Active Site Residues and Amino Acid Specificity of the Ubiquitin Carrier Protein-binding RING-H2 Finger Domain*

Abstract

EL5 is a rice ubiquitin-protein isopeptide ligase (E3) containing a RING-H2 finger domain that interacts with Oryza sativa (Os) UBC5b, a rice ubiquitin carrier protein. We introduced point mutations into the EL5 RING-H2 finger so that residues that functionally interact with OsUBC5b could be identified when assayed for ubiquitination activity in vitro. The residue positions were selected based on the results of an EL5 RING-H2 finger/OsUBC5b NMR titration experiment. These RING-H2 finger residues form or are adjacent to a shallow groove that is recognized by OsUBC5b. The E3 activity of EL5 is shown to be dependent on a Trp located at the center of the groove. We classified rice RING fingers according to the type of metal-chelating motif, i.e. RING-H2 or RING-HC, and according to the presence or absence of a conserved EL5-like Trp. We discuss the probable relationship between E3 activity and the conserved Trp.The RING finger motif, found in many functionally distinct proteins, was first identified as the protein product of the human gene RING1 (Really Interesting New Gene 1) (1). The RING finger motif is defined by the consensus sequence Cys-X 2 -Cys-X 9 -39 -Cys-X 1-3 -His-X 2-3 -(Cys/ His)-X 2 -Cys-X 4 -48 -Cys-X 2 -Cys, where X is any amino acid and the number of X residues varies in different fingers. Two types of RING finger motifs are distinguished by a cysteine (RING-HC) or histidine (RING-H2) as the fifth metal-chelating residue. A RING finger typically binds two zinc atoms, with its Cys and/or His side chains in a unique "cross-brace" arrangement. The nearly invariant spacing between the second and third pairs of Cys/His residues probably conserves the distance between the two metal-chelating sites (2). RING fingers are commonly found in proteins that are involved in cell growth and differentiation (3). Some RING fingers may be required for protein association, e.g. homo-or heterodimerization, whereas others are needed for ubiquitination (4 -6). The ubiquitination product is an isopeptide bond between the C-terminal carboxyl of ubiquitin (Gly 76 ) and a substrate lysine ⑀-amino group. Ubiquitination requires three sequential enzymatic reactions: (i) a ubiquitin-activating enzyme (E1) 2 forms a thiol ester between one of its cysteines and the ubiquitin Gly 76 carboxyl; (ii) then a conjugating enzyme (ubiquitin carrier protein (E2)) transiently carries the ubiquitin (as a thiol ester); and (iii) a ubiquitin-protein isopeptide ligase (E3) helps to transfer the activated ubiquitin from E2 to the substrate Lys. Generally, eukaryotic cells contain a single type of E1, multiple types of E2, and many different E3 enzymes. Efficient and targeted ubiquitination depends on E3. All known E3 enzymes have one of two E2-binding domains: the RING finger domain or the HECT domain (7). The three-dimensional structures of E3-type RING fingers have been determined by NMR spectroscopy (8 -13) or x-ray diffraction (14 -16). These structures all have a groove formed by the first zincbinding loop (N-loop; Cys 134 -Cys 137 of the...

The prolific ATL family of RING-H2 ubiquitin ligases

“…[17][18][19][20] The structural basis of the E2-E3 recognition has been elucidated for EL5, a rice ATL. The three-dimensional structure of the RING-H2 finger domain in solution, as determined by NMR spectroscopy, basically demonstrated the same structural features of previously characterized RING domains.…”

“…36 EL5 has been extensively investigated, and the RING-H2 finger domain has been used as a model to analyze three-dimensional structures as well as interactions with the E2 enzyme. 17,21 EL5 localizes to the plasma membrane and is detectable in the microsomal fraction, suggesting that it is anchored in the plasma membrane. 37 Ectopic expression of EL5 in transgenic rice plants as well as attempts to suppress EL5 by RNAi constructs produces lines with no apparent phenotypic alteration.…”

EL5 is involved in root development as an anti-cell death ubiquitin ligase

“…9 An NMR titration experiment and an in vitro ubiquitination assay using a series of EL5-RFD mutants with a rice E2, OsUBC5b, identified the amino acid residues involved in OsUBC5b binding. 10 The mutated RFDs showed varying decreased E3 activity depending on the degree of contribution imparted upon the interaction with E2. 10 The transcript of OsUBC5b is upregulated by N-acetylchitoo ligosaccharide elicitor as well as EL5, suggesting that OsUBC5b is a physiological partner of EL5.…”

“…10 The mutated RFDs showed varying decreased E3 activity depending on the degree of contribution imparted upon the interaction with E2. 10 The transcript of OsUBC5b is upregulated by N-acetylchitoo ligosaccharide elicitor as well as EL5, suggesting that OsUBC5b is a physiological partner of EL5. 6 Maltose binding protein (MBP), which carries 36 Lys residues, is used as an artificial substrate for in vitro ubiquitination assays.…”

“…11 Replacement of the conserved Cys153, which causes the conformational disruption of EL5-RFD, or of Try165, which is an E2 recognition residue, with Ala (EL5C153A and EL5W165A, respectively) resulted in the loss of its E3 activity in vitro. 10 Rice plants overexpressing EL5 showed no phenotypic changes. On the other hand, plants expressing EL5C153A or EL5W165A were rootless resulting from necrosis in root apical meristems (RAM), where EL5 is expressed, and were not recovered by exogenous auxin.…”