dissociation. Specifically, PtdIns(4,5)P2 induces the membrane penetration of the N-terminal alpha-helix of the ENTH domain, which slows the membrane dissociation of the domain and triggers the membrane deformation. These results provide the biophysical explanation for the membrane bending activity of epsin and its ENTH domain.     

Sequence analysis of Arabidopsis thaliana E/ANTH-domain-containing proteins: membrane tethers of the clathrin-dependent vesicle budding machinery

Summary. The epsin N-terminal homology (ENTH) domain is a conserved protein module present in cytosolic proteins which are required in clathrin-mediated vesicle budding processes. A highly similar, yet unique module is the AP180 N-terminal homology (ANTH) domain, which is present in a set of proteins that also support clathrin-dependent endocytosis. Both ENTH and ANTH (E/ANTH) domains bind to phospholipids and proteins, in order to support the nucleation of clathrin coats on the plasma membrane or the trans-Golgi-network membrane. Therefore, E/ANTH proteins might be considered as universal tethering components of the clathrin-mediated vesicle budding machinery. Since the E/ANTH protein family appears to be crucial in the first steps of clathrin-coated vesicle budding, we performed data base searches of the Arabidopsis thaliana genome. Sequence analysis revealed three proteins containing the ENTH signature motif and eight proteins containing the ANTH signature motif. Another six proteins were found that do not contain either motif but seem to have the same domain structure and might therefore be seen as VHS-domain-containing plant proteins. Functional analysis of plant E/ANTH proteins are rather scarce, since only one ANTH homolog from A. thaliana, At-AP180, has been characterized so far. At-AP180 displays conserved functions as a clathrin assembly protein and as an α-adaptin binding partner, and in addition shows features at the molecular level that seem to be plant-specific. Correspondence and reprints: Cell Biology, Heidelberg Institute for Plant Sciences, Im Neuenheimer Feld 230, 69120 Heidelberg, Federal Republic of Germany.     

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.     

BAR, F-BAR (EFC) and ENTH/ANTH domains in the regulation of membrane-cytosol interfaces and membrane curvature

BAR and ENTH domains are families of alpha-helical lipid bilayer binding modules found in proteins that function in endocytosis, actin regulation and signaling. Several members of these families not only bind the bilayer, but also participate in the regulation of its curvature. These properties are thought to play physiological roles at sites of membrane budding and at other sites where narrow tubular membranes occur in vivo. Studies of BAR and ENTH domains and of their flanking regions have provided new insights into mechanisms of membrane deformation and curvature sensing, and have emphasized the importance of amphipathic helices, thought to intercalate in one of the leaflets of the lipid bilayer, in the generation of membrane curvature. Structural studies and database searches are rapidly expanding the BAR and ENTH domains families, with the identification of new related domains and subfamilies, such as F-BAR (also called EFC) domains and ANTH domains, respectively. Here we present a short overview of the properties of these domains based on evidence obtained from genetics, cell biology, biochemistry and structural biology.     

Recognizing and defining true Ras binding domains II: in silico prediction based on homology modelling and energy calculations

Considering the large number of putative Ras effector proteins, it is highly desirable to develop computational methods to be able to identify true Ras binding molecules. Based on a limited sequence homology among members of the Ras association (RA) and Ras binding (RB) sub-domain families of the ubiquitin super-family, we have built structural homology models of Ras proteins in complex with different RA and RB domains, using the FOLD-X software. A critical step in our approach is to use different templates of Ras complexes, in order to account for the structural variation among the RA and RB domains. The homology models are validated by predicting the effect of mutating hot spot residues in the interface, and residues important for the specificity of interaction with different Ras proteins. The FOLD-X calculated energies of the best-modelled complexes are in good agreement with previously published experimental data and with new data reported here. Based on these results, we can establish energy thresholds above, or below which, we can predict with 96% confidence that a RA/RB domain will or will not interact with Ras. This study shows the importance of in depth structural analysis, high quality force-fields and modelling for correct prediction. Our work opens the possibility of genome-wide prediction for this protein family and for others, where there is enough structural information.     

The structure of the PDZ3-SH3-GuK tandem of ZO-1 protein suggests a supramodular organization of the membrane-associated guanylate kinase (MAGUK) family scaffold protein core

Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tethering membrane receptors, adhesion molecules, and macromolecular signaling complexes for tissue developments, cell-cell communications, and intracellular signal transductions. The defining feature of the MAGUK family scaffolds is that each member contains a conserved core consisting of a PSD-95/Dlg/ZO-1 (PDZ) domain, an Src homology 3 (SH3) domain, and a catalytically inactive guanylate kinase (GuK) domain arranged in tandem, although the structural features and functional implications of the PDZ-SH3-GuK tandem arrangement are unclear. The structure of the ZO-1 PDZ3-SH3-GuK tandem solved in this study reveals that the PDZ domain directly interacts with the SH3-GuK module, forming a structural supramodule with distinct target binding properties with respect to the isolated domains. Structure-based sequence analysis suggests that the PDZ-SH3-GuK tandems of other members of the MAGUK family also form supramodules.     

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.     

Membrane binding and subcellular targeting of C2 domains

C2 domains are a ubiquitous structural module and many of them function in Ca2+ -dependent membrane binding and thereby serve as Ca2+ effectors for divergent Ca2+ -mediated cellular processes. Extensive structural, biochemical, biophysical, and cellular studies of C2 domains and host proteins in the past decade have shown that due to their structural diversity C2 domains have disparate Ca2+ sensitivity, lipid selectivity and membrane binding mechanisms. This review summarizes the basic structural and functional properties of C2 domains as well as recent findings on Ca2+ and membrane binding, lipid selectivity, and subcellular localization of C2 domains and their host proteins.     

Computational study of the human dystrophin repeats: interaction properties and molecular dynamics

Dystrophin is a large protein involved in the rare genetic disease Duchenne muscular dystrophy (DMD). It functions as a mechanical linker between the cytoskeleton and the sarcolemma, and is able to resist shear stresses during muscle activity. In all, 75% of the dystrophin molecule consists of a large central rod domain made up of 24 repeat units that share high structural homology with spectrin-like repeats. However, in the absence of any high-resolution structure of these repeats, the molecular basis of dystrophin central domain's functions has not yet been deciphered. In this context, we have performed a computational study of the whole dystrophin central rod domain based on the rational homology modeling of successive and overlapping tandem repeats and the analysis of their surface properties. Each tandem repeat has very specific surface properties that make it unique. However, the repeats share enough electrostatic-surface similarities to be grouped into four separate clusters. Molecular dynamics simulations of four representative tandem repeats reveal specific flexibility or bending properties depending on the repeat sequence. We thus suggest that the dystrophin central rod domain is constituted of seven biologically relevant sub-domains. Our results provide evidence for the role of the dystrophin central rod domain as a scaffold platform with a wide range of surface features and biophysical properties allowing it to interact with its various known partners such as proteins and membrane lipids. This new integrative view is strongly supported by the previous experimental works that investigated the isolated domains and the observed heterogeneity of the severity of dystrophin related pathologies, especially Becker muscular dystrophy.     

Structural basis for interaction of DivIVA/GpsB proteins with their ligands

DivIVA proteins and their GpsB homologues are late cell division proteins found in Gram‐positive bacteria. DivIVA/GpsB proteins associate with the inner leaflet of the cytosolic membrane and act as scaffolds for other proteins required for cell growth and division. DivIVA/GpsB proteins comprise an N‐terminal lipid‐binding domain for membrane association fused to C‐terminal domains supporting oligomerization. Despite sharing the same domain organization, DivIVA and GpsB serve different cellular functions: DivIVA plays diverse roles in division site selection, chromosome segregation and controlling peptidoglycan homeostasis, whereas GpsB contributes to the spatiotemporal control of penicillin‐binding protein activity. The crystal structures of the lipid‐binding domains of DivIVA from Bacillus subtilis and GpsB from several species share a fold unique to this group of proteins, whereas the C‐terminal domains of DivIVA and GpsB are radically different. A number of pivotal features identified from the crystal structures explain the functional differences between the proteins. Herein we discuss these structural and functional relationships and recent advances in our understanding of how DivIVA/GpsB proteins bind and recruit their interaction partners, knowledge that might be useful for future structure‐based DivIVA/GpsB inhibitor design.     

High-throughput fluorescence assay for membrane-protein interaction

Membrane-protein interaction plays key roles in a wide variety of biological processes. Although various methods have been employed to measure membrane binding of soluble proteins, a robust high-throughput assay that is universally applicable to all proteins is lacking at present. Here we report a new fluorescence quenching assay utilizing enhanced green fluorescence protein (EGFP)-fusion proteins and a lipid containing a dark quencher, N-dimethylaminoazobenzenesulfonyl-phosphatidylethanolamine (dabsyl-PE). The EGFP fluorescence emission intensity showed a large decrease (i.e., >50%) when EGFP-fusion proteins bound the vesicles containing 5 mol% dabsyl-PE. This simple assay, which can be performed using either a cuvette-based spectrofluorometer or a fluorescence plate reader, allowed rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid binding domains, including two pleckstrin homology domains, an epsin N-terminal homology domain, and a phox homology domain. The assay can also be applied to high-throughput screening of small molecules that modulate membrane binding of proteins.     

The molecular basis of differential subcellular localization of C2 domains of protein kinase C-alpha and group IVa cytosolic phospholipase A2

The C2 domain is a Ca(2+)-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. C2 domains are unique among membrane targeting domains in that they show a wide range of lipid selectivity for the major components of cell membranes, including phosphatidylserine and phosphatidylcholine. To understand how C2 domains show diverse lipid selectivity and how this functional diversity affects their subcellular targeting behaviors, we measured the binding of the C2 domains of group IVa cytosolic phospholipase A(2) (cPLA(2)) and protein kinase C-alpha (PKC-alpha) to vesicles that model cell membranes they are targeted to, and we monitored their subcellular targeting in living cells. The surface plasmon resonance analysis indicates that the PKC-alpha C2 domain strongly prefers the cytoplasmic plasma membrane mimic to the nuclear membrane mimic due to high phosphatidylserine content in the former and that Asn(189) plays a key role in this specificity. In contrast, the cPLA(2) C2 domain has specificity for the nuclear membrane mimic over the cytoplasmic plasma membrane mimic due to high phosphatidylcholine content in the former and aromatic and hydrophobic residues in the calcium binding loops of the cPLA(2) C2 domain are important for its lipid specificity. The subcellular localization of enhanced green fluorescent protein-tagged C2 domains and mutants transfected into HEK293 cells showed that the subcellular localization of the C2 domains is consistent with their lipid specificity and could be tailored by altering their in vitro lipid specificity. The relative cell membrane translocation rate of selected C2 domains was also consistent with their relative affinity for model membranes. Together, these results suggest that biophysical principles that govern the in vitro membrane binding of C2 domains can account for most of their subcellular targeting properties.     

Membrane and juxtamembrane targeting by PH and PTB domains

Modular pleckstrin homology (PH) and phospho-tyrosine binding (PTB) domains are present in a remarkably large number of proteins from yeast to humans. With a common core fold, these domain families have evolved to recognize membrane embedded phospholipids, in particular phosphoinositides, peripheral membrane proteins, and peptide motifs in juxtamembrane regions of integral membrane proteins. As the result of intensive investigation using biochemical, biophysical, and structural approaches, common ligand recognition principles have emerged along with insights into the structural variations that account for the diversity of ligand specificities. Analyses of membrane targeting in cells have revealed additional determinants beyond the primary ligand binding sites. In this review, we highlight unifying recognition principles and further illustrate with examples how divergent mechanisms contribute to membrane and juxtamembrane targeting by PH and PTB domains.     

The use of functional domains to improve transmembrane protein topology prediction

Transmembrane proteins affect vital cellular functions and pathogenesis, and are a focus of drug design. It is difficult to obtain diffraction quality crystals to study transmembrane protein structure. Computational tools for transmembrane protein topology prediction fill in the gap between the abundance of transmembrane proteins and the scarcity of known membrane protein structures. Their prediction accuracy is still inadequate: TMHMM, the current state-of-the-art method, has less than 52% accuracy in topology prediction on one set of transmembrane proteins of known topology. Based on the observation that there are functional domains that occur preferentially internal or external to the membrane, we have extended the model of TMHMM to incorporate functional domains, using a probabilistic approach originally developed for computational gene finding. Our extension is better than TMHMM in predicting the topology of transmembrane proteins. As prediction of functional domain improves, our system's prediction accuracy will likely improve as well.     

The order of PDZ3 and TrpCage in fusion chimeras determines their properties—a biophysical characterization

Most of the structural proteins known today are composed of domains that carry their own functions while keeping their structural properties. It is supposed that such domains, when taken out of the context of the whole protein, can retain their original structure and function to a certain extent. Information on the specific functional and structural characteristics of individual domains in a new context of artificial fusion proteins may help to reveal the rules of internal and external domain communication. Moreover, this could also help explain the mechanism of such communication and address how the mutual allosteric effect plays a role in a such multi‐domain protein system. The simple model system of the two‐domain fusion protein investigated in this work consisted of a well‐folded PDZ3 domain and an artificially designed small protein domain called Tryptophan Cage (TrpCage). Two fusion proteins with swapped domain order were designed to study their structural and functional features as well as their biophysical properties. The proteins composed of PDZ3 and TrpCage, both identical in amino acid sequence but different in composition (PDZ3‐TrpCage, TrpCage‐PDZ3), were studied using circualr dichroism (CD) spectrometry, analytical ultracentrifugation, and molecular dynamic simulations. The biophysical analysis uncovered different structural and denaturation properties of both studied proteins, revealing their different unfolding pathways and dynamics.     

pH-dependent binding of the Epsin ENTH domain and the AP180 ANTH domain to PI(4,5)P2-containing bilayers

Epsin and AP180 are essential components of the endocytotic machinery, which controls internalization of protein receptors and other macromolecules at the cell surface. Epsin and AP180 are recruited to the plasma membrane by their structurally and functionally related N-terminal ENTH and ANTH domains that specifically recognize PtdIns(4,5)P₂. Here, we show that membrane anchoring of the ENTH and ANTH domains is regulated by the acidic environment. Lowering the pH enhances PtdIns(4,5)P₂ affinity of the ENTH and ANTH domains reinforcing their association with lipid vesicles and monolayers. The pH dependency is due to the conserved histidine residues of the ENTH and ANTH domains, protonation of which is necessary for the strong PtdIns(4,5)P₂ recognition, as revealed by liposome binding, surface plasmon resonance, NMR, monolayer surface tension and mutagenesis experiments. The pH sensitivity of the ENTH and ANTH domains is reminiscent to the pH dependency of the FYVE domain suggesting a common regulatory mechanism of membrane anchoring by a subset of the PI-binding domains.     

Membrane binding of lipidated Ras peptides and proteins — The structural point of view

Biological membranes are interesting interfaces, at which important biological processes occur. In addition to integral membrane proteins, a number of proteins bind to the membrane surface and associate with it. Posttranslational lipid modification is one important mechanism, by which soluble molecules develop a propensity towards the membrane and reversibly bind to it. Membrane binding by insertion of hydrophobic lipid moieties is relevant for up to 10% of all cellular proteins. A particular interesting lipid-modified protein is the small GTPase Ras, which plays a key role in cellular signal transduction. Until recently, the structural basis for membrane binding of Ras was not well-defined. However, with the advent of new synthesis techniques and the advancement of several biophysical methods, a number of structural and dynamical features about membrane binding of Ras proteins have been revealed. This review will summarize the chemical biology of Ras and discuss in more detail the biophysical and structural features of the membrane bound C-terminus of the protein.     

Membrane binding of lipidated Ras peptides and proteins--the structural point of view

Biological membranes are interesting interfaces, at which important biological processes occur. In addition to integral membrane proteins, a number of proteins bind to the membrane surface and associate with it. Posttranslational lipid modification is one important mechanism, by which soluble molecules develop a propensity towards the membrane and reversibly bind to it. Membrane binding by insertion of hydrophobic lipid moieties is relevant for up to 10% of all cellular proteins. A particular interesting lipid-modified protein is the small GTPase Ras, which plays a key role in cellular signal transduction. Until recently, the structural basis for membrane binding of Ras was not well-defined. However, with the advent of new synthesis techniques and the advancement of several biophysical methods, a number of structural and dynamical features about membrane binding of Ras proteins have been revealed. This review will summarize the chemical biology of Ras and discuss in more detail the biophysical and structural features of the membrane bound C-terminus of the protein.