Hsp90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes. In this study, we identify a function for the chaperone in RNA processing and maintenance. This functionality of Hsp90 involves two recently identified interactors of the chaperone: Tah1 and Pih1/Nop17. Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein. Tah1 and Pih1 bind to the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which we demonstrate is required for the correct accumulation of box C/D small nucleolar ribonucleoproteins. Together with the Tah1 cofactor, Hsp90 functions to stabilize Pih1. As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions. Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs.

The highly conserved, 300-kDa cylindrical protease ClpP is an important component of the cellular protein quality machinery. It consists of 14 subunits arranged into two heptameric rings that enclose a large chamber containing the protease active sites. ClpP associates with ClpX and ClpA ATPases that unfold and translocate substrates into the protease catalytic chamber through axial pores located at both ends of the ClpP cylinder. Although the pathway of substrate delivery is well established, the pathway of product release is unknown. Here, we use recently developed transverse relaxation optimized spectroscopy (TROSY) of methyl groups to show that the interface between the heptameric rings exchanges between two structurally distinct conformations. The conformational exchange process has been quantified by magnetization exchange and methyl TROSY relaxation dispersion experiments recorded between 0.5°C and 40°C, so that the thermodynamic properties for the transition could be obtained. Restriction of the observed motional freedom in ClpP through the introduction of a cysteine linkage results in a protease where substrate release becomes significantly slowed relative to the rate observed in the reduced enzyme, suggesting that the observed motions lead to the formation of transient side pores that may play an important role in product release. methyl transverse relaxation optimized spectroscopy ͉ protein dynamics ͉ ClpP protease C lpP (1) is a representative member of the family of cylindrical self-compartmentalizing proteases (2, 3) that include the bacterial protein HslV (4) as well as the archaeal and eukaryotic 20S proteasomes (5, 6). The proteolytic active sites of these proteases are located in an enclosed catalytic chamber separated from the cellular milieu. Proteins targeted for degradation are recognized by AAA ϩ chaperones that unfold and translocate substrates in an ATP-dependent manner into the lumen of the protease. In the case of ClpP (Fig. 1 a-c), it is well established that ClpX and ClpA deliver substrates through axial pores into the protease catalytic chamber for subsequent degradation (Fig. 1a) (1,7,8). In contrast, the pathway of product release remains controversial. For ClpP (1, 9, 10) and for the proteasome (11), it has been proposed that the entrance pores might also function as product exit sites. However, this proposal is problematic, because it requires the chaperones, which can bind to both ends of the protease simultaneously, to interrupt translocation to allow product release (12).Recently, it has been shown that the ClpP handle region that forms the main interface between two ClpP rings (Fig. 1b) can be deleted without disrupting the oligomeric state of the protein. Furthermore, the x-ray structure of an A153P mutation of Streptococcus pneumoniae ClpP (13) shows that the mutation causes two turns of helix E to become unstructured. Nevertheless, the mutant protein remains a double heptamer, which suggests a high plasticity for the handle region (Fig. 1d). Here, we used recently develo...

ClpP is a conserved serine-protease with two heptameric rings that enclose a large chamber containing the protease active sites. Each ClpP subunit can be divided into a handle region, which mediates ring-ring interactions, and a head domain. ClpP associates with the hexameric ATPases ClpX and ClpA, which can unfold and translocate substrate proteins through the ClpP axial pores into the protease lumen for degradation. We have determined the x-ray structure of Streptococcus pneumoniae ClpP(A153P) at 2.5 Å resolution. The structure revealed two novel features of ClpP which are essential for ClpXP and ClpAP functional activities. First, the Ala 3 Pro mutation disrupts the handle region, resulting in an altered ring-ring dimerization interface, which, in conjunction with biochemical data, demonstrates the unusual plasticity of this region. Second, the structure shows the existence of a flexible Nterminal loop in each ClpP subunit. The loops line the axial pores in the ClpP tetradecamer and then protrude from the protease apical surface. The sequence of the N-terminal loop is highly conserved in ClpP across all kingdoms of life. These loops are essential determinants for complex formation between ClpP and ClpX/ClpA. Mutation of several amino acid residues in this loop or the truncation of the loop impairs ClpXP and ClpAP complex formation and prevents the coupling between ClpX/ClpA and ClpP activities.

No abstract

The major outer membrane protein of Acinetobacter baumannii is the heat-modifiable protein HMP-AB, a porin with a large pore size allowing the penetration of solutes having a molecular weight of up to approximately 800 Da. Cross-linking experiments with glutardialdehyde failed to show any cross-linking between the monomers, a fact that proves again that this porin protein functions as a monomeric porin. The specific activity of this porin was found to be similar to that of other monomeric porins. Tryptic digestion of the outer membrane yielded a 23-kDa fragment of the HMP-AB protein that was resistant to further trypsin treatment. This observation indicates that HMP-AB is assembled in the membrane in a manner similar to monomeric porins. Cloning of the HMP-AB gene revealed an open reading frame of 1038 bp encoding a protein of 346 amino acids and a calculated molecular mass of 35,636 Da. The amino acid sequence and composition were typical of gram-negative bacterial porins: a highly negative hydropathy index, absence of hydrophobic residue stretches, a slightly negative total charge, low instability index, high glycine content, and an absence of cysteine residues. Sequence comparison of HMP-AB with other outer membrane proteins revealed a clear homology with the monomeric outer membrane proteins, outer membrane protein A (OmpA) of Enterobacteria, and outer membrane protein F (OprF) of Pseudomonas sp. Secondary structure analysis indicated that HMP-AB has a 172-amino acid N-terminal domain that spans the outer membrane by eight amphiphilic beta strands and a C-terminal domain that apparently serves as an anchoring protein to the peptidoglycan layer. The results also indicate that HMP-AB belongs to the eight transmembrane beta-strand family of outer membrane proteins.

The ClpXP ATPase-protease complex is a major component of the protein quality control machinery in the cell. A ClpX subunit consists of an N-terminal zinc binding domain (ZBD) and a C-terminal AAA þ domain. ClpX oligomerizes into a hexamer with the AAA þ domains forming the base of the hexamer and the ZBDs extending out of the base. Here, we report that ClpX switches between a capture and a feeding conformation. ZBDs in ClpX undergo large nucleotide-dependent block movement towards ClpP and into the AAA þ ring. This motion is modulated by the ClpX cofactor, SspB. Evidence for this movement was initially obtained by the surprising observation that an N-terminal extension on ClpX is clipped by bound ClpP in functional ClpXP complexes. Protease-protection, crosslinking, and light scattering experiments further support these findings.

The outer membrane protein of Photobacterium damsela (OMP-PD) and the gene encoding for this porin protein were isolated and characterized. The deduced amino acid sequence of the OMP-PD monomer has 338 amino acids and a calculated molecular weight of 36,951 Da. This sequence includes a 22-amino acid signal peptide at the N-terminal, which is not found when the monomer is located in the outer membrane. Native OMP-PD protein forms a trimeric structure of approximately 110 kDa. It exhibits resistance to proteases, and it can be cleaved only following denaturation by SDS. The degree of identity of the OMP-PD amino acid sequence to porins from the Enterobacteriaceae was only 24%. Identity to Vibrio or Photobacterium porins was 38% and 48%, respectively. Nevertheless, the multiple alignment of this sequence with other structurally defined Enterobacteria porins demonstrated that the location of the 16 beta-strands and eight external loops, including a larger external L3 loop, are conserved in OMP-PD. These results, together with the previously known ability of OMP-PD to form an ion channel in artificial liposomes, strongly support its role as a porin in P. damsela and will help further investigations into the role of OMP-PD in P. damsela pathogenicity.

A protein oligomer with an approximate molecular weight of its 37-kDa monomer form was purified from the cell envelope fraction of Vibrio damsela cells. This oligomer exhibited strong porin activity when reconstituted into proteoliposomes with phosphatidyl choline. The functional properties for the 37-kDa protein suggest that it is a nonspecific or general porin, with an apparent pore size of 1.6 nm. This porin allows penetration of a variety of hydrophilic solutes according to their molecular mass. After electroelution, the oligomer was partially dissociated into monomers, whereas treatment with EDTA did not affect its dissociation. The monomers of the 37-kDa protein were not active in the reconstitution assay. The effect of culture media on the composition of the outer membrane protein of V. damsela was examined. Only one outer membrane protein with an apparent molecular weight of 37 kDa (37-kDa protein) was formed in cells grown in 3% NaCl-BHI broth and in 3% NaCl-nutrient broth with the addition of 2% glucose. Three outer membrane proteins, with apparent molecular weights of 37 kDa, 40 kDa, and 46 kDa, were produced in cells grown in 3% NaCl-nutrient broth. An additional outer membrane protein with an apparent molecular weight of 44 kDa (44-kDa protein) was found in cells grown in 3% NaCl-nutrient broth with the addition of 2% maltose. This protein was found to exhibit specificity to maltose derivatives. The results obtained in this study confirm the porin-like character of discussed proteins and give a basis for advanced study of those proteins.