Ubiquitination functions as a sorting signal for lysosomal degradation of cell-surface proteins by facilitating their internalization from the plasma membrane and incorporation into lumenal vesicles of multivesicular bodies (MVBs). Ubiquitin may also mediate sorting of proteins from the trans-Golgi network (TGN) to the endosome, thereby preventing their appearance on the cell surface and hastening their degradation in the lysosome-vacuole. Substantiation of a direct ubiquitin-dependent TGN sorting pathway relies in part on identifying candidate machinery that may function as a ubiquitin-sorting 'receptor'at the TGN. Members of the GGA family of coat proteins localize to the TGN and promote the incorporation of proteins into clathrin-coated vesicles destined for transport to endosomes. We show that the GGA coat proteins bind directly to ubiquitin through their GAT domain and demonstrate that this interaction is required for the ubiquitin-dependent sorting of the Gap1 amino acid transporter from the TGN to endosomes. Thus, GGA proteins fulfill the role of ubiquitin sorting receptors at the TGN.

Ubiquitin (Ub) attachment to cell surface proteins causes their lysosomal degradation by incorporating them into lumenal membranes of multivesicular bodies (MVBs). Two yeast endosomal protein complexes have been proposed as Ub-sorting “receptors,” the Vps27-Hse1 complex and the ESCRT-I complex. We used NMR spectroscopy and mutagenesis studies to map the Ub-binding surface for Vps27 and Vps23. Mutations in Ub that ablate only Vps27 binding or Vps23 binding blocked the ability of Ub to serve as an MVB sorting signal, supporting the idea that both the Vps27-Hse1 and ESCRT-I complexes interact with ubiquitinated cargo. Vps27 also bound Vps23 directly via two PSDP motifs present within the Vps27 COOH terminus. Loss of Vps27-Vps23 association led to less efficient sorting into the endosomal lumen. However, sorting of vacuolar proteases or the overall biogenesis of the MVB were not grossly affected. In contrast, disrupting interaction between Vps27 and Hse1 caused severe defects in carboxy peptidase Y sorting and MVB formation. These results indicate that both Ub-sorting complexes are coupled for efficient recognition of ubiquitinated cargo.

The orientation and dynamics of an 18-residue antimicrobial peptide, ovispirin, has been investigated using solid-state NMR spectroscopy. Ovispirin is a cathelicidin-like model peptide (NH(2)-KNLRRIIRKIIHIIKKYG-COOH) with potent, broad-spectrum bactericidal activity. (15)N NMR spectra of oriented ovispirin reconstituted into synthetic phospholipids show that the helical peptide is predominantly oriented in the plane of the lipid bilayer, except for a small portion of the helix, possibly at the C-terminus, which deviates from the surface orientation. This suggests differential insertion of the peptide backbone into the lipid bilayer. (15)N spectra of both oriented and unoriented peptides show a reduced (15)N chemical shift anisotropy at room temperature compared with that of rigid proteins, indicating that the peptide undergoes uniaxial rotational diffusion around the bilayer normal with correlation times shorter than 10(-4) s. This motion is frozen below the gel-to-liquid crystalline transition temperature of the lipids. Ovispirin interacts strongly with the lipid bilayer, as manifested by the significantly reduced (2)H quadrupolar splittings of perdeuterated palmitoyloleoylphosphatidylcholine acyl chains upon peptide binding. Therefore, ovispirin is a curved helix residing in the membrane-water interface that executes rapid uniaxial rotation. These structural and dynamic features are important for understanding the antimicrobial function of this peptide.

The CD spectra of SMAP-29, an antimicrobial peptide from sheep, showed disordered structure in aqueous buffers, and significant helicity in membrane-like environments, including SDS micelles, lipopolysaccharide (LPS) dispersions, and trifluoroethanol buffer systems. A structure determined by NMR in 40% perdeuterated trifluoroethanol indicated that residues 8-17 were helical, residues 18-19 formed a hinge, and residues 20-28 formed an ordered, hydrophobic segment. SMAP-29 was flexible in 40% trifluoroethanol, forming two sets of conformers that differed in the relative orientation of the N-terminal domain. We used a chromogenic Limulus assay to determine the EC 50 of the peptide (the concentration that bound 50% of the added LPS). Studies with full-length and truncated SMAP-29 molecules revealed that each end of the holopeptide contained an LPS-binding domain. The higher affinity LPS-binding domain was situated in the flexible N-terminal portion. LPS binding to full-length SMAP-29 showed positive cooperativity, so the EC 50 of the peptide (2.6 lM) was considerably lower than that of the individual LPS-binding domains. LPS-binding studies with a mixture of truncated peptides revealed that this cooperativity was primarily intramolecular (i.e. involving the N-and C-terminal LPS-binding sites of the same peptide molecule). , an antimicrobial cathelicidin peptide of rabbits, resembled SMAP-29 in that it contained N-and C-terminal LPS-binding domains, had an EC 50 of 2.5 lM, and bound LPS with positive cooperativity. We conclude that the presence of multiple binding sites that function cooperatively allow peptides such as SMAP-29 and CAP-18 to bind LPS with high affinity.

Human beta-defensin-2 (HBD-2) is a member of the defensin family of antimicrobial peptides. HBD-2 was first isolated from inflamed skin where it is posited to participate in the killing of invasive bacteria and in the recruitment of cells of the adaptive immune response. Static light scattering and two-dimensional proton nuclear magnetic resonance spectroscopy have been used to assess the physical state and structure of HBD-2 in solution. At concentrations of < or = 2.4 mM, HBD-2 is monomeric. The structure is amphiphilic with a nonuniform surface distribution of positive charge and contains several key structural elements, including a triple-stranded, antiparallel beta-sheet with strands 2 and 3 in a beta-hairpin conformation. A beta-bulge in the second strand occurs at Gly28, a position conserved in the entire defensin family. In solution, HBD-2 exhibits an alpha-helical segment near the N-terminus that has not been previously ascribed to solution structures of alpha-defensins or to the beta-defensin BNBD-12. This novel structural element may be a factor contributing to the specific microbicidal or chemokine-like properties of HBD-2.