SummaryClearance of misfolded and aggregated proteins is central to cell survival. Here, we describe a new pathway for maintaining protein homeostasis mediated by the proteasome shuttle factor UBQLN2. The 26S proteasome degrades polyubiquitylated substrates by recognizing them through stoichiometrically bound ubiquitin receptors, but substrates are also delivered by reversibly bound shuttles. We aimed to determine why these parallel delivery mechanisms exist and found that UBQLN2 acts with the HSP70-HSP110 disaggregase machinery to clear protein aggregates via the 26S proteasome. UBQLN2 recognizes client-bound HSP70 and links it to the proteasome to allow for the degradation of aggregated and misfolded proteins. We further show that this process is active in the cell nucleus, where another system for aggregate clearance, autophagy, does not act. Finally, we found that mutations in UBQLN2, which lead to neurodegeneration in humans, are defective in chaperone binding, impair aggregate clearance, and cause cognitive deficits in mice.

Bacteria surround their cytoplasmic membrane with an essential, stress-bearing peptidoglycan (PG) layer. Growing and dividing cells expand their PG layer by using membrane-anchored PG synthases, which are guided by dynamic cytoskeletal elements. In Escherichia coli, growth of the mainly single-layered PG is also regulated by outer membrane-anchored lipoproteins. The lipoprotein LpoB is required for the activation of penicillin-binding protein (PBP) 1B, which is a major, bifunctional PG synthase with glycan chain polymerizing (glycosyltransferase) and peptide cross-linking (transpeptidase) activities. Here, we report the structure of LpoB, determined by NMR spectroscopy, showing an N-terminal, 54-aalong flexible stretch followed by a globular domain with similarity to the N-terminal domain of the prevalent periplasmic protein TolB. We have identified the interaction interface between the globular domain of LpoB and the noncatalytic UvrB domain 2 homolog domain of PBP1B and modeled the complex. Amino acid exchanges within this interface weaken the PBP1B-LpoB interaction, decrease the PBP1B stimulation in vitro, and impair its function in vivo. On the contrary, the N-terminal flexible stretch of LpoB is required to stimulate PBP1B in vivo, but is dispensable in vitro. This supports a model in which LpoB spans the periplasm to interact with PBP1B and stimulate PG synthesis. P eptidoglycan (PG) is an essential component of the bacterial cell envelope, required for cell shape and stability. It is composed of glycan chains that are connected by short peptides, and forms a net-like, elastic structure, called the sacculus, which encases the cytoplasmic/inner membrane (IM) (1). In Gramnegative bacteria, such as Escherichia coli, the sacculus is mainly single-layered and is firmly attached to the outer membrane (OM) by abundant OM proteins. Some of the most effective antibiotic agents, such as the β-lactams and glycopeptides, inhibit PG biosynthesis, resulting in cell lysis.Bacteria enlarge their sacculus by polymerizing new PG from lipid II precursor at the outer face of the IM and incorporating the newly made material into the existing PG layer. At the same time, a significant amount of old material is released. For synthesis and hydrolysis to be coupled, the corresponding enzymes have to be tightly regulated and coordinate their actions (2). How does this happen? The current view is that PG synthases [penicillin-binding proteins (PBPs)] and hydrolases form membrane-anchored multienzyme complexes, which are driven by cytoskeletal elements. More recently, it was established that dedicated regulators tightly control the activities of PG synthases and hydrolases and/or couple it to other cell envelope processes (3-6).PG synthesis requires glycosyltransferases (GTases) to polymerize the glycan chains and transpeptidases (TPases) to form peptide cross-links. Most bacteria carry several PG synthases, which can perform one or both enzymatic reactions. In E. coli, the bifunctional GTase/TPases PBP1A and PBP1B provide the main PG...

The control of mRNA stability is an important component of regulation in bacteria. Processing and degradation of mRNAs are initiated by an endonucleolytic attack, and the cleavage products are processively degraded by exoribonucleases. In many bacteria, these RNases, as well as RNA helicases and other proteins, are organized in a protein complex called the RNA degradosome. In Escherichia coli, the RNA degradosome is assembled around the essential endoribonuclease E. In Bacillus subtilis, the recently discovered essential endoribonuclease RNase Y is involved in the initiation of RNA degradation. Moreover, RNase Y interacts with other RNases, the RNA helicase CshA, and the glycolytic enzymes enolase and phosphofructokinase in a degradosome-like complex. In this work, we have studied the domain organization of RNase Y and the contribution of the domains to protein-protein interactions. We provide evidence for the physical interaction between RNase Y and the degradosome partners in vivo. We present experimental and bioinformatic data which indicate that the RNase Y contains significant regions of intrinsic disorder and discuss the possible functional implications of this finding. The localization of RNase Y in the membrane is essential both for the viability of B. subtilis and for all interactions that involve RNase Y. The results presented in this study provide novel evidence for the idea that RNase Y is the functional equivalent of RNase E, even though the two enzymes do not share any sequence similarity. mRNAs transmit the genetic information from DNA to proteins. In contrast to the high stability of tRNA and rRNA, bacterial mRNAs are readily degraded. This crucial feature ensures the ability of bacteria to respond quickly to changing environmental conditions by adding an extra layer of control, in addition to the regulation of transcription and enzymatic activities of proteins (1). Moreover, mRNA processing allows the adjustment of different expression levels for genes encoded in one operon, as observed for the ilv operon and the glycolytic gapA operon of the Gram-positive soil bacterium Bacillus subtilis (24,40,45).The enzymes that process and degrade RNAs are called RNases. In the two model organisms Escherichia coli and B. subtilis, a set of about 20 RNases has been described thus far, but only a small fraction of these are common to both bacteria (12). Some of these RNases (e.g., RNase M5 or RNase-Mini III in B. subtilis) are necessary for the maturation of one specific target, whereas others (e.g., RNase J1 or RNase Y in B. subtilis) are more promiscuous and have a more global impact on RNA metabolism (12, 43, 58). The individual RNases differ not only in their respective amino acid sequences and in their number of substrates but also in their enzymatic mode of action. Two different classes of RNases can be defined: exoribonucleases, which remove RNA nucleotides one at a time from either the 3Ј or the 5Ј end, and endoribonucleases, which cleave within the RNA molecule. For the degradation of mRNAs, the conc...

The outer membrane (OM) of gram-negative bacteria is an unusual asymmetric bilayer with an external monolayer of lipopolysaccharide (LPS) and an inner layer of phospholipids. The LPS layer is rigid and stabilized by divalent cation cross-links between phosphate groups on the core oligosaccharide regions. This means that the OM is robust and highly impermeable to toxins and antibiotics. During their biogenesis, OM proteins (OMPs), which function as transporters and receptors, must integrate into this ordered monolayer while preserving its impermeability. Here we reveal the specific interactions between the trimeric porins of Enterobacteriaceae and LPS. Isolated porins form complexes with variable numbers of LPS molecules, which are stabilized by calcium ions. In earlier studies, two high-affinity sites were predicted to contain groups of positively charged side chains. Mutation of these residues led to the loss of LPS binding and, in one site, also prevented trimerization of the porin, explaining the previously observed effect of LPS mutants on porin folding. The high-resolution X-ray crystal structure of a trimeric porin-LPS complex not only helps to explain the mutagenesis results but also reveals more complex, subtle porin-LPS interactions and a bridging calcium ion.gram-negative bacteria | lipopolysaccharide | X-ray crystal structure | porin | outer membrane

We have investigated the potential of sedimentation velocity analytical ultracentrifugation for the measurement of the second virial coefficients of proteins, with the goal of developing a method that allows efficient screening of different solvent conditions. This may be useful for the study of protein crystallization. Macromolecular concentration distributions were modeled using the Lamm equation with the approximation of linear concentration dependencies of the diffusion constant, D = D(o) (1 + k(D)c), and the reciprocal sedimentation coefficient s = s(o)/(1 + k(s)c). We have studied model distributions for their information content with respect to the particle and its non-ideal behavior, developed a strategy for their analysis by direct boundary modeling, and applied it to data from sedimentation velocity experiments on halophilic malate dehydrogenase in complex aqueous solvents containing sodium chloride and 2-methyl-2,4-pentanediol, including conditions near phase separation. Using global modeling for three sets of data obtained at three different protein concentrations, very good estimates for k(s) and s degrees and also for D degrees and the buoyant molar mass were obtained. It was also possible to obtain good estimates for k(D) and the second virial coefficients. Modeling of sedimentation velocity profiles with the non-ideal Lamm equation appears as a good technique to investigate weak inter-particle interactions in complex solvents and also to extrapolate the ideal behavior of the particle.

Noncatalytic carbohydrate binding modules (CBMs) are components of glycoside hydrolases that attack generally inaccessible substrates. CBMs mediate a two-to fivefold elevation in the activity of endoacting enzymes, likely through increasing the concentration of the appended enzymes in the vicinity of the substrate. The function of CBMs appended to exo-acting glycoside hydrolases is unclear because their typical endo-binding mode would not fulfill a targeting role. Here we show that the Bacillus subtilis exo-acting β-fructosidase SacC, which specifically hydrolyses levan, contains the founding member of CBM family 66 (CBM66). The SacC-derived CBM66 (BsCBM66) targets the terminal fructosides of the major fructans found in nature. The crystal structure of BsCBM66 in complex with ligands reveals extensive interactions with the terminal fructose moiety (Fru-3) of levantriose but only limited hydrophobic contacts with Fru-2, explaining why the CBM displays broad specificity. Removal of BsCBM66 from SacC results in a ∼100-fold reduction in activity against levan. The truncated enzyme functions as a nonspecific β-fructosidase displaying similar activity against β-2,1-and β-2,6-linked fructans and their respective fructooligosaccharides. Conversely, appending BsCBM66 to BT3082, a nonspecific β-fructosidase from Bacteroides thetaiotaomicron, confers exolevanase activity on the enzyme. We propose that BsCBM66 confers specificity for levan, a branched fructan, through an "avidity" mechanism in which the CBM and the catalytic module target the termini of different branches of the same polysaccharide molecule. This report identifies a unique mechanism by which CBMs modulate enzyme function, and shows how specificity can be tailored by integrating nonspecific catalytic and binding modules into a single enzyme.isothermal titration calorimetry | X-ray crystallography | prebiotics | biofuels | lectins C omplex carbohydrates represent a major nutrient for numerous microbial ecosystems, exemplified by bacterial and fungal communities established in the rumen and large bowel of mammals, where they play an important role in animal nutrition and human health, respectively (1, 2). It is also evident that these composite structures are of increasing industrial significance, particularly in the bioenergy and bioprocessing sectors (3). The enzymes that catalyze the degradation of these complex carbohydrates, primarily glycoside hydrolases but also polysaccharide lyases, are grouped into sequence-based families on the continuously updated CAZy database (4). Members of the same family display a common fold, and the catalytic mechanism and catalytic apparatus are also conserved (5). Substrate specificity within glycoside hydrolase families (GHs), however, can vary significantly (6). Glycanases that attack inaccessible substrates often contain noncatalytic carbohydrate binding modules (CBMs; see ref. 7 for review) that are also grouped into sequence-based families in the CAZy database. Against recalcitrant substrates, CBMs enhance the activit...

RNase J is an essential enzyme in Bacillus subtilis with unusual dual endonuclease and 5'-to-3' exonuclease activities that play an important role in the maturation and degradation of mRNA. RNase J is also a component of the recently identified "degradosome" of B. subtilis. We report the crystal structure of RNase J1 from B. subtilis to 3.0 Å resolution, analysis of which reveals it to be in an open conformation suitable for binding substrate RNA. RNase J is a member of the β-CASP family of zinc-dependent metallo-β-lactamases. We have exploited this similarity in constructing a model for an RNase J1:RNA complex. Analysis of this model reveals candidate-stacking interactions with conserved aromatic side chains, providing a molecular basis for the observed enzyme activity. Comparisons of the B. subtilis RNase J structure with related enzymes reveal key differences that provide insights into conformational changes during catalysis and the role of the C-terminal domain.

Background: The structure of GH115 glucuronidases that remove glucuronic acid from xylan chains is unknown.Results: Bacteroides ovatus GH115 glucuronidase is a dimeric enzyme that contains a flexible active site pocket.Conclusion: The assembly of the catalytic apparatus of the glucuronidase requires substantial conformational changes.Significance: Conformational changes are highly unusual in glycoside hydrolases.