Activity: Verified in Poly-ubiquitin Chain Synthesis Assay.
Verified Applications: In vitro, recombinant NleL E3 ligase accepts activated ubiquitin from E2 conjugating enzymes UBE2D1 or UBE2L3, in a reaction that also requires Ubiquitin Activating Enzyme 1 (an E1). The charged E3 generates K6- and K48-linked poly-ubiquitin chains. Appropriate enzyme concentrations are specific to the application.
2 μg UBA1 run on 4-12% SDS-PAGE gel under reducing conditions, then visualized with Colloidal Coomassie Blue Stain.
Formulation: 40 mM HEPES, 100 mM NaCl, 10% Glycerol, 1 mM EDTA, 1 mM TCEP, pH 7.6
Shipping: The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Storage and Stability: Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Aliquot and store ≤ -70°C (stable for 24 months from date of receipt).
Protein Sequence: GPLGSQGRACLSKAELTADLIWLSANRTGEESAEELNYSGCDLSGLSLVGLNLSSVNFSGAVLDDTDLRMSDLSQAVLENCSFKNSILNECNFCYANLSNCIIRALFENSNFSNSNLKNASFKGSSYIQYPPILNEADLTGAIIIPGMVLSGAILGDVKELFSEKSNTINLGGCYIDLSDIQENILSVLDNYTKSNKSILLTMNTSDDKYNHDKVRAAEELIKKISLDELAAFRPYVKMSLADSFSIHPYLNNANIQQWLEPICDDFFDTIMSWFNNSIMMYMENGSLLQAGMYFERHPGAMVSYNSSFIQIVMNGSRRDGMQERFRELYEVYLKNEKVYPVTQQSDFGLCDGSGKPDWDDDSDLAYNWVLLSSQDDGMAMMCSLSHMVDMLSPNTSTNWMSFFLYKDGEVQNTFGYSLSNLFSESFPIFSIPYHKAFSQNFVSGILDILISDNELKERFIEALNSNKSDYKMIADDQQRKLACVWNPFLDGWELNAQHVDMIMGSHVLKDMPLRKQAEILFCLGGVFCKYSSSDMFGTEYDSPEILRRYANGLIEQAYKTDPQVFGSVYYYNDILDRLQGRNNVFTCTAVLTDMLTEHAKESFPEIFSLYYPVAWR
Background Information and Alternate Names
Species & domain architecture.
Originating from E. coli pathotypes EHEC and EPEC, NleL comprises an N-terminal β-helix formed by pentapeptide repeats (residues 1–250) and a C-terminal bilobed catalytic domain typifying the bacterial HECT (bHECT) fold (residues 251-548); key motifs include the catalytic Cys387 and an E2-binding patch.
UPS pathway role.
Once translocated via the T3SS, NleL positions itself downstream of host E2 enzymes, directly catalyzing ubiquitin transfer to host substrates or building unanchored chains that act as signaling decoys; it functions analogously to eukaryotic HECT E3s yet employs a distinct β-helix scaffold for substrate recruitment.
Relevance to TPD & disease biology.
By ubiquitinating JNK and suppressing MAP-kinase activation, NleL enhances formation of attaching-and-effacing (A/E) lesions, a hallmark of EHEC virulence. Its compact size, rapid kinetics, and unique linkage bias make it an attractive scaffold for engineering synthetic degraders or studying linkage-restricted ubiquitin biology relevant to cancer and neuro-inflammation, where K6/K48 chains modulate proteostasis.
References
Lin, D. Y-W., et al. (2011) J Biol Chem 2011 286:441-449. PMID 20980253
Piscatelli, H., et al., (2011) PLoS ONE 2011 6:e19331. PMID 21541301
Lin, D. Y-W., et al., (2012) PNAS 109:1925-1930. PMID 22308380
Hospenthal, M. K., et al., (2013) Nat. Struct. Mol. Biol. 20:555-565. PMID 23563141
Riling, C., et al., (2015) J Biol Chem 290:23875-87. PMID 26245901
Sheng, X., et al., (2017 ) PLoS Pathog 13:e1006534. PMID 28753655
