Multidrug-efflux transporters demonstrate an unusual ability to recognize multiple structurally dissimilar toxins. A comparable ability to bind diverse hydrophobic cationic drugs is characteristic of the Bacillus subtilis transcription regulator BmrR, which upon drug binding activates expression of the multidrug transporter Bmr. Crystal structures of the multidrug-binding domain of BmrR (2.7 A resolution) and of its complex with the drug tetraphenylphosphonium (2.8 A resolution) revealed a drug-induced unfolding and relocation of an alpha helix, which exposes an internal drug-binding pocket. Tetraphenylphosphonium binding is mediated by stacking and van der Waals contacts with multiple hydrophobic residues of the pocket and by an electrostatic interaction between the positively charged drug and a buried glutamate residue, which is the key to cation selectivity. Similar binding principles may be used by other multidrug-binding proteins.

Hemoglobin has been a long-standing paradigm for understanding protein allostery. Here, the x-ray structures of two chemically crosslinked, fully liganded hemoglobins, ␣ 2 ␤ 82 CA 82 ␤ and ␣ 2 ␤ 82 ND 82 ␤, are described at 2.3 Å and 2.6 Å resolution, respectively. Strikingly, these crosslinked hemoglobins assume intermediate conformations that lie between those of R and the controversial liganded hemoglobin state R2 rather than between R and T. Thus, these structures support only a T 7 R 7 R2 allosteric pathway and underscore the physiological importance of the R2 conformation.The quaternary end-state structures of human hemoglobin have long been accepted as the unliganded T and liganded R conformations (1-7). However, the appearance of a second fully liganded conformation, R2 (8-10), has stirred debate as to whether this conformation is an intermediate that lies between T and R (8, 11), an off-pathway structure (12), or the physiologically relevant end state (11, 13). On the basis of the dislocation of the imidazole side chain of residue ␤ 2 His-97 from the ␣ 1 C-helix, the R2 conformation was first proposed as an intermediate between the R and T states because it suggested a mechanism by which this residue switches from its T to R state position (8). However, the results of the calculated trajectory of the atomic coordinates in transiting from the T to R2 structure casted doubt on the validity of this proposal (13). Specifically, that trajectory was shown to pass close to the R conformation and thereby suggested R2 might be the physiologically relevant liganded end-state conformation. Clearly, the relevance of the R2 conformation and its position along hemoglobin's allosteric pathway is critical to our complete understanding of the function of hemoglobin. Here, we present the structures of two chemically crosslinked, fully liganded hemoglobins that capture R 7 R2 conformational intermediates and thus clarify the relevance of the R2 state.

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The ferric uptake regulator, Fur, represses iron uptake and siderophore biosynthetic genes under ironreplete conditions. Here we report in vitro solution studies on Vibrio anguillarum Fur binding to the consensus 19-bp Escherichia coli iron box in the presence of several divalent metals. We found that V. anguillarum Fur binds the iron box in the presence of Mn 2؉ , Co 2؉ , Cd 2؉ , and to a lesser extent Ni 2؉ but, unlike E. coli Fur, not in the presence of Zn 2؉ . We also found that V. anguillarum Fur contains a structural zinc ion that is necessary yet alone is insufficient for DNA binding.Iron is essential for survival and virulence of bacterial pathogens; however, its concentration in host tissues is limited. To acquire iron, bacterial pathogens rely on iron uptake systems that consist of an iron siderophore (a small-molecule chelator) and membrane transport proteins, which import Fe 3ϩ -siderophore complexes into the cell (7, 18). These systems are regulated by the DNA-binding protein Fur (ferric uptake regulator) in response to iron availability (3,11,20). When the intracellular concentration of iron increases above a certain level, Fur represses the transcription of genes encoding components of the membrane transport system as well as enzymes involved in siderophore biosynthesis.To date, the most extensive biochemical studies have been limited to the Escherichia coli Fur protein, which in the presence of various divalent metals that act as corepressors binds a conserved 19-bp operator sequence, the iron box, which is located in the promoter region of iron uptake genes (4, 5, 9). Moreover, these studies have indicated that E. coli Fur possesses two metal ion-binding sites. One, the corepressor binding site, uses histidines and carboxylate ligands to coordinate binding of Fe 2ϩ (and two of its functional mimics, Mn 2ϩ and Co 2ϩ ) (1). This event promotes a conformational change that leads to DNA binding. The other cation-binding site is involved in protein structure and stability and binds Zn 2ϩ with high affinity, using the thiols of Cys92 and Cys95 as two of the four coordinating ligands (2,3,14). These cysteines are essential for E. coli Fur activity both in vivo and in vitro (6).Fur homologues have been identified in multiple bacteria, where they also regulate iron acquisition. In the fish pathogen Vibrio anguillarum strain 775, Fur represses production of a siderophore, anguibactin, and of Fe 3ϩ -anguibactin transport proteins in response to abundant iron (21). V. anguillarum Fur is closely related to its counterparts from other Vibrio species, including Vibrio cholerae (93% sequence identity), Vibrio vulnificus (91%), and Vibrio parahaemolyticus (91%) (Fig. 1). Yet V. anguillarum Fur is less homologous to E. coli Fur (76% sequence identity), and the two proteins have different numbers of cysteines and histidines (Fig. 1) The V. anguillarum fur gene subcloned into the isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible pT7-5 vector (21) was kindly provided by A. M. Wertheimer. E. coli BL21(DE3) cells t...