Phosphatidylinositol-4,5-bisphosphate is required for endocytic coated vesicle formation

Receptor-mediated endocytosis via clathrin-coated vesicles has been extensively studied and, while many of the protein players have been identified, much remains unknown about the regulation of coat assembly and the mechanisms that drive vesicle formation [1]. Some components of the endocytic machinery interact with inositol polyphosphates and inositol lipids in vitro, implying a role for phosphatidylinositols in vivo [2] [3]. Specifically, the adaptor protein complex AP2 binds phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), PtdIns(3)P, PtdIns(3,4,5)P3 and inositol phosphates. Phosphatidylinositol binding regulates AP2 self-assembly and the interactions of AP2 complexes with clathrin and with peptides containing endocytic motifs [4] [5]. The GTPase dynamin contains a pleckstrin homology (PH) domain that binds PtdIns(4,5)P2 and PtdIns(3,4,5)P3 to regulate GTPase activity in vitro [6] [7]. However, no direct evidence for the involvement of phosphatidylinositols in clathrin-mediated endocytosis exists to date. Using well-characterized PH domains as high affinity and high specificity probes in combination with a perforated cell assay that reconstitutes coated vesicle formation, we provide the first direct evidence that PtdIns(4,5)P2 is required for both early and late events in endocytic coated vesicle formation.     

Canine parvovirus capsid structure, analyzed at 2.9 A resolution

The DNA-containing capsid of canine parvovirus (CPV) is analyzed following atomic refinement at 2.9 A resolution. The capsid contains 60 copies of the capsid protein related by icosahedral symmetry. The atomic model has been extended from the first residue (Gly37) of the unrefined 3.25 A structure towards the N terminus. The electron density shows that approximately 87% of the capsid proteins have N termini on the inside of the capsid, but for approximately 13%, the polypeptide starts on the outside and runs through one of the pores surrounding each 5-fold axis, explaining apparently conflicting antigenic data. Analysis of potential hydrogen bonds reveals approximately 50% more secondary structure than previously apparent. Most of the additional secondary structure are in the 71 and 221 residue-long loop insertions between beta-strands E and F and G and H, forming subunit-bridging sheets that likely add specificity to assembly interactions. Structural analysis of the extensive subunit interactions around the 3-fold axes shows that assembly is a multistep process with loops intertwining following initial contact. Estimated free energies of association suggest that the formation of 3 and 5-fold contacts likely takes precedence over 2-fold interactions. Energies for initial association into trimers or pentamers would be similar, but the intertwining of loops about the 3-fold axis adds an additional large activation barrier to dissociation. Analysis of the surfaces of the assembled capsid shows a surprising lack of basic amino acids that might have been expected to interact with the negatively charged phosphoribose backbone of the DNA. Instead, uncharged polar and van der Waal's interactions predominate in the packaging of single-stranded DNA into the capsid.     

Probing the structure of complex macromolecular interactions by homolog specificity scanning: the P1 and P7 plasmid partition systems

The P1 plasmid partition locus, P1 par, actively distributes plasmid copies to Escherichia coli daughter cells. It encodes two DNA sites and two proteins, ParA and ParB. Plasmid P7 uses a similar system, but the key macromolecular interactions are species specific. Homolog specificity scanning (HSS) exploits such specificities to map critical contact points between component macromolecules. The ParA protein contacts the par operon operator for operon autoregulation, and the ParB contacts the parS partition site during partition. Here, we refine the mapping of these contacts and extend the use of HSS to map protein-protein contacts. We found that ParB participates in autoregulation at the operator site by making a specific contact with ParA. Similarly, ParA acts in partition by making a specific contact with ParB bound at parS. Both these interactions involve contacts between a C-terminal region of ParA and the extreme N-terminus of ParB. As a single type of ParA-ParB complex appears to be involved in recognizing both DNA sites, the operator and the parS sites may both be occupied by a single protein complex during partition. The general HSS strategy may aid in solving the three-dimensional structures of large complexes of macromolecules.     

配合物NH4[Ln（H2O）9][（VⅣO）2（μ2-O）（nta）2]（Ln=Sm,Y）的合成及钐配合物的晶体结构

在水溶液中合成了两个钒-稀土配合物NH4[Ln(H2O)9][(VIVO)2(μ2-O)(nta)2](Ln=Sm,Y),其中钒-钐配合物为单晶体,钒-钇配合物为多晶态粉末。单晶X-射线衍射分析表明,钒-钐配合物属单斜晶系,C2/c空间群,晶胞参数a=2.336(4),b=1.0907(9),c=1.0823(10)nm,β=93.21(2)°,V=2.753(6)nm3,Z=4。配合物包含了一个二核的钒阴离子[(VIVO)2(μ2-O)(nta)2]4-(H3nta=氨三乙酸)和一个九配位的钐阳离子[Sm(H2O)9]3+,两者通过氢键连接成三维超分子结构。     

Molecular characterization of GCP170, a 170-kDa protein associated with the cytoplasmic face of the Golgi membrane

We have isolated a cDNA clone encoding a protein (designated GCP170) of 1530 amino acid residues with a calculated molecular mass of 170 kDa that is localized to the Golgi complex. Hydropathy analysis shows that GCP170 contains no NH2-terminal signal sequence nor a hydrophobic domain sufficient for participating in membrane localization. It is also predicted that GCP170 has characteristic secondary structures including an extremely long alpha-helical domain that likely forms a coiled-coil between non-coil domains at the NH2 and COOH termini, suggesting that the protein is organized as a globular head, a stalk, and a tail. Immunocytochemical observations revealed that GCP170 was localized to the Golgi complex and the cytoplasm, consistent with biochemical data indicating that the protein exits as a membrane-associated form and a soluble form. GCP170 was dissociated from the Golgi membrane in response to brefeldin A as rapidly as a coat protein complex of non-clathrin-coated vesicles (beta-COP, a subunit of coatomer), but did not co-localize with beta-COP on the Golgi membrane when examined by immunoelectron microscopy. The protein was detected as phosphorylated and unphosphorylated forms, of which the unphosphorylated form was more tightly associated with the Golgi membrane. When cells were extracted with 1% Triton X-100 under microtubule-stabilizing conditions, GCP170 remained in the cells in association with the Golgi complex. These results indicate that GCP170 is a peripheral membrane protein with a long coiled-coil domain that may be involved in the structural organization or stabilization of the Golgi complex.     

Inhibition of the interaction between tyrosine-based motifs and the medium chain subunit of the AP-2 adaptor complex by specific tyrphostins

Several intracellular membrane trafficking events are mediated by tyrosine-containing motifs found within the cytosolic domains of certain integral membrane proteins. Many of these tyrosine motifs conform to the consensus YXXPhi (where Phi represents a bulky hydrophobic residue). This YXXPhi motif has been shown to interact with the medium chain subunits of adaptor complexes that generally link relevant integral membrane protein cytosolic domains to the clathrin coat involved in vesicle formation. The motif YXXPhi is also very similar to motifs that are targets for phosphorylation by tyrosine kinases. Tyrosine kinase inhibitors known as tyrphostins are structural analogues of tyrosine, and so it is possible that tyrphostins could also inhibit interactions between medium chains and YXXPhi motifs. TGN38 is a type I integral membrane protein containing a tyrosine motif, YQRL, within the cytosolic domain. We have previously shown that this motif interacts directly with the medium chain subunit of the plasma membrane localized AP-2 adaptor complex (mu2). We have investigated a range of tyrphostins and demonstrated a specific inhibition of the interaction between mu2 and the TGN38 cytosolic domain by tyrphostin A23 through in vitro analysis and the yeast two-hybrid system. These data raise the exciting possibility that different membrane traffic events could be inhibited by specific tyrphostins.     

Three-dimensional structure of an evolutionarily conserved N-terminal domain of syntaxin 1A

Syntaxin 1A plays a central role in neurotransmitter release through multiple protein-protein interactions. We have used NMR spectroscopy to identify an autonomously folded N-terminal domain in syntaxin 1A and to elucidate its three-dimensional structure. This 120-residue N-terminal domain is conserved in plasma membrane syntaxins but not in other syntaxins, indicating a specific role in exocytosis. The domain contains three long alpha helices that form an up-and-down bundle with a left-handed twist. A striking residue conservation is observed throughout a long groove that is likely to provide a specific surface for protein-protein interactions. A highly acidic region binds to the C2A domain of synaptotagmin I in a Ca2+-dependent interaction that may serve as an electrostatic switch in neurotransmitter release.     

The structure of the membrane protein squalene-hopene cyclase at 2.0 A resolution

Squalene cyclases catalyze a cationic cyclization cascade, which is homologous to a key step in cholesterol biosynthesis. The structure of the enzyme from Alicyclobacillus acidocaldarius has been determined in a new crystal form at 2.0 A resolution (1 A=0.1 nm) and refined to an R-factor of 15.3 % (Rfree=18.7 %). The structure indicates how the initial protonation and the final deprotonation of squalene occur and how the transient carbocations are stabilized. The pathways of the flexible educt squalene from the membrane interior to the active center cavity and of the rigid fused-ring product hopene in the reverse direction are discussed. The enzyme contains eight so-called QW-sequence repeats that fortify the alpha/alpha-barrels by an intricate interaction network. They are unique to the known triterpene cyclases and are presumed to shield these enzymes against the released enthalpy of the highly exergonic catalyzed reaction. The enzyme is a monotopic membrane protein, the membrane-binding interactions of which are described and compared with those of two prostaglandin-H2 synthase isoenzymes, the only other structurally characterized proteins of this type. In the crystals the membrane-binding regions face each other, suggesting a micelle-type detergent structure between them.     

ADP-ribosylation factor and phosphatidic acid levels in Golgi membranes during budding of coatomer-coated vesicles

The finding that ADP-ribosylation factor (ARF) can activate phospholipase D has led to debate as to whether ARF recruits coat proteins through direct binding or indirectly by catalytically increasing phosphatidic acid production. Here we test critical aspects of these hypotheses. We find that Golgi membrane phosphatidic acid levels do not rise-in fact they decline-during cell-free budding reactions. We confirm that the level of membrane-bound ARF can be substantially reduced without compromising coat assembly [Ktistakis, N. T., Brown, H. A., Waters, M. G., Sternweis, P. C. & Roth, M. G. (1996) J. Cell Biol. 134, 295-306], but find that under all conditions, ARF is present on the Golgi membrane in molar excess over bound coatomer. These results do not support the possibility that the activation of coat assembly by ARF is purely catalytic, and they are consistent with ARF forming direct interactions with coatomer. We suggest that ARF, like many other G proteins, is a multifunctional protein with roles in trafficking and phospholipid signaling.     

Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments

The repressor protein of bacteriophage P22 binds to DNA as a homodimer. This dimerization is absolutely required for DNA binding. Dimerization is mediated by interactions between amino acids in the carboxyl (C)-terminal domain. We have constructed a plasmid, p22CT-1, which directs the overproduction of just the C-terminal domain of the P22 repressor (P22CT-1). Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex. Cross-linking experiments show that P22CT-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT-1 heterodimers. We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repressors and have created chimeric proteins to help identify amino acid regions required for dimerization specificity. Our results indicate that the dimerization specificity region of these proteins is concentrated in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors. We also show that the set of amino acids that forms the cooperativity interface of the P22 repressor may be distinct from those that form its dimer interface. Furthermore, cooperativity studies of the wild-type and chimeric proteins suggest that the location of cooperativity interface in the 434 repressor may also be distinct from that of its dimerization interface. Interestingly, changes in the dimer interface decreases the ability of the 434 repressor to discriminate between its wild-type binding sites, O(R)1, O(R)2, and O(R)3. Since 434 repressor discrimination between these sites depends in large part on the ability of this protein to recognize sequence-specific differences in DNA structure and flexibility, this result indicates that the C-terminal domain is intimately involved in the recognition of sequence-dependent differences in DNA structure and flexibility.     

Mechanisms of virus assembly probed by Raman spectroscopy: the icosahedral bacteriophage P22

A microdialysis flow cell has been developed for time-resolved Raman spectroscopy of biological macromolecules and their assemblies. The flow cell permits collection of Raman spectra concurrent with the efflux of small solute molecules into a solution of macromolecules and facilitates real-time spectroscopic detection of structural transitions induced by the effluent. Additionally, the flow cell is well suited to the investigation of hydrogen-isotope exchange phenomena that can be exploited as dynamic probes of viral protein folding and solvent accessibility along the assembly pathway. Here, we describe the application of the Raman dynamic probe to the maturation of the icosahedral capsid of bacteriophage P22, a double-stranded DNA virus. The P22 virion is constructed from a capsid precursor (procapsid) consisting of 420 coat subunits (gp5) in an outer shell and a few hundred scaffolding subunits (gp8) within. Capsid maturation involves expulsion of scaffolding subunits coupled with shell expansion at the time of DNA packaging. Raman static and dynamic probes reveal that the scaffolding subunit is highly alpha-helical and highly thermolabile, and lacks a typical hydrophobic core. When bound within the procapsid, the alpha-helical fold of gp8 is thermostabilized; however, this stabilization confers no apparent protection against peptide NH-->ND exchange. A molten globule model is proposed for the native scaffolding subunit that functions in procapsid assembly. Accompanying capsid expansion, a small conformational change (alpha-helix-->beta-strand) is also observed in the coat subunit. Domain movement mediated by hinge bending is proposed as the mechanism of capsid expansion. On the basis of these results, a molecular model is proposed for assembly of the P22 procapsid.     

Reciprocal effect of Waardenburg syndrome mutations on DNA binding by the Pax-3 paired domain and homeodomain

The Pax-3 protein contains two DNA-binding domains, a paired domain and a homeodomain. Mutations in Pax-3 cause Waardenburg syndrome (WS) in humans and the mouse Splotch (Sp) phenotype. In the Sp-delayed mouse, a mutation in the Pax-3 paired domain (G9R) abrogates the DNA-binding activity of both the paired domain and the homeodomain, suggesting that they may functionally interact. To investigate this possibility further, we have analyzed the DNA-binding properties of additional point mutants in the Pax-3 paired domain and homeodomain that occur in WS patients (F12L, N14H, G15S, P17L, R23L, G48A, S51F and G66D in the paired domain, V47F and R53G in the homeodomain), the Pax-1 un mutation (G15A) and a substitution associated with Peters' anomaly in the PAX-6 gene (R23G). Within the paired domain, seven of 10 mutations were found to abrogate DNA-binding by the paired domain. Remarkably, these seven mutations also affected DNA binding by the homeodomain, causing either a complete loss (P17L and G66D), a reduction (R23G, R23L, G15S and G15A) or an increase in DNA-binding activity (N14H). In addition, the effect of paired domain mutations occurred at the level of monomer formation by the homeodomain, while the dimerization potential of this domain seemed unaffected in mutants where it could be analyzed. Furthermore, while both homeodomain mutations were found to abolish DNA binding by this domain, the R53G mutation also abrogated DNA binding by the paired domain. The important observation that independent mutations in either domain can affect DNA binding by the other in the intact Pax-3 protein strongly suggests that the two domains are not functionally independent but bind DNA through cooperative interactions. Modeling the deleterlous mutations on the three-dimensional structure of the paired domain of Drosophila Prd shows that these mutations cluster at the DNA interface, thus suggesting that a series of DNA contacts are essential for DNA binding by both the paired domain and the homeodomain of Pax-3.     

Mutations affecting lysine-35 of gpNu1, the small subunit of bacteriophage lambda terminase, alter the strength and specificity of holoterminase interactions with DNA

The small subunit of lambda terminase, gpNu1, contains a low-affinity ATPase activity that is stimulated by nonspecific dsDNA. The location of the gpNu1 ATPase center is suggested by a sequence match between gpNu1 (29-VLRGGGKG-36) and the phosphate-binding loop, or P-loop (GXXXXGKT/S), of known ATPase. The proposed P-loop of gpNu1 is just downstream of a putative helix-turn-helix DNA-binding motif, located between residues 5 and 24. Published work has shown that changing lysine-35 of the proposed P-loop of gpNu1 alters the response of the ATPase activity to DNA, as follows. The changes gpNu1 k35A and gpNu1 K35D increase the level of DNA required for maximal stimulation of the gpNu1 ATPase by factors of 2- and 10-fold, respectively. The maximally stimulated ATPase activities of the mutant enzymes are indistinguishable from that of the wild-type enzyme. In the present work, the effects of changing lysine-35 on the cos-cleavage and DNA-packaging activities of terminase were examined. In vitro, the gpNu1 K35A enzyme cleaved cos as efficiently as the wild-type enzyme, but required a 2-fold increased level of substrate DNA for saturation, suggesting a slight reduction in DNA affinity. In a crude DNA-packaging system using cleaved lambda DNA as substrate, the gpNu1 K35A enzyme had a 10-fold defect. In vivo, lambda Nu1 K35A showed a 2-fold reduction in cos cleavage, but no packaged DNA was detected. The primary defect of the gpNu1 K35A enzyme was concluded to be in a post-cos-cleavage step of DNA packaging. In in vitro cos-cleavage experiments, the gpNu1 K35D enzyme had a 10-fold increased requirement for saturation by substrate DNA. Furthermore, the cos-cleavage activity of gpNu1 K35D enzyme was strongly inhibited by the presence of nonspecific DNA, indicating that the gpNu1 K35D enzyme is unable to discriminate effectively between cos and nonspecific DNA. No cos cleavage was observed in vivo for lambda Nu1 K35D, a result consistent with the discrimination defect found in vitro for the gpNu1 K35D enzyme. In a crude packaging system the gpNu1 K35D enzyme had a 200-fold defect; in a purified packaging system, the gpNu1 K35D enzyme was found to be unable to discriminate between lambda DNA and nonspecific phage T7 DNA, a result indicating that the gpNu1 K35D enzyme is also defective in discriminating between lambda DNA and nonspecific DNA during DNA packaging.     

Boundary of pRNA functional domains and minimum pRNA sequence requirement for specific connector binding and DNA packaging of phage phi29

Bacteriophage phi29 utilizes a viral-encoded 120-base RNA (pRNA) to accomplish dsDNA packaging into a preformed procapsid. Six pRNAs bind to the procapsid and work sequentially. The pRNA contains two functional domains, one for binding to the DNA translocating connector, and the other for interacting with another component of the DNA packaging machinery during DNA translocation. By UV crosslinking, the pRNA was found to bind to the connector specifically and not to the capsid or scaffolding proteins. When purified connectors were incubated with pRNA, rosette-like connector oligomers were observed. These oligomers were found to contain pRNA. A series of deletion mutants of the pRNA were constructed and their ability to perform various tasks involved in phi29 assembly were assayed. The minimum sizes of the pRNA needed for the following activities have been determined: (1) specific binding to procapsid or to connectors; (2) connector or procapsid binding with full efficiency compared with wild-type pRNA; and (3) genomic DNA packaging. In summary, bases 37-91 (55 nt) comprised the minimum sequence required for specific connector binding, although with lower efficiency; bases 6-113 (105 nt with the additional deletion of two nonessential bases, C109 and A106) comprised the minimum sequence required for full connector binding activity; and bases 1-117 comprised the minimum sequence needed for full DNA packaging activity. These data indicate clearly that the helical region composed of bases 1-6 and 113-117 plays a crucial role in DNA translocation, but is dispensable for connector binding. A model for the role of the pRNA in DNA packaging was also presented.