The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) 1 catalyzes the uptake and phosphorylation of carbohydrates (1). It is further involved in signal transduction (2), e.g. catabolite repression (3), chemotaxis (4), and allosteric regulation of metabolic enzymes and transporters in response to the availability of carbohydrates (5, 6). The PTS is widely distributed in eubacteria and absent from eukaryotes. It generally consists of two central phosphoryl carriers, EI and HPr, and depending on the species, between 1 and 20 peripheral phosphotransferases, the integral membrane components that recognize and transport hexoses, hexitols, and disaccharides. EI and HPr are the proteins at the top of this divergent phosphorylation cascade (7). EI is a 64-kDa two-domain protein (8, 9). In the aminoterminal domain (EIN, residues 1-250) is located His-189, the amino acid transiently phosphorylated by PEP. The threedimensional structure of EIN has been elucidated (10, 11) and its mode of interaction with HPr characterized by NMR spectroscopy (12, 13). It is composed of a HPr binding ␣-helical subdomain and an ␣/ subdomain, structurally similar to the phosphohistidine swivel domain of pyruvate phosphate dikinase (PPDK). Phospho-EIN transfers the phosphoryl group to HPr, but it requires the presence of the carboxyl-terminal domain (EIC) to become phosphorylated by PEP (8,14). EIC contains the PEP binding site (15) and plays a crucial role in dimerization (16,17). It also confers species and subunit specificity during the phosphoryl transfer to the next phosphocarrier protein (14). EIC is proteolytically unstable and flexible. Its structure is not yet known. However, the amino acid sequence similarity between EIC and the PEP binding domain of PPDK of Clostridium symbiosum points toward similar fold and structure. There is also sequence similarity with PEP synthase (18).The mode of action of EI and the way its activity is controlled are not yet fully understood. The protein dimerizes in a temperature-dependent manner (19). The dissociation constant has been reported to shift from 20 M at 6°C to 0.9 M at 30°C. The presence of divalent cations (Mg 2ϩ or Mn 2ϩ ) and PEP also affects the equilibrium and the association and dissociation rate constants (20,21). The association rate constant is 2-3 orders of magnitude slower than in other dimeric proteins, * This work was supported by Grant 31-45838.95 from the Swiss National Science Foundation and a fellowship from the Secretaría de Estado de Educación y Universidades, Spain (to L. F. G.-A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ To whom correspondence should be addressed. Tel.: 41-0-31-631-3792; Fax: 41-0-31-631-4887; E-mail:garcia@ibc.unibe.ch.1 The abbreviations used are: PTS, phosphoenolpyruvate:glucose phosphotransferase system; Z-and E-Cl-PEP, (Z)-3-and (E)-3-chlorophos...