Identification of domain required for catalytic activity of auxilin in supporting clathrin uncoating by Hsc70

During clathrin-mediated endocytosis Hsc70, supported by the J-domain protein auxilin, uncoats clathrin-coated vesicles. Auxilin contains both a clathrin-binding domain and a J-domain that binds Hsc70, and it has been suggested that these two domains are both necessary and sufficient for auxilin activity. To test this hypothesis, we created a chimeric protein consisting of the J-domain of auxilin linked to the clathrin-binding domain of the assembly protein AP180. This chimera supported uncoating, but unlike auxilin it acted stoichiometrically rather than catalytically because, like Hsc70, it remained associated with the uncoated clathrin. This observation supports our proposal that Hsc70 chaperones uncoated clathrin by inducing formation of a stable Hsc70-clathrin-AP complex. It also shows that Hsc70 acts by dissociating individual clathrin triskelions rather than cooperatively destabilizing clathrin-coated vesicles. Because the chimera lacks the C-terminal subdomain of the auxilin clathrin-binding domain, it seemed possible that this subdomain is required for auxilin to act catalytically, and indeed its deletion caused auxilin to act stoichiometrically. In contrast, deletion of the N-terminal subdomain weakened auxilin-clathrin binding and prevented auxilin from polymerizing clathrin. Therefore the C-terminal subdomain of the clathrin-binding domain of auxilin is required for auxilin to act catalytically, whereas the N-terminal subdomain strengthens auxilin-clathrin binding.     

Crystal structure of a major secreted protein of Mycobacterium tuberculosis-MPT63 at 1.5-A resolution

MPT63 is a small, major secreted protein of unknown function from Mycobacterium tuberculosis that has been shown to have immunogenic properties and has been implicated in virulence. A BLAST search identified that MPT63 has homologs only in other mycobacteria, and is therefore mycobacteria specific. As MPT63 is a secreted protein, mycobacteria specific, and implicated in virulence, MPT63 is an attractive drug target against the deadliest infectious disease, tuberculosis (TB). As part of the TB Structural Genomics Consortium, the X-ray crystal structure of MPT63 was determined to 1.5-Angstrom resolution with the hope of yielding functional information about MPT63. The structure of MPT63 is an antiparallel beta-sandwich immunoglobulin-like fold, with the unusual feature of the first beta-strand of the protein forming a parallel addition to the small antiparallel beta-sheet. MPT63 has weak structural similarity to many proteins with immunoglobulin folds, in particular, Homo sapiens beta2-adaptin, bovine arrestin, and Yersinia pseudotuberculosis invasin. Although the structure of MPT63 gives no conclusive evidence to its function, structural similarity suggests that MPT63 could be involved in cell-host interactions to facilitate endocytosis/phagocytosis.     

Physical descriptions of experimental selectivity measurements in ion channels

Three experiments that quantify the amount of selectivity exhibited by a biological ion channel are examined with Poisson-Nernst-Planck (PNP) theory. Conductance ratios and the conductance mole fraction experiments are examined by considering a simple model ion channel for which an approximate solution to the PNP equations with Donnan boundary conditions is derived. A more general result is derived for the Goldman-Hodgkin-Katz permeability ratio. The results show that (1) the conductance ratio measures the ratio of the diffusion coefficients of the ions inside the channel, (2) the mole fraction experiment measures the difference of the excess chemical potentials of the ions inside the channel, and (3) the permeability ratio measures both diffusion coefficients and excess chemical potentials. The results are used to divide selectivity into two components: partitioning, an equilibrium measure of how well the ions enter the channel, and diffusion, a nonequilibrium measure of how well the ions move through the channel.     

GXXXG and AXXXA: common alpha-helical interaction motifs in proteins, particularly in extremophiles

The GXXXG motif is a frequently occurring sequence of residues that is known to favor helix-helix interactions in membrane proteins. Here we show that the GXXXG motif is also prevalent in soluble proteins whose structures have been determined. Some 152 proteins from a non-redundant PDB set contain at least one alpha-helix with the GXXXG motif, 41 +/- 9% more than expected if glycine residues were uniformly distributed in those alpha-helices. More than 50% of the GXXXG-containing alpha-helices participate in helix-helix interactions. In fact, 26 of those helix-helix interactions are structurally similar to the helix-helix interaction of the glycophorin A dimer, where two transmembrane helices associate to form a dimer stabilized by the GXXXG motif. As for the glycophorin A structure, we find backbone-to-backbone atomic contacts of the C alpha-H...O type in each of these 26 helix-helix interactions that display the stereochemical hallmarks of hydrogen bond formation. These glycophorin A-like helix-helix interactions are enriched in the general set of helix-helix interactions containing the GXXXG motif, suggesting that the inferred C alpha-H...O hydrogen bonds stabilize the helix-helix interactions. In addition to the GXXXG motif, some 808 proteins from the non-redundant PDB set contain at least one alpha-helix with the AXXXA motif (30 +/- 3% greater than expected). Both the GXXXG and AXXXA motifs occur frequently in predicted alpha-helices from 24 fully sequenced genomes. Occurrence of the AXXXA motif is enhanced to a greater extent in thermophiles than in mesophiles, suggesting that helical interaction based on the AXXXA motif may be a common mechanism of thermostability in protein structures. We conclude that the GXXXG sequence motif stabilizes helix-helix interactions in proteins, and that the AXXXA sequence motif also stabilizes the folded state of proteins.     

Multicopy crystallographic refinement of a relaxed glutamine synthetase from Mycobacterium tuberculosis highlights flexible loops in the enzymatic mechanism and its regulation

The crystal structure of glutamine synthetase (GS) from Mycobacterium tuberculosis determined at 2.4 A resolution reveals citrate and AMP bound in the active site. The structure was refined with strict 24-fold noncrystallographic symmetry (NCS) constraints and has an R-factor of 22.7% and an R-free of 25.5%. Multicopy refinement using 10 atomic models and strict 24-fold NCS constraints further reduced the R-factor to 20.4% and the R-free to 23.2%. The multicopy model demonstrates the range of atomic displacements of catalytic and regulatory loops in glutamine synthesis, simulating loop motions. A comparison with loop positions in substrate complexes of GS from Salmonella typhimurium shows that the Asp50 and Glu327 loops close over the active site during catalysis. These loop closures are preceded by a conformational change of the Glu209 beta-strand upon metal ion or ATP binding that converts the enzyme from a relaxed to a taut state. We propose a model of the GS regulatory mechanism based on the loop motions in which adenylylation of the Tyr397 loop reverses the effect of metal ion binding, and regulates intermediate formation by preventing closure of the Glu327 loop.     

Computational methods of analysis of protein-protein interactions

Computational methods play an important role at all stages of the process of determining protein-protein interactions. They are used to predict potential interactions, to validate the results of high-throughput interaction screens and to analyze the protein networks inferred from interaction databases.     

MHC class II-bound self peptides from autoimmune MRL/lpr mice reveal potential T cell epitopes for autoantibody production in murine systemic lupus erythematosus

The systemic lupus erythematosus-like syndrome in MRL/lpr mice involves high-titered IgG autoantibodies, particularly antinuclear Abs that target histones, DNA, and RNA particles. Although T cell help is required for the generation of antinuclear Abs, the epitopes recognized by such helper T cells are unknown. To address this question, we isolated and sequenced self peptides bound by MHC class II molecules from MRL/lpr mice. We identified a number of peptides that are not seen in similar preparations from nonautoimmune C3H animals. The "abnormal" peptide donors include histone, a protein component of a small nuclear ribonucleoprotein, ribosomal proteins, and RNA processing enzymes. We postulate that the peptides from these donors are T cell epitopes required for the generation of the most frequent antinuclear Abs specificities seen in MRL/lpr mice.     

DIP: the database of interacting proteins

The Database of Interacting Proteins (DIP; http://dip.doe-mbi.ucla.edu) is a database that documents experimentally determined protein-protein interactions. This database is intended to provide the scientific community with a comprehensive and integrated tool for browsing and efficiently extracting information about protein interactions and interaction networks in biological processes. Beyond cataloging details of protein-protein interactions, the DIP is useful for understanding protein function and protein-protein relationships, studying the properties of networks of interacting proteins, benchmarking predictions of protein-protein interactions, and studying the evolution of protein-protein interactions.