Itay Levin



2000  B.Sc. in Biology, Faculty of Life Sciences, Tel Aviv University.

2002 M.Sc in Biotechnology, Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel Aviv University.   “In vivo Expression of Genes that Encode for Dihydrofolate Reductases from Halophilic and Mesophilic Microorganisms in Escherichia coli and Haloferax volcanii.” Graduated with honors.

2007 Ph.D. under the supervision of Prof Moshe Mevarech, department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel Aviv University.  “Tetrahydrofolate Biosynthesis in Microorganisms.”

Current Research:

My main research focus is the function of the Salmonella typhimurium effector protein SspH2. SspH2 is a secreted toxin of the Salmonella pathogenicity island (SPI-2) type III secretion system of Salmonella enterica serovar Typhimurium, it is conserved in all pathogenic Salmonella species.   Recently it has been reported that IpaH9.8, a Shigella SsPH2 homolog, have an E3 like ubiquitin ligase activity in vitro (Rohde et al Cell Host Microbe. 2007). To date the full machinery necessary for ubiquitination was found exclusively in Eukaryotes, which consistsof an enzyme cascade consisting of E1, E2 and E3 enzymes. Ubiquitination (Ub) can have several distinct effects on substrate proteins, the most common being degradation by the 26S proteasome. However, ubiquitination also regulates several other cellular processes, such as DNA repair, signaling, endocytosis, vesicular trafficking, and cell-cycle progression. Thus, since this class of E3s has a unique structure bacterial ubiquitin ligases might have a novel mechanism of  ubiquitination but will have similar physiological effects. My research in collaboration with Dr. Peter Brzovic from Prof. Rachel Klevit’s group at the University of Washington has shown that although SspH2 utilize the mammalian ubiquitination machinery, its activity involves a novel mechanism; as opposed to mammalian E3s that bind E2s which are not charged with ubiquitin, SspH2 selectively binds the human E2 ∼ Ub conjugate recognizing regions of both the E2 (UbcH5c) and Ub surface. Surprisingly, intermediates in SspH2-directed reactions are activated poly-Ub chains directly tethered to the UbcH5 active site (UbcH5 ∼ Ubn) (Levin et al PNAS 2010).  This mechanistic model was speculated but never demonstrated before for ubiquitin ligases.

SspH2 selectively binds the UbcH5 ∼ Ub conjugate

SspH2 selectively binds the UbcH5~Ub conjugate. Overlay of 1H, 15N- TROSY spectra of (A) free 15N-UbcH5 and 15N-Ub in the absence (Black) and presence (Green) of SspH2 catalytic domain , (B) 15N-UbcH5-O-Ub ( a more stable Ub charged UbcH5) in the absence (Black) and presence (Red) SspH2 catalytic domain, (C) 15N-UbcH5-O-Ub in the absence (Black) and presence (Cyan) of  Catalytic inactive mutant of the sspH2 catalytic domain  C580S-SspH2, and (D) 15N-A96D-UbcH5-O-Ub UbcH5 ( UbcH5 mutant that does not bind mammalian ubiquitin ligases),  in the absence (Black) and presence (Magenta) of  SspH2 catalytic domain  SspH2.

In A and D the overlaid spectra are nearly identical indicating little or no interaction whereas significant perturbations are observed in B and C indicating interaction.

Resonances of 15N-labeled UbcH5-O-Ub significantly affected by addition of SspH2 catalytic domain (B) are mapped in red onto a ribbon structure (E) and a surface representation (F) of a model of UbcH5~ Ub based upon the Ubc13 ~Ub conjugate (PDB ID 2 gmi). Topological features of UbcH5 in E.  represents Residues that correspond to the canonical eukaryotic E3 binding surface located in Helix-1, Loop 4, and Loop 7 and are shown in light pink. The region on UbcH5 where eukaryotic E3 and SspH2 binding surfaces overlap is shown in orange.


Detection of polyubiquitin chains on UbcH5c active cystein.

SspH2 generates poly-Ub chains linked to the active site of UbcH5. Western analysis with α-UbcH5c antibodies performed on a reaction mixture containing purified 37 μM UbcH5-S ~ Ubwt and 0.6 μM SspH2 Catalytic domain. Samples were collected at 0, 1, 2,3, and 4 minutes after addition of SspH2 catalytic domain  at 30°C. Products were separated by SDS–PAGE under nonreducing (−DTT) and reducing (+DTT) conditions.

Higher molecular weight species of UbcH5~Ubn are observed only in the presence of SspH2477–788 and under nonreducingconditions.


Levin I, Eakin C, Blanc MP, Klevit RE, Miller SI, Brzovic PS (2010) Identification of an unconventional E3 binding surface on then UbcH5 ∼ Ub conjugate recognized by a pathogenic bacterial E3 ligase. Proc Natl Acad Sci U S A. 2010 Feb 16;107(7):2848-53. Epub 2010 Feb 1

Levin I, Mevarech M, Palfey BA. (2007) Characterization of a novel bi-functional dihydropteroate synthase/dihydropteroate reductase enzyme from Helicobacter pylori. J Bacteriol. 189(11): 4062–4069. 2004

Levin I*, Giladi M*, Altman-Price N, Ortenberg R, Mevarech M. (2004) An alternative pathway for reduced folate biosynthesis in bacteria and halophilic archaea. Mol Microbiol. ;54(5):1307-18

*Equally contributed to the paper.

Giladi M, Altman-Price N, Levin I, Levy L, Mevarech M. (2003) FolM, a new chromosomally encoded dihydrofolate reductase in Escherichia coli. J Bacteriol. :185(23):7015-8.