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Philip R Evans

Publications and source records attributed to Philip R Evans.

15 recordsLinked to original sources
PubMed2006

An introduction to stereochemical restraints.

At the resolution available from most macromolecular crystals, the X-ray data alone are insufficient to lead to a chemically reasonable structure, so stereochemical restraints are essential. These usually restrain bond lengths, bond angles, planes and chiral volumes. The definition of these restraints and where the values come from are described. A dictionary entry contains information about the atom types, their connectivity and all the appropriate restraints. Torsion angles are not usually restrained, but they do have optimum values. In the special case of flexible five- and six-membered rings, including pentose and hexose sugars, the ring pucker is defined by combinations of torsion angles and the pucker affects the position of substituents.

Carbon↗
PubMed2006

Mechanism of endophilin N-BAR domain-mediated membrane curvature.

Endophilin-A1 is a BAR domain-containing protein enriched at synapses and is implicated in synaptic vesicle endocytosis. It binds to dynamin and synaptojanin via a C-terminal SH3 domain. We examine the mechanism by which the BAR domain and an N-terminal amphipathic helix, which folds upon membrane binding, work as a functional unit (the N-BAR domain) to promote dimerisation and membrane curvature generation. By electron paramagnetic resonance spectroscopy, we show that this amphipathic helix is peripherally bound in the plane of the membrane, with the midpoint of insertion aligned with the phosphate level of headgroups. This places the helix in an optimal position to effect membrane curvature generation. We solved the crystal structure of rat endophilin-A1 BAR domain and examined a distinctive insert protruding from the membrane interaction face. This insert is predicted to form an additional amphipathic helix and is important for curvature generation. Its presence defines an endophilin/nadrin subclass of BAR domains. We propose that N-BAR domains function as low-affinity dimers regulating binding partner recruitment to areas of high membrane curvature.

Acyltransferases↗
PubMed2006

Structural analysis of the interaction between the SNARE Tlg1 and Vps51.

Membrane fusion in cells involves the interaction of SNARE proteins on apposing membranes. Formation of SNARE complexes is preceded by tethering events, and a number of protein complexes that are thought to mediate this have been identified. The VFT or GARP complex is required for endosome-Golgi traffic in yeast. It consists of four subunits, one of which, Vps51, has been shown to bind specifically to the SNARE Tlg1, which participates in the same fusion event. We have determined the structure of the N-terminal domain of Tlg1 bound to a peptide from the N terminus of Vps51. Binding depends mainly on residues 18-30 of Vps51. These form a short helix which lies in a conserved groove in the three-helix bundle formed by Tlg1. Surprisingly, although both Vps51 and Tlg1 are required for transport to the late Golgi from endosomes, removal of the Tlg1-binding sequences from Vps51 does not block such traffic in vivo. Thus, this particular interaction cannot be crucial to the process of vesicle docking or fusion.

Amino Acid Sequence↗
PubMed2005

Non-canonical YXXGPhi endocytic motifs: recognition by AP2 and preferential utilization in P2X4 receptors.

During clathrin-mediated endocytosis, proteins on the cell surface are selected for inclusion in clathrin-coated vesicles by clathrin adaptors, mainly the adaptor complex AP2. The P2X4 subtype of ATP-gated ion channel has in its C-terminus two putative endocytic motifs: a canonical YXXPhi motif and a non-canonical YXXGPhi motif (YEQGL). We demonstrate that endocytosis of P2X4 receptors is mediated preferentially by the YXXGPhi motif because the YXXPhi motif is inaccessible to AP2 owing to the structure of the channel. The crystal structure of a complex between residues 160-435 of the mu2 subunit of AP2 and a P2X4 C-terminal peptide showed that the YEQGL motif binds to mu2 at the same site as YXXPhi motifs. Y and Phi residues are accommodated in the same hydrophobic pockets in mu2 with the extra residue between them being accommodated by changes in the peptide's backbone configuration, when compared to YXXPhi motifs. These data demonstrate that the family of potential tyrosine-based endocytic signals must be expanded to include motifs with an additional glycine at Y+3 (YXXGPhi).

Adaptor Protein Complex 2↗
PubMed2004

Two distinct interaction motifs in amphiphysin bind two independent sites on the clathrin terminal domain beta-propeller.

During the assembly of clathrin-coated vesicles, many peripheral membrane proteins, including the amphiphysins, use LLDLD-type clathrin-box motifs to interact with the N-terminal beta-propeller domain (TD) of clathrin. The 2.3 A-resolution structure of the clathrin TD in complex with a TLPWDLWTT peptide from amphiphysin 1 delineates a second clathrin-binding motif, PWXXW (the W box), that binds at a site on the TD remote from the clathrin box-binding site. The presence of both sequence motifs within the unstructured region of the amphiphysins allows them to bind more tightly to free TDs than do other endocytic proteins that contain only clathrin-box motifs. This property, along with the propensity of the N-terminal BAR domain to bind curved membranes, will preferentially localize amphiphysin and its partner, dynamin, to the periphery of invaginated clathrin lattices.

Amino Acid Motifs↗
PubMed2004

Adaptors for clathrin coats: structure and function.

Clathrin-coated vesicles (CCVs) are responsible for the transport of proteins between various compartments of the secretory and endocytic systems. Clathrin forms a scaffold around these vesicles that is linked to membranes by clathrin adaptors. The adaptors simultaneously bind to clathrin and to transmembrane proteins and/or phospholipids and can also interact with each other and with other components of the CCV formation machinery. The result is a collection of proteins that can make multiple, moderate strength (microM Kd) interactions and thereby establish the dynamic regulatable networks to drive vesicle genesis at the correct time and place in the cell. This review focuses on the structure of clathrin adaptors and how these structures provide functional information on the mechanism of CCV formation.

Adaptor Proteins, Vesicular Transport↗
PubMed2003

BAR domains as sensors of membrane curvature: the amphiphysin BAR structure.

The BAR (Bin/amphiphysin/Rvs) domain is the most conserved feature in amphiphysins from yeast to human and is also found in endophilins and nadrins. We solved the structure of the Drosophila amphiphysin BAR domain. It is a crescent-shaped dimer that binds preferentially to highly curved negatively charged membranes. With its N-terminal amphipathic helix and BAR domain (N-BAR), amphiphysin can drive membrane curvature in vitro and in vivo. The structure is similar to that of arfaptin2, which we find also binds and tubulates membranes. From this, we predict that BAR domains are in many protein families, including sorting nexins, centaurins, and oligophrenins. The universal and minimal BAR domain is a dimerization, membrane-binding, and curvature-sensing module.

ADP-Ribosylation Factors↗
PubMed2003

EpsinR: an AP1/clathrin interacting protein involved in vesicle trafficking.

EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.

Adaptor Proteins, Vesicular Transport↗
PubMed2003

A conformational switch between transcriptional repression and replication initiation in the RepA dimerization domain.

Plasmids are natural vectors for gene transfer. In Gram-negative bacteria, plasmid DNA replication is triggered when monomers of an initiator protein (Rep) bind to direct repeats at the origin sequence. Rep dimers, which are inactive as initiators, bind to an inverse repeat operator, repressing transcription of the rep gene. Rep proteins are composed of N-terminal dimerization and C-terminal DNA-binding domains. Activation of Rep is coupled to dimer dissociation, converting the dimerization domain into a second origin-binding module. Although the structure of the monomeric F plasmid initiator (mRepE) has been determined, the molecular nature of Rep activation remains unknown. Here we report the crystal structure of the dimeric N-terminal domain of the pPS10 plasmid initiator (dRepA). dRepA has a winged-helix fold, as does its homologous domain in mRepE. However, dimerization transforms an interdomain loop and beta-strand (monomeric RepE) into an alpha-helix (dimeric RepA). dRepA resemble the C terminus of eukaryotic and archaeal Cdc6, giving clues to the phylogeny of DNA replication initiators.

Amino Acid Sequence↗
PubMed2002

Curvature of clathrin-coated pits driven by epsin.

Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P(2) in conjunction with clathrin polymerization. We have discovered that formation of an amphipathic alpha-helix in epsin is coupled to PtdIns(4,5)P(2) binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is sufficient to facilitate the formation of clathrin-coated invaginations.

Adaptor Proteins, Vesicular Transport↗
PubMed2002

Influence of cytokine gene polymorphisms on the development of prostate cancer.

Polymorphisms in the promoter regions of cytokine genes may influence prostate cancer (PC) development via regulation of the antitumor immune response and/or pathways of tumor angiogenesis. PC patients (247) and 263 controls were genotyped for interleukin (IL)-1beta-511, IL-8-251, IL-10-1082, tumor necrosis factor-alpha-308, and vascular endothelial growth factor (VEGF)-1154 single nucleotide polymorphisms. Patient control comparisons revealed that IL-8 TT and VEGF AA genotypes were decreased in patients compared with controls [23.9 versus 32.3%; P = 0.04, odds ratio (OR) = 0.66, 95% confidence interval (CI) 0.44-0.99 and 6.3 versus 12.9%; P = 0.01, OR = 0.45, 95% CI 0.24-0.86, respectively], whereas the IL-10 AA genotype was significantly increased in patients compared with controls (31.6 versus 20.6%; P = 0.01, OR = 1.78, 95% CI 1.14-2.77). Stratification according to prognostic indicators showed association between IL-8 genotype and log prostate-specific antigen level (P = 0.05). These results suggest that single nucleotide polymorphisms associated with differential production of IL-8, IL-10, and VEGF are risk factors for PC, possibly acting via their influence on angiogenesis.

Aged↗
PubMed2002

Molecular architecture and functional model of the endocytic AP2 complex.

AP2 is the best-characterized member of the family of heterotetrameric clathrin adaptor complexes that play pivotal roles in many vesicle trafficking pathways within the cell. AP2 functions in clathrin-mediated endocytosis, the process whereby cargo enters the endosomal system from the plasma membrane. We describe the structure of the 200 kDa AP2 "core" (alpha trunk, beta2 trunk, mu2, and sigma2) complexed with the polyphosphatidylinositol headgroup mimic inositolhexakisphosphate at 2.6 A resolution. Two potential polyphosphatidylinositide binding sites are observed, one on alpha and one on mu2. The binding site for Yxxphi endocytic motifs is buried, indicating that a conformational change, probably triggered by phosphorylation in the disordered mu2 linker, is necessary to allow Yxxphi motif binding. A model for AP2 recruitment and activation is proposed.

Adaptor Protein Complex 1↗
PubMed2002

Endocytosis and vesicle trafficking.

Several common themes have emerged from recent structural and functional studies of proteins involved in the formation of coated vesicles. For example, inositol polyphosphate lipid headgroups are bound specifically by a variety of different domains in ways appropriate to domain function. Another theme is the recognition of short sequence motifs in structureless regions of other coat components, allowing dynamic multicomponent networks to be established.

Binding Sites↗
PubMed2002

Gamma-adaptin appendage domain: structure and binding site for Eps15 and gamma-synergin.

The AP1 complex is one of a family of heterotetrameric clathrin-adaptor complexes involved in vesicular trafficking between the Golgi and endosomes. The complex has two large subunits, gamma and beta1, which can be divided into trunk, hinge, and appendage domains. The 1.8 A resolution structure of the gamma appendage is presented. The binding site for the known gamma appendage ligand gamma-synergin is mapped through creation of point mutations designed on the basis of the structure. We also show that Eps15, a protein believed to be involved in vesicle formation at the plasma membrane, is also a ligand of gamma appendage and binds to the same site as gamma-synergin. This observation explains the demonstrated brefeldinA (BFA)-sensitive colocalization of Eps15 and AP1 at the Golgi complex.

Adaptor Protein Complex 1↗