Biochemical Analysis of a Prokaryotic Deubiquitinase from Escherichia Coli

Cameron Wade, Purdue University

Abstract

ElaD is a cysteine protease found in Escherichia coli (E. coli ) and has been shown to function as a deubiquitinating enzyme (DUB). However, ubiquitin and the ubiquitination system are exclusive to eukaryotic cells. This indicates that ElaD may be used when the bacteria come in contact with a eukaryotic cell. This explains its presence in the intestinally infective strains of E. coli. For the invasive strains of E. coli, membrane fusion and phagosome formation is used for entry of the infective cell into the host cell. In order to counteract this, ubiquitination, the eukaryotic cell’s first defense mechanism, is used to signal for degradation of the phagosome. The phagosome is signaled for degradation by the host via adornment of the phagosome with Lys63-linked ubiquitin chains. We hypothesize that ElaD is used to neutralize ubiquitination of the phagosome and prevent destruction of the bacteria. With this hypothesis in mind we seek to examine the substrate preference of the DUB using diubiquitin substrates of defined linkage types. In addition we wish to understand the role of the active-site loop in determining selectivity of the DUB for ubiquitin over a ubiquitin-like modifier Nedd8. Through biochemical analysis of diubiquitin chain types we are able to establish a preference for Lys63 and Lys11-linked diubiquitin over Lys48-linked diubiquitin. The ability to deubiquitinate polyubiquitinated green fluorescent protein (GFP-Ubn) shows ElaD can completely remove ubiquitin from ubiquitinated proteins. The preference for Lys63-linked diubiquitin and the ability to cleave ubiquitin chains from GFP-Ubn is consistent with ElaDs deubiquitination activity at the phagosome. A previously studied bacterial DUB, SdeA from Legionella Pneumophilia, has both deubiquitinating and deneddylating abilities. Unlike SdeA, ElaD shows a strong preference for ubiquitinated substrates over neddylated substrates. The crystal structure of SdeA shows that a seven residue loop (L6), adjacent to the active site cleft, interacts with substrates, via a conserved residue between ubiquitin and Nedd8 (Gln40). ElaD, however, has a predicted two residue loop at an equivalent location. This gives rise to a hypothesis that the loop in ElaD may play a role in substrate selection. To gather insight into the possible role of the loop in substrate recognition and selectivity, a chimeric mutant of ElaD, containing the L6 loop of SdeA (ElaD L6), is compared with wild type ElaD (ElaD wt). The activity toward a Nedd8 substrate was increased in ElaD L6 relative to ElaD wt, although the overall activity for diubiquitin was reduced in ElaD L6. This indicates that the L6 loop insertion contributes to Nedd8 binding. To probe the validity of this, a mutation was made at the conserved Gln40 of ubiquitin in a diubiquitin substrate. The activity of ElaD L6 toward the mutant substrate was examined relative to the wild type substrate. ElaD L6 exhibited more activity toward the mutant substrate than to the wild type substrate suggesting that the inserted loop does in fact make contact with ubiquitin, therefore possibly Nedd8. Together all these data indicate that the small loop in ElaD contributes to substrate affinity and selectivity.

Degree

M.S.

Advisors

Das, Purdue University.

Subject Area

Chemistry|Biochemistry

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