EL5, a RING-H2 finger protein, is rapidly induced by N-acetylchitooligosaccharides in rice cell. We expressed the EL5 RING-H2 finger domain in Escherichia coli and determined its structure in solution by NMR spectroscopy. The EL5 RING-H2 finger domain consists of twostranded ␤-sheets (␤1, Ala 147 -Phe 149 ; ␤2, Gly 156 -His 158 ), one ␣-helix (Cys 161 -Leu 166 ), and two large N-and C-terminal loops. It is stabilized by two tetrahedrally coordinated zinc ions. This structure is similar to that of other RING finger domains of proteins of known function. From structural analogies, we inferred that the EL5 RING-H2 finger is a binding domain for ubiquitin-conjugating enzyme (E2). The binding site is probably formed by solvent-exposed hydrophobic residues of the N-and C-terminal loops and the ␣-helix. We demonstrated that the fusion protein with EL5-(96 -181) and maltose-binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and a rice E2 protein, OsUBC5b. This supported the idea that the EL5 RING finger domain is essential for ubiquitin-ligase activity of EL5. By NMR titration experiments, we identified residues that are critical for the interaction between the EL5 RING-H2 finger and OsUBC5b. We conclude that the RING-H2 finger domain of EL5 is the E2 binding site of EL5.Upon sensing the invasion of microorganisms, plants evoke a variety of defense reactions, including the synthesis of antimicrobial compounds (phytoalexins) and proteins. Many of these biochemical reactions are based on the activation of defenserelated genes. In some cases, the level of protein accumulation and the rapidity of gene induction in the host plant are correlated to the degree of its disease resistance. Therefore, it might be possible to control disease resistance by modifying the regulatory factors for the expression of defense-related genes.Such regulatory factors could be elements of signal transduction pathways leading from the recognition of invading pathogens to the activation of defense-related genes. Most of the defense responses are reproducible in suspension-cultured cells treated with specific substances called elicitor (1). Chitin fragments (N-acetylchitooligosaccharides) can act as elicitors (2), which induce the transient expression of several "early responsive" genes, such as EL5 (3). EL5 is a RING finger protein, which is structurally related to proteins of the Arabidopsis ATL family. These proteins are characterized by a transmembrane domain (domain I), basic domain (domain II), conserved domain (domain III), and RING-H2 finger domain (domain IV) followed by the C-terminal region with highly diverse amino acid sequences (4). Although some ATL family genes resemble EL5 in being induced in early stages of the defense responses (5), their biochemical function is obscure. Recently, it was shown that the fusion protein of EL5 with maltose-binding protein (MBP) 1 was polyubiquitinated by incubation with ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2). ...

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