The plug domain of FepA, a TonB-dependent transport protein from Escherichia coli, binds its siderophore in the absence of the transmembrane barrel domain.

FepA, an outer membrane iron siderophore transporter from Escherichia coli, is composed of a 22-stranded membrane-spanning beta barrel with a globular N-terminal "plug" domain of 148 residues that folds up inside the barrel and completely occludes the barrel's interior (1). We have overexpressed and purified this plug domain by itself and find that it behaves in vitro as a predominantly unfolded yet soluble protein, as determined by circular dichroism, thermal denaturation, and NMR studies. Despite its unfolded state, the isolated domain binds ferric enterobactin, the siderophore ligand of FepA, with an affinity of 5 microM, just 100-fold reduced from that of intact FepA. These findings argue against the hypothesis that the plug domain is pulled intact from the barrel during transport in vivo but may be consistent either with a model where the plug rearranges within the barrel to create a channel large enough to allow transport or with a model where the plug unfolds and comes out of the barrel.

Metabolic effects of mislocalized mitochondrial and peroxisomal citrate synthases in yeast Saccharomyces cerevisiae.

Genes CIT1 and CIT2 from Saccharomyces cerevisiae encode mitochondrial and peroxisomal citrate synthases involved in the Krebs tricarboxylic acid (TCA) cycle and glyoxylate pathway, respectively. A Deltacit1 mutant does not grow on acetate, despite the presence of Cit2p that could, in principle, bypass the resulting block in the TCA cycle. To elucidate this absence of cross-complementation, we have examined the ability of Cit1p to function in the cytosol, and that of Cit2p to function in mitochondria. A cytosolically localized form of Cit1p was also incompetent for restoration of growth of a Deltacit1 strain on acetate, suggesting that mitochondrial localization of Cit1p is essential for its function in the TCA cycle. Cit2p was able, when mislocalized in mitochondria, to restore a wild-type phenotype in a strain lacking Cit1p. We have purified these two isoenzymes as well as mitochondrial malate dehydrogenase, Mdh1p, and have shown that Cit2p was also able to mimic Cit1p in its in vitro interaction with Mdh1p. Models of Cit1p and Cit2p structures generated on the basis of that of pig citrate synthase indicate very high structural and electrostatic surface potential similarities between the two yeast isozymes. Altogether, these data indicate that metabolic functions may require structural as well as catalytic roles for the enzymes.

Three-dimensional structure of AmpC beta-lactamase from Escherichia coli bound to a transition-state analogue: possible implications for the oxyanion hypothesis and for inhibitor design.

The structures of AmpC beta-lactamase from Escherichia coli, alone and in complex with a transition-state analogue, have been determined by X-ray crystallography. The native enzyme was determined to 2.0 A resolution, and the structure with the transition-state analogue m-aminophenylboronic acid was determined to 2.3 A resolution. The structure of AmpC from E. coli resembles those previously determined for the class C enzymes from Enterobacter cloacae and Citrobacter freundii. The transition-state analogue, m-aminophenylboronic acid, makes several interactions with AmpC that were unexpected. Perhaps most surprisingly, the putative "oxyanion" of the boronic acid forms what appears to be a hydrogen bond with the backbone carbonyl oxygen of Ala318, suggesting that this atom is protonated. Although this interaction has not previously been discussed, a carbonyl oxygen contact with the putative oxyanion or ligand carbonyl oxygen appears in most complexes involving a beta-lactam recognizing enzyme. These observations may suggest that the high-energy intermediate for amide hydrolysis by beta-lactamases and related enzymes involves a hydroxyl and not an oxyanion, although the oxyanion form certainly cannot be discounted. The involvement of the main-chain carbonyl in ligand and transition-state recognition is a distinguishing feature between serine beta-lactamases and serine proteases, to which they are often compared. AmpC may use the interaction between the carbonyl of Ala318 and the carbonyl of the acylated enzyme to destabilize the ground-state intermediate, this destabilization energy might be relieved in the transition state by a hydroxyl hydrogen bond. The structure of the m-aminophenylboronic acid adduct also suggests several ways to improve the affinity of this class of inhibitor and points to the existence of several unusual binding-site-like features in the region of the AmpC catalytic site.

Crystal structures of CheY from Thermotoga maritima do not support conventional explanations for the structural basis of enhanced thermostability.

The crystal structure of CheY protein from Thermotoga maritima has been determined in four crystal forms with and without Mg++ bound, at up to 1.9 A resolution. Structural comparisons with CheY from Escherichia coli shows substantial similarity in their folds, with some concerted changes propagating away from the active site that suggest how phosphorylated CheY, a signal transduction protein in bacterial chemotaxis, is recognized by its targets. A highly conserved segment of the protein (the "y-turn loop," residues 55-61), previously suggested to be a rigid recognition determinant, is for the first time seen in two alternative conformations in the different crystal structures. Although CheY from Thermotoga has much higher thermal stability than its mesophilic counterparts, comparison of structural features previously proposed to enhance thermostability such as hydrogen bonds, ion pairs, compactness, and hydrophobic surface burial would not suggest it to be so.

alpha-Fluoro acid and alpha-fluoro amide analogs of acetyl-CoA as inhibitors of citrate synthase: effect of pKa matching on binding affinity and hydrogen bond length.

An alpha-fluoro acid analog and an alpha-fluoro amide analog of acetyl-CoA have been synthesized. The ternary complexes of these inhibitors with oxaloacetate and citrate synthase have been crystallized and their structures analyzed at 1.7 A resolution. The structures are similar to those reported for the corresponding non-fluorinated analogs (Usher et al., 1994), with all forming unusually short hydrogen bonds to Asp 375. The alpha-fluoro amide analog binds with an affinity 1.5-fold lower than that of a previously described amide analog lacking the alpha-fluoro group. The alpha-fluoro acid analog binds with a 50-fold decreased affinity relative to the corresponding unfluorinated analog. The binding affinities are consistent with increased strengths of hydrogen bonds to Asp 375 with closer matching of pKa values between hydrogen bond donors and acceptors. The results do not support any direct correlation between hydrogen bond strength and hydrogen bond length in enzyme-inhibitor complexes.

A very short hydrogen bond provides only moderate stabilization of an enzyme-inhibitor complex of citrate synthase.

Two extremely potent inhibitors of citrate synthase, carboxyl and primary amide analogues of acetyl coenzyme A, have been synthesized. The ternary complexes of these inhibitors with oxaloacetate and citrate synthase have been crystallized and their structures analyzed at 1.70- and 1.65-A resolution, respectively. The inhibitors have dissociation constants in the nanomolar range, with the carboxyl analogue binding more tightly (Ki = 1.6 nM at pH 6.0) than the amide analogue (28 nM), despite the unfavorable requirement for proton uptake by the former. The carboxyl group forms a shorter hydrogen bond with the catalytic Asp 375 (distance < 2.4 A) than does the amide group (distance approximately 2.5 A). Particularly with the carboxylate inhibitor, the very short hydrogen bond distances measured suggest a low barrier or short strong hydrogen bond. However, the binding constants differ by only a factor of 20 at pH 6.0, corresponding to an increase in binding energy for the carboxyl analogue on the enzyme of about 2 kcal/mol more than the amide analogue, much less than has been proposed for short strong hydrogen bonds based on gas phase measurements [> 20 kcal/mol (Gerlt & Gassman, 1993a,b)]. The inhibitor complexes support proposals that Asp 375 and His 274 work in concert to form an enolized form of acetyl-coenzyme A as the first step in the reaction.