Structural and Biochemical Characterization of Deubiquitinating Enzymes, Sst2 and Ssel

Rashmi Kalicharan Shrestha, Purdue University

Abstract

molecule with SH3 domain of molecule (STAM), or AMSH, is a zinc metallo deubiquitinase (DUB) with endosomal-lysosomal receptor trafficking. AMSH is responsible for downregulation and lysosomal degradation of cell-surface receptors (cargo) or autophagic cargo mediated by the endosomal sorting complexes required for transport (ESCRT) machinery. AMSH displays high specificity towards Lys63-linked ubiquitin chains, which acts as a signal for ESCRT machinery. This specificity is due to the simultaneous binding of AMSH to both the distal and proximal ubiquitin groups in a Lys63-linked diubiquitin substrate. The same specificity precludes it from accepting mono-ubiquitinated cargos as substrates; thus, it can disassemble a Lys63-linked polyubiquitin chain attached to a cargo but not completely deubiquitinate it. Therefore, the recruitment of AMSH at ESCRT-0 has led to the speculation that it may modulate the ubiquitin chains of the targeted endosomal cargo and facilitate the transfer of cargo from ESCRT-0 to subsequent complexes and eventually to its degradation. In an attempt to crystallographically characterize activation of AMSH at ESCRT-0, the crystal structures of the catalytic domain of AMSH orthologue Sst2 from fission yeast, its ubiquitin (product)-bound form, and its Lys63-linked diubiquitin (substrate)-bound form at 1.45 Å, 1.7 Å, and 2.3 Å, respectively were determined. The structural analysis provided insight into the mode of substrate recognition and revealed that the distal ubiquitin can remain bound to the enzyme after cleaving Lys63-linked diubiquitin substrate, resulting in the formation of product bound form. A comparable value of KD (∼10 μM) for ubiquitin binding to the enzyme and KM of the enzyme catalyzing hydrolysis of Lys63-linked diubiquitin substrate (∼20 μM) and that the intracellular concentration of free ubiquitin (approximately 20 μM) exceeds that of Lys63-linked polyubiquitin chains, suggested that the free, cytosolic form of the enzyme remains inhibited by being tightly bound to free ubiquitin. Comparison of all the Sst2 structures with that of the substrate-bound form suggested the importance of dynamics of a flexible flap near the active site in catalysis, including product release. Structural analysis of AMSH and its homologs, AMSH-LP (AMSH-like protein) and Sst2, revealed a conserved Phe residue in the flap, which may be critical for substrate binding and/or catalysis. Mutation of this Phe residue to Ala and Trp in Sst2 (Phe403) revealed that the Phe residue in the flap contributes key interactions during the transition state but not to substrate binding Interestingly, molecular dynamics simulations indicated that the Trp mutant was quite flexible, allowing almost free rotation of the indole side-chain which may affect the transition-state stabilization of the enzyme. The ability of Trp mutant to cleave Lys48-linked and Lys11-linked diubiquitin better than the wild-type enzyme also indicated altered mobility and hence reduced selectivity. Since mutations in AMSH lead to Microcephaly-Capillary (MIC-CAP) syndrome, mutational studies in Sst2 or AMSH were performed using various biophysical tools, which revealed that most of the mutations lead to a local change around the site of mutation. As a result, either substrate binding or integrity and stability of the enzyme were affected, which may have profound functional effect leading to the disease. The project was then extended to include another disease DUB, Salmonella-secreted factor L, or Ssel which is a prokaryotic DUB secreted by Salmonella typhimurium. It is responsible for slowing down the lysosomal degradation of selective autophagy. Ssel acts as a virulence factor that completely removes ubiquitin from bacteria-containing vesicles and protein aggregates, and prevents them from autophagic clearance; thus, favoring the intracellular replication of Salmonella. In an effort to understand the mechanism of ubiquitin recognition by Ssel, different constructs of Ssel (Full-length Ssel, SselΔ121 and SselΔ157) were generated and the crystal structure of the shortest construct was determined. Surprisingly, both SselΔ121 and SselΔ157 constructs showed pronounced defect in the catalytic activity of the enzyme compared to full-length Ssel. The full-length Ssel showed strong preference for Lys63-linked diubiquitin over Lys48- and Lys11-linked diubiquitin substrate. Moreover, this prokaryotic DUB also also uses Glu40 patch in binding to its substrate as seen in SdeA from Legionella pneumophila. To further understand the catalytic inefficiency of the shorter constructs, ITC experiments were performed, which confirmed that the N-terminal residues of Ssel may be required to bind ubiquitin. According to Pruneda et al., three variable regions and one constant region are responsible for ubiquitin binding. Although, SselΔ121 construct consisted of all the regions for ubiquitin binding, it still showed impaired activity. Taken together, these data suggests that Ssel may bind its substrate in at least two steps: First, the VHS domain which has higher affinity for ubiquitin (KD: 5.26 μM) may be used to capture the substrate. Then, through protein dynamics (for example, the flexible loop that links VHS domain to the catalytic domain), VHS domain would present ubiquitin to the catalytic domain for catalysis.

Degree

Ph.D.

Advisors

Das, Purdue University.

Subject Area

Biochemistry

COinS