Crystal structure of the Homer 1 family conserved region reveals the interaction between the EVH1 domain and own proline-rich motif

PSD-Zip45 (also named Homer 1c/Vesl-1L) is a synaptic scaffolding protein, which interacts with neurotransmitter receptors and other scaffolding proteins to target them into post-synaptic density (PSD), a specialized protein complex at the synaptic junction. Binding of the PSD-Zip45 to the receptors and scaffolding proteins results in colocalization and clustering of its binding partners in PSD. It has an Ena/VASP homology 1 (EVH1) domain in the N terminus for receptor binding, two leucine zipper motifs in the C terminus for clustering, and a linking region whose function is unclear despite the high level of conservation within the Homer 1 family. The X-ray crystallographic analysis of the largest fragment of residues 1-163, including an EVH1 domain reported here, demonstrates that the EVH1 domain contains an alpha-helix longer than that of the previous models, and that the linking part included in the conserved region of Homer 1 (CRH1) of the PSD-Zip45 interacts with the EVH1 domain of the neighbour CRH1 molecule in the crystal. The results suggest that the EVH1 domain recognizes the PPXXF motif found in the binding partners, and the SPLTP sequence (P-motif) in the linking region of the CRH1. The two types of binding are partly overlapped in the EVH1 domain, implying a mechanism to regulate multimerization of Homer 1 family proteins.     

Dual epitope recognition by the VASP EVH1 domain modulates polyproline ligand specificity and binding affinity

The Ena-VASP family of proteins act as molecular adaptors linking the cytoskeletal system to signal transduction pathways. Their N-terminal EVH1 domains use groups of exposed aromatic residues to specifically recognize 'FPPPP' motifs found in the mammalian zyxin and vinculin proteins, and ActA protein of the intracellular bacterium Listeria monocytogenes. Here, evidence is provided that the affinities of these EVH1-peptide interactions are strongly dependent on the recognition of residues flanking the core FPPPP motifs. Determination of the VASP EVH1 domain solution structure, together with peptide library screening, measurement of individual K(d)s by fluorescence titration, and NMR chemical shift mapping, revealed a second affinity-determining epitope present in all four ActA EVH1-binding motifs. The epitope was shown to interact with a complementary hydrophobic site on the EVH1 surface and to increase strongly the affinity of ActA for EVH1 domains. We propose that this epitope, which is absent in the sequences of the native EVH1-interaction partners zyxin and vinculin, may provide the pathogen with an advantage when competing for the recruitment of the host VASP and Mena proteins in the infected cell.     

High-resolution structural analysis of mammalian profilin 2a complex formation with two physiological ligands: the formin homology 1 domain of mDia1 and the proline-rich domain of VASP

Profilins are small proteins capable of binding actin, poly-l-proline and other proline-rich sequences, and phosphatidylinositol (4,5)-bisphosphate. A number of proline-rich ligands for profilin have been characterised, including proteins of the Ena/VASP and formin families. We have determined the high-resolution crystal structures of mouse profilin 2a in complex with peptides from two functionally important ligands from different families, VASP and mDia1. The structures show that the binding mode of the peptide ligand is strongly affected by the non-proline residues in the sequence, and the peptides from VASP and mDia1 bind to profilin 2a in distinct modes. The high resolution of the crystallographic data allowed us to detect conserved CH-π hydrogen bonds between the peptide and profilin in both complexes. Furthermore, both peptides, which are shown to have micromolar affinity, induced the dimerisation of profilin, potentially leading to functionally different ligand-profilin-actin complexes. The peptides did not significantly affect actin polymerisation kinetics in the presence or in the absence of profilin 2a. Mutant profilins were tested for binding to poly-l-proline and the VASP and mDia1 peptides, and the F139A mutant bound proline-rich ligands with near-native affinity. Peptide blotting using a series of designed peptides with profilins 1 and 2a indicates differences between the two profilins towards proline-rich peptides from mDia1 and VASP. Our data provide structural insights into the mechanisms of mDia1 and VASP regulated actin polymerisation.     

1.15 A crystal structure of the X. tropicalis Spred1 EVH1 domain suggests a fourth distinct peptide-binding mechanism within the EVH1 family

The recently described Spred protein family has been implicated in the modulation of receptor tyrosine kinase signalling. We report the crystal structure of the Enabled/vasodilator-stimulated phosphoprotein homology-1 (EVH1) domain from Xenopus tropicalis Spred1, solved to 1.15 A resolution. This structure confirms that the Spred EVH1 adopts the pleckstrin-homology fold, with a similar secondary structure to Enabled. A translation of one of the peptide-binding groove beta-strands narrows this groove, whilst one end of the groove shows structural flexibility. We propose that Spred1 will bind peptides that are less proline-rich than other EVH1 domains, with conformational changes indicating an induced fit.     

A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family.

The ActA protein of the intracellular pathogen Listeria monocytogenes induces a dramatic reorganization of the actin-based cytoskeleton. Two profilin binding proteins, VASP and Mena, are the only cellular proteins known so far to bind directly to ActA. This interaction is mediated by a conserved module, the EVH1 domain. We identify E/DFPPPPXD/E, a motif repeated 4-fold within the primary sequence of ActA, as the core of the consensus ligand for EVH1 domains. This motif is also present and functional in at least two cellular proteins, zyxin and vinculin, which are in this respect major eukaryotic analogs of ActA. The functional importance of the novel protein-protein interaction was examined in the Listeria system. Removal of EVH1 binding sites on ActA reduces bacterial motility and strongly attenuates Listeria virulence. Taken together we demonstrate that ActA-EVH1 binding is a paradigm for a novel class of eukaryotic protein-protein interactions involving a proline-rich ligand that is clearly different from those described for SH3 and WW/WWP domains. This class of interactions appears to be of general importance for processes dependent on rapid actin remodeling.     

Requirements of basic amino acid residues within the lectin-like domain of LOX-1 for the binding of oxidized low-density lipoprotein.

Lectin-like OxLDL receptor-1 (LOX-1) was identified as the major receptor for oxidized low-density lipoprotein (OxLDL) in aortic endothelial cells. LOX-1 is a type II membrane protein that structurally belongs to the C-type lectin family. Here, we found that the lectin-like domain of LOX-1 is essential for ligand binding, but the neck domain is not. In particular, the large loop between the third and fourth cysteine of the lectin-like domain plays a critical role for OxLDL binding as well as C-terminal end residues. Alanine-directed mutagenesis of the basic amino acid residues around this region revealed that all of the basic residues are involved in OxLDL binding. Simultaneous mutations of these basic residues almost abolished the OxLDL-binding activity of LOX-1. Electrostatic interaction between basic residues in the lectin-like domain of LOX-1 and negatively charged OxLDL is critical for the binding activity of LOX-1.     

Structure of EVH1, a novel proline-rich ligand-binding module involved in cytoskeletal dynamics and neural function.

The Ena-VASP homology (EVH1) domain is a protein interaction module found in several proteins that are involved in transducing migratory and morphological signals into cytoskeletal reorganization. EVH1 specifically recognizes proline-rich sequences in its binding partners and directs the localization and formation of multicomponent assemblies involved in actin-based motile processes and neural development. The structure of the complex between an EVH1 domain and the target peptide sequence EFPPPPT identifies the interactions responsible for recognition and distinguishes it from other proline-rich binding modules, including SH3 and WW domains. Surprisingly, the EVH1 domain has structural similarity to pleckstrin homology (PH), phosphotyrosine-binding (PTB) and ran-binding (RanBD) domains.     

The WH1 and EVH1 domains of WASP and Ena/VASP family members bind distinct sequence motifs

A complex of N-WASP and WASP-interacting protein (WIP) plays an important role in actin-based motility of vaccinia virus and the formation of filopodia. WIP is also required to maintain the integrity of the actin cytoskeleton in T and B lymphocytes and is essential for T cell activation. However, in contrast to many other N-WASP binding proteins, WIP does not stimulate the ability of N-WASP to activate the Arp2/3 complex. Although the WASP homology 1 (WH1) domain of N-WASP interacts directly with WIP, we still lack the exact nature of its binding site. We have now identified and characterized the N-WASP WH1 binding motif in WIP in vitro and in vivo using Shigella and vaccinia systems. The WH1 domain, which is predicted to have a similar structural fold to the Ena/VASP homology 1 (EVH1) domain, binds to a sequence motif in WIP (ESRFYFHPISD) that is very different from the EVH1 proline-rich DL/FPPPP ligand. Interaction of the WH1 domain of N-WASP with WIP is dependent on the two highly conserved phenylalanine residues in the motif. The WH1 binding motif we have identified is conserved in WIP, CR16, WICH, and yeast verprolin.     

Two mutations of basic residues within the N-terminus of HMG-1 B domain with different effects on DNA supercoiling and binding to bent DNA

High mobility group (HMG) 1 protein and its two homologous DNA-binding domains, A and B ("HMG-boxes"), can bend and supercoil DNA in the presence of topoisomerase I, as well as recognize differently bent and distorted DNA structures, including four-way DNA junctions, supercoiled DNA and DNA modified with anticancer drug cisplatin. Here we show that the lysine-rich part of the linker region between A and B domains of HMG-1, the (85)TKKKFKD(91) sequence that is attached to the N-terminus of the B domain within HMG-1, is a prerequisite for a preferential binding of the B domain to supercoiled DNA. The above sequence is also essential for a high-affinity binding of the B domain to DNA containing a site-specific major 1,2-d(GpG) intrastrand DNA adduct of cisplatin. Mutation of Arg(97), but not Lys(90) [Lys(90) forms a specific cross-link with platinum(II) in major groove of cisplatin-modified DNA; Kane, S. A., and Lippard, S. J. (1996) Biochemistry 35, 2180--2188], to alanine significantly (>40-fold) reduces affinity of the B domain to cisplatin-modified DNA, inhibits the ability of the B domain to bend (ligase-mediated circularization) or supercoil DNA, and results in a loss of the preferential binding of the B domain to supercoiled DNA without affecting the structural-specificity of the HMG-box for four-way DNA junctions. Some of the reported activities of the B domain are enhanced when the B domain is covalently linked to the A domain. We propose that binding of the A/B linker region within the major DNA groove helps the two HMG-1 domains to anchor to the minor DNA groove to facilitate their DNA binding and other activities.     

The interaction of the human GGA1 GAT domain with rabaptin-5 is mediated by residues on its three-helix bundle

GGA proteins regulate clathrin-coated vesicle trafficking by interacting with multiple proteins during vesicle assembly. As part of this process, the GAT domain of GGA is known to interact with both ARF and Rabaptin-5. Particularly, the GAT domains of GGA1 and -2, but not of GGA3, specifically bind with a coiled-coil region of Rabaptin-5. Rabaptin-5 interacts with Rab5 and is an essential component of the fusion machinery for targeting endocytic vesicles to early endosomes. The recently determined crystal structure of the GGA1 GAT domain has provided insights into its interactions with partner proteins. Here, we describe mutagenesis studies on the GAT-Rabaptin-5 interaction. The results demonstrate that a hydrophobic surface patch on the C-terminal three-helix bundle motif of the GAT domain is directly involved in Rabaptin-5 binding. A GGA3-like mutation, N284S, in this Rabaptin-5 binding patch of GGA1 led to a reduced level of Rabaptin-5 binding. Furthermore, a reversed mutation, S293N, in GGA3 partially establishes Rabaptin-5 binding ability in its GAT domain. These results provide a structural explanation for the binding affinity difference among GGA proteins. The current results also suggest that the binding of GAT to Rabaptin-5 is independent of its interaction with ARF.     

Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.     

FBP WW domains and the Abl SH3 domain bind to a specific class of proline-rich ligands.

WW domains are conserved protein motifs of 38-40 amino acids found in a broad spectrum of proteins. They mediate protein-protein interactions by binding proline-rich modules in ligands. A 10 amino acid proline-rich portion of the morphogenic protein, formin, is bound in vitro by both the WW domain of the formin-binding protein 11 (FBP11) and the SH3 domain of Abl. To explore whether the FBP11 WW domain and Abl SH3 domain bind to similar ligands, we screened a mouse limb bud expression library for putative ligands of the FBP11 WW domain. In so doing, we identified eight ligands (WBP3 through WBP10), each of which contains a proline-rich region or regions. Peptide sequence comparisons of the ligands revealed a conserved motif of 10 amino acids that acts as a modular sequence binding the FBP11 WW domain, but not the WW domain of the putative signal transducing factor, hYAP65. Interestingly, the consensus ligand for the FBP11 WW domain contains residues that are also required for binding by the Abl SH3 domain. These findings support the notion that the FBP11 WW domain and the Abl SH3 domain can compete for the same proline-rich ligands and suggest that at least two subclasses of WW domains exist, namely those that bind a PPLP motif, and those that bind a PPXY motif.     

Structural insights into a StART-like domain in Lam4 and its interaction with sterol ligands

Sterols are essential components of cellular membranes and shape their biophysical properties. The recently discovered family of Lipid transfer proteins Anchored at Membrane contact sites (LAMs) has been suggested to carry out intracellular sterol traffic using StART-like domains. Here, we studied the second StART-like domain of Lam4p from S. cerevisiae by NMR. We show that NMR data are consistent with the StART-like domain structure, and that several functionally important regions within the domain exhibit significant conformational dynamics. NMR titration experiments confirm sterol binding to the canonical sterol-binding site and suggest a role of membrane interactions on the thermodynamics and kinetics of sterol binding.     

Glucocorticoid ligands specify different interactions with NF-kappaB by allosteric effects on the glucocorticoid receptor DNA binding domain

Glucocorticoids inhibit inflammation by acting through the glucocorticoid receptor (GR) and powerfully repressing NF-kappaB function. Ligand binding to the C-terminal of GR promotes the nuclear translocation of the receptor and binding to NF-kappaB through the GR DNA binding domain. We sought how ligand recognition influences the interaction between NF-kappaB and GR. Both dexamethasone (agonist) and RU486 (antagonist) promote efficient nuclear translocation, and we show occupancy of the same intranuclear compartment as NF-kappaB with both ligands. However, unlike dexamethasone, RU486 had negligible activity to inhibit NF-kappaB transactivation. This failure may stem from altered co-factor recruitment or altered interaction with NF-kappaB. Using both glutathione S-transferase pull-down and bioluminescence resonance energy transfer approaches, we identified a major glucocorticoid ligand effect on interaction between the GR and the p65 component of NF-kappaB, with RU486 inhibiting recruitment compared with dexamethasone. Using the bioluminescence resonance energy transfer assay, we found that RU486 efficiently recruited NCoR to the GR, unlike dexamethasone, which recruited SRC1. Therefore, RU486 promotes differential protein recruitment to both the C-terminal and DNA binding domain of the receptor. Importantly, using chromatin immunoprecipitation, we show that impaired interaction between GR and p65 with RU486 leads to reduced recruitment of the GR to the NF-kappaB-responsive region of the interleukin-8 promoter, again in contrast to dexamethasone that significantly increased GR binding. We demonstrate that ligand-induced conformation of the GR C-terminal has profound effects on the functional surface generated by the DNA binding domain of the GR. This has implications for understanding ligand-dependent interdomain communication.     

Geometry of interaction of the histidine ring with other planar and basic residues

Among the aromatic residues in protein structures, histidine (His) is unique, as it can exist in the neutral or positively charged form at the physiological pH. As such, it can interact with other aromatic residues as well as form hydrogen bonds with polar and charged (both negative and positive) residues. We have analyzed the geometry of interaction of His residues with nine other planar side chains containing aromatic (residues Phe, Tyr, Trp, and His), carboxylate (Asp and Glu), carboxamide (Asn and Gln) and guanidinium (Arg) groups in 432 polypeptide chains. With the exception of the aspartic (Asp) and glutamic (Glu) acid side-chains, all other residues prefer to interact in a face-to-face or offset-face-stacked orientation with the His ring. Such a geometry is different from the edge-to-face relative orientation normally associated with the aromatic-aromatic interaction. His-His pair prefers to interact in a face-to-face orientation; however, when both the residues bind the same metal ion, the interplanar angle is close to 90 degrees. The occurrence of different interactions (including the nonconventional N-H...pi and C-H...pi hydrogen bonds) have been correlated with the relative orientations between the interacting residues. Several structural motifs, mostly involved in binding metal ions, have been identified by considering the cases where His residues are in contact with four other planar moieties. About 10% of His residues used here are also found in sequence patterns in PROSITE database. There are examples of the amino end of the Lys side chain interacting with His residues in such a way that it is located on an arc around a ring nitrogen atom.     

Doing (F/L)PPPPs: EVH1 domains and their proline-rich partners in cell polarity and migration.

Actin filament assembly is a tightly regulated process that functions in many aspects of cell physiology. Members of the Ena/VASP (Drosophila Enabled/vasodilator-stimulated phosphoprotein) family are key players in regulating actin filament assembly, in many cases through their association with binding partners that display a particular proline-rich motif, FPPPP. Ena/VASP proteins interact with these partners via the highly conserved Ena/VASP homology 1 (EVH1) domain. The diverse array of binding partners for EVH1 domains, including cytoskeletal proteins such as zyxin, transmembrane guidance receptors such as Roundabout, and the T-cell signaling protein Fyb/SLAP, shows that these interactions are likely to be important in a number of cellular processes that require regulated actin filament assembly.     

The phot LOV2 domain and its interaction with LOV1

Phot proteins are homologs of the blue-light receptor phototropin. We report a comparative study of the photocycles of the isolated, light-sensitive domains LOV1 and LOV2 from Chlamydomonas reinhardtii phot protein, as well as the construct LOV1/2 containing both domains. Transient absorption measurements revealed a short lifetime of the LOV2-wt triplet state (500 ns), but a long lifetime (287 micros) of the triplet in the mutant LOV2-C250S, in which the reactive cysteine is replaced by serine. For LOV1, in comparison, corresponding numbers of 800 ns and 4 micros for the two conformers in LOV1-wt, and 27 micros for LOV1-C57S have been reported. The triplet decay kinetics in the mixed domains LOV1/2-wt, LOV1/2-C57S, and LOV1/2-C250S can be analyzed as the superposition of the behavior of the corresponding single domains. The situation is different for the slow, thermal reaction of the photoadduct back to the dark form. Whereas the individual domains LOV1 and LOV2 show two decay components, the double domains LOV1/2-C57S and LOV1/2-C250S both show only a single component. The interaction of the two domains does therefore not manifest itself during the lifetime of the triplet states, but changes the decay behavior of the adduct states.     

NHERF1/EBP50 head-to-tail intramolecular interaction masks association with PDZ domain ligands

Loss of cell polarity is one of the initial alterations in the development of human epithelial cancers. Na(+)/H(+) exchanger regulatory factor (NHERF) homologous adaptors 1 and 2 are membrane-associated proteins composed of two amino (N)-terminal PDZ domains and an ezrin-radixin-moesin (ERM)-binding (EB) carboxyl (C)-terminal region. We describe here an intramolecular conformation of NHERF1/EBP50 (ERM-binding phosphoprotein 50) in which the C-terminal EB region binds to the PDZ2 domain. This novel head-to-tail conformation masked the interaction of both PDZ domains with PDZ domain-specific ligands, such as PTEN and beta-catenin. An EB region composite structure comprising an alpha-helix ending in a PDZ-binding motif imparted opposite effects to NHERF1 associations, mediating binding to ERM proteins and inhibiting binding of PDZ domain ligands. The PDZ domain inhibition was released by prior association of ezrin with the EB region, a condition that occurs in vivo and likely disrupts NHERF1 head-to-tail interaction. In contrast, NHERF2 did not present a regulatory mechanism for protein complex formation. Functionally, NHERF1 is required to organize complexes at the apical membranes of polarized epithelial cells. The regulation of NHERF1 interactions at the apical membrane thus appears to be a dynamic process that is important for maintaining epithelial-tissue integrity.