Alexei Yu. Denisov

Two WXXF-based motifs in NECAPs define the specificity of accessory protein binding to AP-1 and AP-2

The adaptor proteins AP‐2 and AP‐1/GGAs are essential components of clathrin coats at the plasma membrane and trans‐Golgi network, respectively. The adaptors recruit accessory proteins to clathrin‐coated pits, which is dependent on the adaptor ear domains engaging short peptide motifs in the accessory proteins. Here, we perform an extensive mutational analysis of a novel WXXF‐based motif that functions to mediate the binding of an array of accessory proteins to the α‐adaptin ear domain of AP‐2. Using nuclear magnetic resonance and mutational studies, we identified WXXF‐based motifs as major ligands for a site on the α‐ear previously shown to bind the DPW‐bearing proteins epsin 1/2. We also defined the determinants that allow for specific binding of the α‐ear motif to AP‐2 as compared to those that allow a highly related WXXF‐based motif to bind to the ear domains of AP‐1/GGAs. Intriguingly, placement of acidic residues around the WXXF cores is critical for binding specificity. These studies provide a structural basis for the specific recruitment of accessory proteins to appropriate sites of clathrin‐coated vesicle formation.

Connecdenn, A Novel DENN Domain-Containing Protein of Neuronal Clathrin-Coated Vesicles Functioning in Synaptic Vesicle Endocytosis

Clathrin-coated vesicles (CCVs) are responsible for the endocytosis of multiple cargo, including synaptic vesicle membranes. We now describe a new CCV protein, termed connecdenn, that contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a poorly characterized protein module found in multiple proteins of unrelated function and a C-terminal peptide motif domain harboring three distinct motifs for binding the α-ear of the clathrin adaptor protein 2 (AP-2). Connecdenn coimmunoprecipitates and partially colocalizes with AP-2, and nuclear magnetic resonance and peptide competition studies reveal that all three α-ear-binding motifs contribute to AP-2 interactions. In addition, connecdenn contains multiple Src homology 3 (SH3) domain-binding motifs and coimmunoprecipitates with the synaptic SH3 domain proteins intersectin and endophilin A1. Interestingly, connecdenn is enriched on neuronal CCVs and is present in the presynaptic compartment of neurons. Moreover, connecdenn has a uniquely stable association with CCV membranes because it resists extraction with Tris and high-salt buffers, unlike most other CCV proteins, but it is not detected on purified synaptic vesicles. Together, these observations suggest that connecdenn functions on the endocytic limb of the synaptic vesicle cycle. Accordingly, disruption of connecdenn interactions with its binding partners through overexpression of the C-terminal peptide motif domain or knock down of connecdenn through lentiviral delivery of small hairpin RNA both lead to defects in synaptic vesicle endocytosis in cultured hippocampal neurons. Thus, we identified connecdenn as a component of the endocytic machinery functioning in synaptic vesicle endocytosis, providing the first evidence of a role for a DENN domain-containing protein in endocytosis.

The NECAP PHear domain increases clathrin accessory protein binding potential

AP‐2 is a key regulator of the endocytic protein machinery driving clathrin‐coated vesicle (CCV) formation. One critical function, mediated primarily by the AP‐2 α‐ear, is the recruitment of accessory proteins. NECAPs are α‐ear‐binding proteins that enrich on CCVs. Here, we have solved the structure of the conserved N‐terminal region of NECAP 1, revealing a unique module in the pleckstrin homology (PH) domain superfamily, which we named the PHear domain. The PHear domain binds accessory proteins bearing FxDxF motifs, which were previously thought to bind exclusively to the AP‐2 α‐ear. Structural analysis of the PHear domain reveals the molecular surface for FxDxF motif binding, which was confirmed by site‐directed mutagenesis. The reciprocal analysis of the FxDxF motif in amphiphysin I identified distinct binding requirements for binding to the α‐ear and PHear domain. We show that NECAP knockdown compromises transferrin uptake and establish a functional role for NECAPs in clathrin‐mediated endocytosis. Our data uncover a striking convergence of two evolutionarily and structurally distinct modules to recognize a common peptide motif and promote efficient endocytosis.

Characterization of a γ‐adaptin ear‐binding motif in enthoprotin

Enthoprotin, a newly identified component of clathrin‐coated vesicles, interacts with the trans‐Golgi network (TGN) clathrin adapters AP‐1 and GGA2. Here we perform a multi‐faceted analysis of the site in enthoprotin that is responsible for the binding to the γ‐adaptin ear (γ‐ear) domain of AP‐1. Alanine scan mutagenesis and nuclear magnetic resonance (NMR) studies reveal the full extent of the site as well as critical residues for this interaction. NMR studies of the γ‐ear in complex with a synthetic peptide from enthoprotin provide structural details of the binding site for TGN accessory proteins within the γ‐ear.

Recoupling of residual dipolar couplings in single-domain polymer-stabilized liquid crystals undergoing magic-angle spinning.

Measurement of dipolar couplings, chemical shift anisotropies, and quadrupole couplings in oriented media such as liquid crystals are of great importance for extraction of structural parameters in biological macromolecules. Here, we introduce a new technique, SAD-REDOR, that consists of recoupling heteronuclear dipolar couplings in molecules dissolved in a single-domain liquid crystal or other oriented medium through the combined use of magic-angle spinning and rotor-synchronized radiofrequency pulses. This application of the REDOR pulse sequence to oriented media offers several advantages such as selectivity over the type of coupling recovered and tunable scaling of the interaction. The effectiveness of the technique is demonstrated both theoretically and experimentally, using the recently developed polyacrylamide-stabilized Pf1 phage medium and 15N-labeled benzamide as the aligned molecule.

Structure of 2',5'-linked tetraribonucleotide loops: a novel RNA motif.

Abstract We report on the three dimensional structure of an RNA hairpin containing a 2′,5′-linked tetraribonucleotide loop, namely, 5′-rGGAC(UUCG)GUCC-3′ (where UUCG = U2′p5′U2′p5′C2′p5′G2′p5′). We show that the 2′,5′-linked RNA loop adopts a conformation that is quite different from that previously observed for the native 3′,5′-linked RNA loop. The 2′,5′- RNA loop is stabilized by (a) U:G wobble base pairing, with both bases in the anti conformation, (b) extensive base stacking, and (c) sugar–base contacts, all of which contribute to the extra stability of this hairpin structure.