The peptidoglycan biosynthetic pathway is a critical process in the bacterial cell and is exploited as a target for the design of antibiotics. This pathway culminates in the production of the peptidoglycan layer, which is composed of polymerized glycan chains with cross-linked peptide substituents. This layer forms the major structural component of the protective barrier known as the cell wall. Disruption in the assembly of the peptidoglycan layer causes a weakened cell wall and subsequent bacterial lysis. With bacteria responsible for both properly functioning human health (probiotic strains) and potentially serious illness (pathogenic strains), a delicate balance is necessary during clinical intervention. Recent research has furthered our understanding of the precise molecular structures, mechanisms of action, and functional interactions involved in peptidoglycan biosynthesis. This research is helping guide our understanding of how to capitalize on peptidoglycan-based therapeutics and, at a more fundamental level, of the complex machinery that creates this critical barrier for bacterial survival.

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E3 ubiquitin ligases are essential enzymes in the ubiquitination pathway responsible for the recognition of specific E2 conjugating enzymes and for transferring ubiquitin to a substrate targeted for degradation. In autosomal recessive juvenile Parkinson's disease, an early onset form of Parkinson's disease, point mutations in the E3 ligase parkin are one of the most commonly observed traits. Parkin is a multidomain E3 ligase that contains an N-terminal ubiquitin-like domain that interacts with, and effects the ubiquitination of, substrates such as cyclin E, p38 and synphilin. In this work we have examined the folding and structure of the parkin ubiquitin-like domain (Ubld) and of the protein with two causative disease mutations (K48A and R42P). Parallel experiments with the protein ubiquitin were done in order to determine if the same mutations were detrimental to the ubiquitin structure and stability. Despite similar folds between the parkin Ubld and ubiquitin, urea unfolding experiments show that the parkin Ubld is surprisingly approximately 10.6 kJ/mol less stable than ubiquitin. The K48A mutation had little effect on the stability of the parkin Ubld or ubiquitin indicating that this mutation contributes to defective protein-protein interactions. In contrast, the single point mutation R42P in parkin's Ubld caused poor expression and degradation of the protein. To avoid these problems, a GB1-Ubld fusion protein was characterized by NMR spectroscopy to show that the R42P mutation causes the complete unfolding of the parkin Ubld. This observation provides a rationale for the more rapid degradation of parkin carrying the R42P mutation in vivo, and its inability to interact with some substrate proteins. Our work provides the first structural and folding insight into the effects of causative mutations within the ubiquitin-like domain in autosomal recessive juvenile Parkinson's disease.

Parkin is a multidomain E3 ligase associated with autosomal recessive Parkinson disease. The N-terminal ubiquitin-like domain (Ubld) of parkin functions with the S5a proteasomal subunit, positioning substrate proteins for degradation. In addition the parkin Ubld recruits the endocytotic protein Eps15, allowing the E3 ligase to ubiquinate Eps15 distal from its parkininteracting site. The recognition sequences in the S5a subunit and Eps15 for the parkin Ubld are ubiquitin-interacting motifs (UIM). Each protein has two UIM sequences separated by a 50-residue spacer in S5a, but only ϳ5 residues in Eps15. In this work we used NMR spectroscopy to determine how the parkin Ubld recognizes the proteasomal subunit S5a compared with Eps15, a substrate for ubiquitination. We show that Eps15 contains two flexible ␣-helices each encompassing a UIM sequence. The ␣-helix surrounding UIM II is longer than that for UIM I, a situation that is reversed from S5a. Furthermore, we show the parkin Ubld preferentially binds to UIM I in the S5a subunit. This interaction is strongly diminished in a K48A substitution, found near the center of the S5a interacting surface on the parkin Ubld. In contrast to S5a, parkin recruits Eps15 using both its UIM sequences resulting in a larger interaction surface that includes residues from ␤1 and ␤2, not typically known to interact with UIM sequences. These results show that the parkin Ubld uses differential surfaces to recruit UIM regions from the S5a proteasomal subunit compared with Eps15 involved in cell signaling.Modification of proteins by ubiquitin is an essential biochemical process that signals proteins for degradation via the 26 S proteasome and also for non-proteolytic processes such as cell cycle and cell division, protein trafficking, endocytosis, and DNA repair (1-3). Three enzymes in the ubiquitination pathway (E1, E2, and E3) label a targeted protein with ubiquitin. The E3 enzymes are important for mediating the transfer of ubiquitin onto the target protein through their interaction with both the E2 enzyme and substrate and provide the specificity for target protein recognition. Autosomal recessive juvenile parkinsonism (ARJP) 2 is an early-onset familial form of the disease that is clinically indistinguishable from the more prevalent idiopathic form of Parkinson disease. Mutations in several genes have been identified in ARJP patients, although the most commonly mutated gene encodes the E3 ubiquitin-protein ligase parkin (4 -6). Mutations in parkin account for ϳ50% of all ARJP cases. Parkin is a 465-residue multidomain E3 ligase comprising an N-terminal ubiquitin-like domain (Ubld) followed by a unique parkin-specific domain, two RING domains (RING0, RING1), an in-between RING (IBR) domain, and a C-terminal RING domain (RING2) (7,8). Mutations associated with ARJP are found throughout the parkin protein and have profound affects on the folding and functionality of the protein.For example, missense mutations in the C terminus of parkin have been shown to disrupt its function with E2 ...

The ubiquitin signaling pathway consists of hundreds of enzymes that are tightly regulated for the maintenance of cell homeostasis. Parkin is an E3 ubiquitin ligase responsible for conjugating ubiquitin onto a substrate protein, which itself can be ubiquitinated. Ataxin-3 performs the opposing function as a deubiquitinating enzyme that can remove ubiquitin from parkin. In this work, we have identified the mechanism of interaction between the ubiquitin-like (Ubl) domain from parkin and three C-terminal ubiquitin-interacting motifs (UIMs) in ataxin-3. 1H–15N heteronuclear single-quantum coherence titration experiments revealed that there are weak direct interactions between all three individual UIM regions of ataxin-3 and the Ubl domain. Each UIM utilizes the exposed β-grasp surface of the Ubl domain centered around the I44 patch that did not vary in the residues involved or the surface size as a function of the number of ataxin-3 UIMs involved. Further, the apparent dissociation constant for ataxin-3 decreased as a function of the number of UIM regions used in experiments. A global multisite fit of the nuclear magnetic resonance titration data, based on three identical binding ligands, resulted in a KD of 669 ± 62 μM for each site. Our observations support a multivalent ligand binding mechanism employed by the parkin Ubl domain to recruit multiple UIM regions in ataxin-3 and provide insight into how these two proteins function together in ubiquitination–deubiquitination pathways.