RICH-1 has a BIN/Amphiphysin/Rvsp domain responsible for binding to membrane lipids and tubulation of liposomes

RhoGAP interacting with CIP4 homologs-1 (RICH-1) was previously found in a yeast two-hybrid screen for proteins interacting with the SH3 domain of the Cdc42-interacting protein 4 (CIP4). RICH-1 was shown to be a RhoGAP for Cdc42 and Rac. In this study, we show that the BIN/Amphiphysin/Rvsp (BAR) domain in RICH-1 confers binding to membrane lipids, and has the potential to deform spherical liposomes into tubes. In accordance with previous findings for the BAR domains in endophilin and amphiphysin, RICH-1-induced tubes appeared striated. We propose that these striated structures are formed by oligomerization of RICH-1 through a putative coiled-coil region within the BAR domain. In support of this notion, we show that RICH-1 forms oligomers in the presence of the chemical cross-linker BS3. These results point to an involvement of RICH-1 in membrane deformation events.     

An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex

The MBD2-NuRD (Nucleosome Remodeling and Deacetylase) complex is an epigenetic reader of DNA methylation that regulates genes involved in normal development and neoplastic diseases. To delineate the architecture and functional interactions of the MBD2-NuRD complex, we previously solved the structures of MBD2 bound to methylated DNA and a coiled-coil interaction between MBD2 and p66α that recruits the CHD4 nucleosome remodeling protein to the complex. The work presented here identifies novel structural and functional features of a previously uncharacterized domain of MBD2 (MBD2_IDR). Biophysical analyses show that the MBD2_IDR is an intrinsically disordered region (IDR). However, despite this inherent disorder, MBD2_IDR increases the overall binding affinity of MBD2 for methylated DNA. MBD2_IDR also recruits the histone deacetylase core components (RbAp48, HDAC2 and MTA2) of NuRD through a critical contact region requiring two contiguous amino acid residues, Arg²⁸⁶ and Leu²⁸⁷. Mutating these residues abrogates interaction of MBD2 with the histone deacetylase core and impairs the ability of MBD2 to repress the methylated tumor suppressor gene PRSS8 in MDA-MB-435 breast cancer cells. These findings expand our knowledge of the multi-dimensional interactions of the MBD2-NuRD complex that govern its function.     

A coiled-coil domain of melanophilin is essential for Myosin Va recruitment and melanosome transport in melanocytes

Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F-binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.     

Structure and function of PICK1

PICK1 is a peripheral membrane protein conserved from Caenorhabditis elegans to the human. It is expressed in many tissues with high levels in brain and testis. Inside cells, PICK1 is localized at the perinuclear region as well as specialized structures such as synapses of neurons. PICK1 contains a PDZ domain and a BAR domain. The PDZ domain of PICK1 binds to a large number of membrane proteins, especially proteins with C-terminal type II PDZ-binding motifs. The BAR domain of PICK1 binds to lipid molecules, mainly phosphoinositides. While the PDZ domain and the linker region of PICK1 enhance BAR domain's lipid binding, the C-terminal region of PICK1 inhibits its lipid binding. PICK1 regulates the subcellular localization and surface expression of its PDZ-binding partners. Lipid binding of PICK1's BAR domain is important for this regulation. With its PDZ domain interacting with membrane proteins and its BAR domain binding to lipids, the unique structure of PICK1 enables it to couple membrane proteins to protein-trafficking machinery.     

A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor

BACKGROUND: Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. RESULTS: ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. CONCLUSIONS: The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.     

The coiled-coil domain is required for HS1 to bind to F-actin and activate Arp2/3 complex

HS1 (hematopoietic lineage cell-specific protein 1), a substrate of protein tyrosine kinases in lymphocytes, binds to F-actin, and promotes Arp2/3 complex-mediated actin polymerization. However, the mechanism for the interaction between HS1 and F-actin has not yet been fully characterized. HS1 contains 3.5 tandem repeats, a coiled-coil region, and an SH3 domain at the C terminus. Unlike cortactin, which is closely related to HS1 and requires absolutely the repeat domain for F-actin binding, an HS1 mutant with deletion of the repeat domain maintains a significant F-actin binding activity. On the other hand, deletion of the coiled-coil region abolished the ability of HS1 to bind to actin filaments and to activate the Arp2/3 complex for actin nucleation and actin branching. Furthermore, a peptide containing the coiled-coil sequence only was sufficient for F-actin binding. Within cells overexpressing green fluorescent protein-tagged HS1 proteins, wild type HS1 co-localizes with cortical F-actin at the cell leading edge, whereas mutants with deletion of either the coiled-coil region or the repeat domain diffuse in the cytoplasm. Immunoprecipitation analysis reveals that the coiled-coil deletion mutant binds poorly to F-actin, whereas the mutant without the repeat domain fails to bind to both Arp2/3 complex and F-actin. These data suggest that the HS1 coiled-coil region acts synergistically with the repeat domain in the modulation of the Arp2/3 complex-mediated actin polymerization.     

Unique Features of the Anti-parallel,Heterodimeric Coiled-coil Interaction between Methyl-cytosine Binding Domain 2 (MBD2) Homologues and GATA Zinc Finger Domain Containing 2A (GATAD2A/p66α)

The methyl-cytosine binding domain 2 (MBD2)-nucleosome remodeling and deacetylase (NuRD) complex recognizes methylated DNA and silences expression of associated genes through histone deacetylase and nucleosome remodeling functions. Our previous structural work demonstrated that a coiled-coil interaction between MBD2 and GATA zinc finger domain containing 2A (GATAD2A/p66α) proteins recruits the chromodomain helicase DNA-binding protein (CHD4/Mi2β) to the NuRD complex and is necessary for MBD2-mediated DNA methylation-dependent gene silencing in vivo (Gnanapragasam, M. N., Scarsdale, J. N., Amaya, M. L., Webb, H. D., Desai, M. A., Walavalkar, N. M., Wang, S. Z., Zu Zhu, S., Ginder, G. D., and Williams, D. C., Jr. (2011) p66α-MBD2 coiled-coil interaction and recruitment of Mi-2 are critical for globin gene silencing by the MBD2-NuRD complex. Proc. Natl. Acad. Sci. U.S.A. 108, 7487–7492). The p66α-MBD2 interaction differs from most coiled-coils studied to date by forming an anti-parallel heterodimeric complex between two peptides that are largely monomeric in isolation. To further characterize unique features of this complex that drive heterodimeric specificity and high affinity binding, we carried out biophysical analyses of MBD2 and the related homologues MBD3, MBD3-like protein 1 (MBD3L1), and MBD3-like protein 2 (MBD3L2) as well as specific mutations that modify charge-charge interactions and helical propensity of the coiled-coil domains. Analytical ultracentrifugation analyses show that the individual peptides remain monomeric in isolation even at 300 μm in concentration for MBD2. Circular dichroism analyses demonstrate a direct correlation between helical content of the coiled-coil domains in isolation and binding affinity for p66α. Furthermore, complementary electrostatic surface potentials and inherent helical content of each peptide are necessary to maintain high-affinity association. These factors lead to a binding affinity hierarchy of p66α for the different MBD2 homologues (MBD2 ≈ MBD3 > MBD3L1 ≈ MBD3L2) and suggest a hierarchical regulatory model in tissue and life cycle stage-specific silencing by NuRD complexes.     

An atypical BAR domain protein in autophagy

The sorting nexin Atg20 interacts with the selective macroautophagy/autophagy scaffolding protein Atg11, suggesting an important role for Atg20 in the initiation of selective autophagy. To explore this possibility, we recently investigated the structure and function of Atg20 using a variety of biophysical and yeast genetic approaches. Our data demonstrate that the BAR domain of Atg20 interacts with Snx4/Atg24 to form an asymmetric heterodimeric BAR domain complex. Atg20 also contains a long intrinsically disordered N terminus that facilitates binding to Atg11 and a large 89-amino acid insertion in its BAR domain, which we have termed the BAR-GAP. This BAR-GAP region is a unique feature of Atg20 and has not been observed in other BAR domains. Furthermore, the BAR-GAP of Atg20 contains an amphipathic helix which is required for membrane binding, tubulation and autophagy. Our findings demonstrate the important role of this novel region in autophagy.     

NMR studies of the C-terminus of alpha4 reveal possible mechanism of its interaction with MID1 and protein phosphatase 2A

Alpha4 is a regulatory subunit of the protein phosphatase family of enzymes and plays an essential role in regulating the catalytic subunit of PP2A (PP2Ac) within the rapamycin-sensitive signaling pathway. Alpha4 also interacts with MID1, a microtubule-associated ubiquitin E3 ligase that appears to regulate the function of PP2A. The C-terminal region of alpha4 plays a key role in the binding interaction of PP2Ac and MID1. Here we report on the solution structure of a 45-amino acid region derived from the C-terminus of alpha4 (alpha45) that binds tightly to MID1. In aqueous solution, alpha45 has properties of an intrinsically unstructured peptide although chemical shift index and dihedral angle estimation based on chemical shifts of backbone atoms indicate the presence of a transient α-helix. Alpha45 adopts a helix-turn-helix HEAT-like structure in 1% SDS micelles, which may mimic a negatively charged surface for which alpha45 could bind. Alpha45 binds tightly to the Bbox1 domain of MID1 in aqueous solution and adopts a structure consistent with the helix-turn-helix structure observed in 1% SDS. The structure of alpha45 reveals two distinct surfaces, one that can interact with a negatively charged surface, which is present on PP2A, and one that interacts with the Bbox1 domain of MID1.     

cDNA cloning, expression, and characterization of methyl-CpG-binding domain type 2/3 proteins from starfish and sea urchin

Two kinds of cDNAs that are highly homologous to mammalian MBD2 and MBD3 cDNAs were cloned from ovary of the starfish Asterina pectinifera. They are splicing variants and designated sMBD2/3a and sMBD2/3b cDNAs. sMBD2/3a cDNA spans 1378 bp and consists of a 48-bp upstream untranslated region, a 807-bp open reading frame encoding sMBD2/3a, and a 523-bp downstream untranslated region. sMBD2/3a and sMBD2/3b cDNAs encode proteins with predicted molecular weights of 30,724 and 29,635 consisting of 268 and 260 amino acid residues, respectively. The deduced amino acid sequences of these two are identical from residues 1 to 255, but different from residues 256 to the C-terminal ends. sMBD2/3a is expressed in all the tissues of starfish, whereas sMBD2/3b is highly expressed in ovary and oocytes, slightly in testis, but not in somatic cells. As suggested from the whole-genome sequence of Strongylocentrotus purpuratus, a sea urchin MBD2/3 cDNA was cloned from eggs of Hemicentrotus pulcherrimus and designated suMBD2/3 cDNA. It encodes a protein with predicted molecular weight of 30,778 consisting of 274 amino acid residues. All the three echinodermal MBD2/3 proteins consist of a methy-CpG-binding domain (MBD) and a coiled-coil domain, and only sMBD2/3a contains a glutamate-rich C-terminal region, a key mark in vertebrate MBD3. The three MBD2/3 proteins expressed in Escherichia coli and purified to homogeneity were capable to bind specifically to methylated DNA. It was shown that sMBD2/3a exists as dimer or in the monomer-dimer equilibrium, whereas sMBD2/3b and suMBD2/3 exist as monomer and dimer, respectively.     

Spatial Regulation of Cdc42 During Cytokinesis

Cdc42 GTPase plays a critical role in the establishment of cell polarity in most eukaryotic organisms. Cdc42 active state, as that of other GTPases, depends on the bound nucleotide. The protein with GTP is active, and only in this state can it interact with different target effector proteins. The spatio-temporal control of Cdc42 activity is therefore necessary to generate growth polarity. In fission yeast cells, Cdc42 mainly localizes to the division area, and also to the growing tips and to some internal membranes. While the role of Cdc42 in apical growth is well defined, no role has been described for Cdc42 in the process of cell division. Fission yeast Cdc42 activity is regulated by two specific guanidine nucleotide exchange factors (GEFs), Scd1, and Gef1. We discuss here how Hob3, a BAR domain containing protein similar to human BIN3 and S. cerevisiae Rsv161, may be required to recruit Cdc42 to the cell division site as well as for the activation of this GTPase mediated by Gef1. We also discuss the possible role of Cdc42 in the contraction of the actomyosin ring necessary for cytokinesis.     

Mutations in BIN1 Associated with Centronuclear Myopathy Disrupt Membrane Remodeling by Affecting Protein Density and Oligomerization

The regulation of membrane shapes is central to many cellular phenomena. Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are key players for membrane remodeling during endocytosis, cell migration, and endosomal sorting. BIN1, which contains an N-BAR domain, is assumed to be essential for biogenesis of plasma membrane invaginations (T-tubules) in muscle tissues. Three mutations, K35N, D151N and R154Q, have been discovered so far in the BAR domain of BIN1 in patients with centronuclear myopathy (CNM), where impaired organization of T-tubules has been reported. However, molecular mechanisms behind this malfunction have remained elusive. None of the BIN1 disease mutants displayed a significantly compromised curvature sensing ability. However, two mutants showed impaired membrane tubulation both in vivo and in vitro, and displayed characteristically different behaviors. R154Q generated smaller membrane curvature compared to WT N-BAR. Quantification of protein density on membranes revealed a lower membrane-bound density for R154Q compared to WT and the other mutants, which appeared to be the primary reason for the observation of impaired deformation capacity. The D151N mutant was unable to tubulate liposomes under certain experimental conditions. At medium protein concentrations we found ‘budding’ structures on liposomes that we hypothesized to be intermediates during the tubulation process except for the D151N mutant. Chemical crosslinking assays suggested that the D151N mutation impaired protein oligomerization upon membrane binding. Although we found an insignificant difference between WT and K35N N-BAR in in vitro assays, depolymerizing actin in live cells allowed tubulation of plasma membranes through the K35N mutant. Our results provide insights into the membrane-involved pathophysiological mechanisms leading to human disease.     

Domain and functional analysis of a novel breast tumor suppressor protein, SCUBE2

Signal peptide CUB (complement proteins C1r/C1s, Uegf, and Bmp 1)-EGF domain-containing protein 2 (SCUBE2) is a secreted, membrane-associated multidomain protein composed of five recognizable motifs: an NH(2)-terminal signal peptide sequence, nine copies of epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeats, and one CUB domain at the COOH terminus. Our previous clinical study showed that SCUBE2 may act as a novel breast tumor suppressor gene and serve as a useful prognostic marker. However, the specific domain responsible for its tumor suppressor activity and the precise mechanisms of its anti-tumor effect remain unknown. Using a combination of biochemical, molecular, and cell biology techniques, we further dissected the molecular functions and signal pathways mediated by the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain of SCUBE2. Independent overexpression of the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain resulted in suppression of MCF-7 breast cancer cell proliferation and reduced MCF-7 xenograft tumor growth in nude mice. Molecular and biochemical analyses revealed that the COOH-terminal CUB domain could directly bind to and antagonize bone morphogenetic protein activity in an autocrine manner, whereas the NH(2)-terminal EGF-like repeats could mediate cell-cell homophilic adhesions in a calcium-dependent fashion, interact with E-cadherin (a master tumor suppressor), and decrease the β-catenin signaling pathway. Together, our data demonstrate that SCUBE2 has growth inhibitory effects through a coordinated regulation of two distinct mechanisms: antagonizing bone morphogenetic protein and suppressing the β-catenin pathway in breast cancer cells.     

Role of MED1 (MBD4) Gene in DNA repair and human cancer

The human protein MED1, also known as MBD4, was isolated in a yeast two-hybrid screening as an interactor of the mismatch repair protein MLH1. MED1 contains an N-terminal 5-methylcytosine binding domain (MBD), which allows binding to methylated DNA, and a C-terminal catalytic domain with homology to bacterial DNA damage-specific glycosylases/lyases. This suggests that DNA methylation may play a role in human DNA repair. MED1 acts as a mismatch-specific DNA N-glycosylase active on thymine, uracil, 5-fluorouracil and, weakly, 3,N(4)-ethenocytosine paired with guanine. The glycosylase activity of MED1 prefers substrates in which the G:T mismatch is present in the context of methylated or unmethylated CpG sites. Since G:T mismatches can originate via spontaneous deamination of 5-methylcytosine to thymine, MED1 appears to act as a caretaker of genomic fidelity at CpG sites. Mutagenesis caused by these deamination events is a frequent mechanism of genetic instability in cancer; thus, based on the biochemical activity of its gene product, MED1 is a candidate tumor suppressor gene. Indeed, frameshift mutations of the MED1 gene have been reported in human colorectal, gastric, endometrial, and pancreatic cancer. In the future, efforts should be directed toward investigations of the functional role of the MED1 gene in the pathogenesis, prevention, and treatment of human cancer.