David Owen

The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase Gcn5p

The bromodomain is an ∼110 amino acid module found in histone acetyltransferases and the ATPase component of certain nucleosome remodelling complexes. We report the crystal structure at 1.9 Å resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15–29 of histone H4 acetylated at the ζ‐N of lysine 16. We show that this bromodomain preferentially binds to peptides containing an N‐acetyl lysine residue. Only residues 16–19 of the acetylated peptide interact with the bromodomain. The primary interaction is the N‐acetyl lysine binding in a cleft with the specificity provided by the interaction of the amide nitrogen of a conserved asparagine with the oxygen of the acetyl carbonyl group. A network of water‐mediated H‐bonds with protein main chain carbonyl groups at the base of the cleft contributes to the binding. Additional side chain binding occurs on a shallow depression that is hydrophobic at one end and can accommodate charge interactions at the other. These findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed.

A Structural Explanation for the Binding of Multiple Ligands by the α-Adaptin Appendage Domain

Abstract The α subunit of the endocytotic AP2 adaptor complex contains a 30 kDa appendage domain, which is joined to the rest of the protein via a flexible linker. The 1.9 A resolution crystal structure of this domain reveals a single binding site for its ligands, which include amphiphysin, Eps15, and epsin. This domain when overexpressed in COS7 fibroblasts is shown to inhibit transferrin uptake, whereas mutants in which interactions with its binding partners are abolished do not. DPF/W motifs present in appendage domain–binding partners are shown to play a crucial role in their interactions with the domain. A single site for binding multiple ligands would allow for temporal and spatial regulation in the recruitment of components of the endocytic machinery.

The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition.

The structure of a truncated form of the γ‐subunit of phosphorylase kinase (PHKγt) has been solved in a ternary complex with a non‐hydrolysable ATP analogue (adenylyl imidodiphosphate, AMPPNP) and a heptapeptide substrate related in sequence to both the natural substrate and to the optimal peptide substrate. Kinetic characterization of the phosphotransfer reaction confirms the peptide to be a good substrate, and the structure allows identification of key features responsible for its high affinity. Unexpectedly, the substrate peptide forms a short anti‐parallel β‐sheet with the kinase activation segment, the region which in other kinases plays an important role in regulation of enzyme activity. This anchoring of the main chain of the substrate peptide at a fixed distance from the γ‐phosphate of ATP explains the selectivity of PHK for serine/threonine over tyrosine as a substrate. The catalytic core of PHK exists as a dimer in crystals of the ternary complex, and the relevance of this phenomenon to its in vivo recognition of dimeric glycogen phosphorylase b is considered.

Crystal structure of the amphiphysin-2 SH3 domain and its role in the prevention of dynamin ring formation

The amphiphysins are brain‐enriched proteins, implicated in clathrin‐mediated endocytosis, that interact with dynamin through their SH3 domains. To elucidate the nature of this interaction, we have solved the crystal structure of the amphiphysin‐2 (Amph2) SH3 domain to 2.2 Å. The structure possesses several notable features, including an extensive patch of negative electrostatic potential covering a large portion of its dynamin binding site. This patch accounts for the specific requirement of amphiphysin for two arginines in the proline‐rich binding motif to which it binds on dynamin. We demonstrate that the interaction of dynamin with amphiphysin SH3 domains, unlike that with SH3 domains of Grb2 or spectrin, prevents dynamin self‐assembly into rings. Deletion of a unique insert in the n‐Src loop of Amph2 SH3, a loop adjacent to the dynamin binding site, significantly reduces this effect. Conversely, replacing the n‐Src loop of the N‐terminal SH3 domain of Grb2 with that of Amph2 causes it to favour dynamin ring disassembly. Transferrin uptake assays show that shortening the n‐Src loop of Amph2 SH3 reduces the ability of this domain to inhibit endocytosis in vivo. Our data suggest that amphiphysin SH3 domains are important regulators of the multimerization cycle of dynamin in endocytosis.

The structure and function of the β2-adaptin appendage domain

The heterotetrameric AP2 adaptor (α, β2, μ2 and σ2 subunits) plays a central role in clathrin‐mediated endocytosis. We present the protein recruitment function and 1.7 Å resolution structure of its β2‐appendage domain to complement those previously determined for the μ2 subunit and α appendage. Using structure‐directed mutagenesis, we demonstrate the ability of the β2 appendage alone to bind directly to clathrin and the accessory proteins AP180, epsin and eps15 at the same site. Clathrin polymerization is promoted by binding of clathrin simultaneously to the β2‐appendage site and to a second site on the adjacent β2 hinge. This results in the displacement of the other ligands from the β2 appendage. Thus clathrin binding to an AP2–accessory protein complex would cause the controlled release of accessory proteins at sites of vesicle formation.

Clathrin coat construction in endocytosis

Electron cryomicroscopy of the clathrin coat and X-ray crystallography of parts of the clathrin heavy chain combine to give a detailed picture of the clathrin molecule, assembled as a cage. Recently determined domain structures of other components of the endocytic machinery, particularly the mu2 subunit and the alpha-appendage domain of the AP2 adaptor complex, provide important information on the sequence of recognition events involved in the dynamic process of clathrin coat assembly.

ARP, a peptide derived from the stress-associated acetylcholinesterase variant has hematopoietic growth promoting activities

BackgroundPsychological stress induces rapid and long-lasting changes in blood cell composition, implying the existence of stress-induced factors that modulate hematopoiesis. Here we report the involvement of the stress-associated “readthrough” acetylcholinesterase (AChE-R) variant, and its 26 amino acid C-terminal domain (ARP) in hematopoietic stress responses.Materials and MethodsWe studied the effects of stress, cortisol, antisense oligonucleotides to AChE, and synthetic ARP on peripheral blood cell composition and clonogenic progenitor status in mice under normal and stress conditions, and on purified CD341 cells of human origin. We employed in situ hybridization and immunocytochemical staining to monitor gene expression, and 5-bromo-2-deoxyuridine (BrdU), primary liquid cultures, and clonogenic progenitor assays to correlate AChE-R and ARP with proliferation and differentiation of hematopoietic progenitors.ResultsWe identified two putative glucocorticoid response elements in the human ACHE gene encoding AChE. In human CD341 hematopoietic progenitor cells, cortisol elevated AChE-R mRNA levels and promoted hematopoietic expansion. In mice, a small peptide crossreacting with anti-ARP antiserum appeared in serum following forced swim stress. Ex vivo, ARP was more effective than cortisol and equally as effective as stem cell factor in promoting expansion and differentiation of early hematopoietic progenitor cells into myeloid and megakaryocyte lineages.ConclusionsOur findings attribute a role to AChE-R and ARP in hematopoietic homeostasis following stress, and suggest the use of ARP in clinical settings where ex vivo expansion of progenitor cells is required.

γ-Adaptin Appendage Domain: Structure and Binding Site for Eps15 and γ-Synergin

Abstract 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, γ and β1, which can be divided into trunk, hinge, and appendage domains. The 1.8 A resolution structure of the γ appendage is presented. The binding site for the known γ appendage ligand γ-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 γ appendage and binds to the same site as γ-synergin. This observation explains the demonstrated brefeldinA (BFA)-sensitive colocalization of Eps15 and AP1 at the Golgi complex.

Multiple Roles for Cyclin G-Associated Kinase in Clathrin-Mediated Sorting Events

Cyclin G‐associated kinase (GAK), also known as auxilin 2, is a potential regulator of clathrin‐mediated membrane trafficking. It possesses a kinase domain at its N‐terminus that can phosphorylate the clathrin adaptors AP‐1 and AP‐2 in vitro. The GAK C‐terminus can act as a cochaperaone in vitro for Hsc70, a heat‐shock protein required for clathrin uncoating. Here we show that the specificity of GAK is very similar to that of adaptor‐associated kinase 1, another mammalian adaptor kinase. We used siRNA to investigate GAKs in vivo function. We discovered that early stages of clathrin‐mediated endocytosis (CME) were partially inhibited when GAK expression was knocked down. This defect was specifically caused by GAK knockdown because it could be rescued by expressing a rat GAK gene that could not be silenced by one of the siRNAs. To identify the GAK activity required during CME, we mutated the kinase domain and the J domain of the rat gene. Only GAK with a functional J domain could rescue the defect, suggesting that GAK is important for clathrin uncoating. Furthermore, we demonstrated that GAK plays a role in the clathrin‐dependent trafficking from the trans Golgi network.

Structural insights into clathrin-mediated endocytosis.

The process of clathrin-mediated endocytosis from the plasma membrane has been the subject of many biological and biochemical investigations. Recent atomic resolution structures determined by X-ray crystallography now enable the molecular basis for the interactions of some components of the endocytic machinery to be understood in detail.