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Most of the non-lysosomal proteolysis that occurs in eukaryotic cells is performed by a nonspecific and abundant barrel-shaped complex called the 20S proteasome. Substrates access the active sites, which are sequestered in an internal chamber, by traversing a narrow opening (alpha-annulus) that is blocked in the unliganded 20S proteasome by amino-terminal sequences of alpha-subunits. Peptide products probably exit the 20S proteasome through the same opening. 11S regulators (also called PA26 (ref. 4), PA28 (ref. 5) and REG) are heptamers that stimulate 20S proteasome peptidase activity in vitro and may facilitate product release in vivo. Here we report the co-crystal structure of yeast 20S proteasome with the 11S regulator from Trypanosoma brucei (PA26). PA26 carboxy-terminal tails provide binding affinity by inserting into pockets on the 20S proteasome, and PA26 activation loops induce conformational changes in alpha-subunits that open the gate separating the proteasome interior from the intracellular environment. The reduction in processivity expected for an open conformation of the exit gate may explain the role of 11S regulators in the production of ligands for major histocompatibility complex class I molecules.

Proteasomes can be markedly activated by associating with 19S regulatory complexes to form the 26S protease or by binding 11S protein complexes known as REG or PA28. Three REG subunits, alpha, beta, and gamma, have been expressed in Escherichia coli, and each recombinant protein can activate human proteasomes. Combining PCR mutagenesis with an in vitro activity assay, we have isolated and characterized 36 inactive, single-site mutants of recombinant REGalpha. Most are monomers that produce functional proteasome activators when mixed with REGbeta subunits. Five REGalpha mutants that remain inactive in the mixing assay contain amino acid substitutions clustered between Arg-141 and Gly-149. The crystal structure of the REGalpha heptamer shows that this region forms a loop at the base of each REGalpha subunit. One mutation in this loop (N146Y) yields a REGalpha heptamer that binds the proteasome as tightly as wild-type REGalpha but does not activate peptide hydrolysis. Corresponding amino acid substitutions in REGbeta (N135Y) and REGgamma (N151Y) produce inactive proteins that also bind the proteasome and inhibit proteasome activation by their normal counterparts. Our studies clearly demonstrate that REG binding to the proteasome can be separated from activation of the enzyme. Moreover, the dominant negative REGs identified here should prove valuable for elucidating the role(s) of these proteins in antigen presentation.

The specificity of the 20S proteasome, which degrades many intracellular proteins, is regulated by protein complexes that bind to one or both ends of the cylindrical proteasome structure. One of these regulatory complexes, the 11S regulator (known as REG or PA28), stimulates proteasome peptidase activity and enhances the production of antigenic peptides for presentation by class I molecules of the major histocompatibility complex (MHC). The three REG subunits that have been identified, REGalpha, REGbeta and REGgamma (also known as the Ki antigen), share extensive sequence similarity, apart from a highly variable internal segment of 17-34 residues which may confer subunit-specific properties. REGalpha and REGbeta preferentially form a heteromeric complex, although purified REGalpha forms a heptamer in solution and has biochemical properties similar to the heteromeric REGalpha/REGbeta complex. We have now determined the crystal structure of human recombinant REGalpha at 2.8 A resolution. The heptameric barrel-shaped assembly contains a central channel that has an opening of 20 A diameter at one end and another of 30 A diameter at the presumed proteasome-binding surface. The binding of REG probably causes conformational changes that open a pore in the proteasome alpha-subunits through which substrates and products can pass.

Full-length cDNAs for three human proteasome activator subunits, called REGalpha, REGbeta, and REGgamma, have been expressed in Escherichia coli, and the purified recombinant proteins have been characterized. Recombinant alpha or gamma subunits form heptameric species; recombinant beta subunits are found largely as monomers or small multimers. Each recombinant REG stimulates cleavage of fluorogenic peptides by human red cell proteasomes. The pattern of activated peptide hydrolysis is virtually identical for REGalpha and REGbeta. These two subunits, alone or in combination, stimulate cleavage after basic, acidic, and most hydrophobic residues in many peptides. Recombinant alpha and beta subunits bind each other with high affinity, and the REGalpha/beta heteromeric complex activates hydrolysis of LLVY-methylcoumaryl-7-amide (LLVY-MCA) and LLE-beta-nitroanilide (LLE-betaNA) more than REGalpha or REGbeta alone. Using filter binding and gel filtration assays, recombinant REGgamma subunits were shown to bind themselves but not alpha or beta subunits. REGgamma differs from REGalpha and REGbeta in that it markedly stimulates hydrolysis of peptides with basic residues in the P1 position but only modestly activates cleavage of LLVY-MCA or LLE-betaNA by the proteasome. REGgamma binds the proteasome with higher affinity than REGalpha or REGbeta yet with lower affinity than complexes containing both REGalpha and REGbeta. In summary, each of the three REG homologs is a proteasome activator with unique biochemical properties.

The crystal structure and site-directed mutagenesis analyses of Aquifex aeolicus transcription factor NusG show domain-swapped dimers and exhibit a 'ball-and-socket' junction, which participates in the intermolecular interactions within the oligomers. A spring-loaded state is proposed for the functional state of NusG.

N-utilization factor G (NusG) frorn Aquifex aeolicus (Aa) was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapor-diffusion technique. The drops consisted of 2.5 µl protein solution (∼30 mg ml[sup -1] in 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 2 mM EDTA and 10 mM DTT) and 2.5 µl reservoir solution (0.085 M Na HEPES pH 7.5, 15% glycerol, 11% 2-propanol and 20% PEG 4000) derived from condition number 41 of the Hampton Cryo Screen. The crystals grew at 291 ± l K and reached dimensions of 0.2 × 0.1 × 0.05 mm in 5-7 d. The crystals, which diffracted to 2.45 Å resolution, belonged to space group C222[sub 1], with unit-cell parameters a = 65.95, b = 124.58, c = 83.60 Å. One AaNusG molecule is present in the asymmetric unit, corresponding to a solvent content of 59.80% (Matthews coefficient = 3.06 ų Da[sup -1]). Crystal structure determination is in progress. [ABSTRACT FROM AUTHOR]

To test whether it is practical to use phage display coupled with proteolysis for protein design, we used this approach to convert a partially unfolded four-helix bundle protein, apocytochrome b562, to a stably folded four-helix bundle protein. Four residues expected to form a hydrophobic core were mutated. One residue was changed to Trp to provide a fluorescence probe for studying the protein''s physical properties and to partially fill the void left by the heme. The other three positions were randomly mutated. In addition, another residue in the region to be redesigned was substituted with Arg to provide a specific cutting site for protease Arg-c. This library of mutants was displayed on the surface of phage and challenged with protease Arg-c to select stably folded proteins. The consensus sequence that emerged from the selection included hydrophobic residues at only one of the three positions and non-hydrophobic residues at the other two. Nevertheless, the selected proteins were thermodynamically very stable. The structure of a selected protein was characterized using multi-dimensional NMR. All four helices were formed in the structure. Further, site-directed mutagenesis was used to change one of the two non-hydrophobic residues to a hydrophobic residue, which increased the stability of the protein, indicating that the selection result was not based solely on the protein''s global stability and that local structural characteristics may also govern the selection. This conclusion is supported by the crystal structure of another mutant that has two hydrophobic residues substituted for the two non-hydrophobic residues. These results suggest that the hydrophobic interactions in the core are not sufficient to dictate the selection and that the location of the cutting site of the protease also influences the selection of structures. [Copyright &y& Elsevier]