The PpL-Fab complex shows the first interaction of a bacterial virulence factor with a Fab light chain outside the conventional combining site. Structural comparison with two other bacterial proteins interacting with the Fab heavy chain shows that PpL, structurally homologous to streptococcal SpG domains, shares with the latter a similar binding mode. These two bacterial surface proteins interact with their respective immunoglobulin regions through a similar beta zipper interaction.

Inositol monophosphatase is a key enzyme of the phosphatidylinositol signalling pathway and the putative target of the mood-stabilizing drug lithium. The crystal structure of bovine inositol monophosphatase has been determined at 1.4 Å resolution in complex with the physiological magnesium ion ligands. Three magnesium ions are octahedrally coordinated at the active site of each of the two subunits of the inositol monophosphatase dimer and a detailed three-metal mechanism is proposed. Ligands to the three metals include the side chains of Glu70, Asp90, Asp93 and Asp220, the backbone carbonyl group of Ile92 and several solvent molecules, including the proposed nucleophilic water molecule (W1) ligated by both Mg-1 and Mg-3. Modelling of the phosphate moiety of inositol monophosphate to superpose the axial phosphate O atoms onto three active-site water molecules orientates the phosphoester bond for in-line attack by the nucleophilic water which is activated by Thr95. Modelling of the pentacoordinate transition state suggests that the 6-OH group of the inositol moiety stabilizes the developing negative charge by hydrogen bonding to a phosphate O atom. Modelling of the post-reaction complex suggests a role for a second water molecule (W2) ligated by Mg-2 and Asp220 in protonating the departing inositolate. This second water molecule is absent in related structures in which lithium is bound at site 2, providing a rationale for enzyme inhibition by this simple monovalent cation. The higher resolution structural information on the active site of inositol monophosphatase will facilitate the design of substrate-based inhibitors and aid in the development of better therapeutic agents for bipolar disorder (manic depression).

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Protein misfolding plays a role in the pathogenesis of many diseases. ␣ 1 -Antitrypsin misfolding leads to the accumulation of long chain polymers within the hepatocyte, reducing its plasma concentration and predisposing the patient to emphysema and liver disease. In order to understand the misfolding process, it is necessary to examine the folding of ␣ 1 -antitrypsin through the different structures involved in this process. In this study we have used a novel technique in which unique cysteine residues were introduced at various positions into ␣ 1 -antitrypsin and fluorescently labeled with N,N-dimethyl-N-(iodoacetyl)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-ethylenediamine. The fluorescence properties of each protein were studied in the native state and as a function of guanidine hydrochloride-mediated unfolding. The studies found that ␣ 1 -antitrypsin unfolded through a series of intermediate structures. From the position of the fluorescence probes, the fluorescence quenching data, and the molecular modeling, we show that unfolding of ␣ 1 -antitrypsin occurs via disruption of the A and C ␤-sheets followed by the B ␤-sheet. The implications of these data on both ␣ 1 -antitrypsin function and polymerization are discussed.Protein folding is the process by which the primary sequence is translated into tertiary structure. Modification of this sequence via in vivo mutation or in vitro mutagenesis often alters the ability of the protein to fold correctly. Misfolding of proteins during their synthesis within the cell can lead to loss of protein function (1), an example being ␣ 1 -antitrypsin (␣ 1 -AT) 1 deficiency (2-4). Many well characterized ␣ 1 -AT variants are associated with an ␣ 1 -AT plasma deficiency; in these cases, the variants aggregate at their site of synthesis within the liver cell (5, 6). This aggregation is via an ordered polymerization process that is initiated by protein misfolding. In the case of the ␣ 1 -AT Z mutation (Glu-342 3 Lys), the rate of protein folding is much slower than in the native state, leading to the accumulation of an intermediate, which then polymerizes (4). It has recently been shown that the heat-induced polymerization of ␣ 1 -AT also involves the formation of an unfolding intermediate (7). The actual process of polymerization has been well studied, and two mechanisms have been proposed: the loop-A-sheet and loop-C-sheet mechanisms (8 -11). Both processes involve the insertion of the reactive center loop residues of the donating ␣ 1 -AT molecule into either the A ␤-sheet (loop-A-sheet) or the C ␤-sheet (loop-C-sheet) of the acceptor molecule (11). There is evidence for the occurrence of both of these mechanisms; indeed, in vitro ␣ 1 -AT has been shown to undergo both depending upon the buffer used (12-14).␣ 1 -AT is a member of the serine proteinase inhibitor (serpin) superfamily and is composed of 394 amino acid residues arranged into three ␤-sheets (A, B, and C) and nine ␣-helices (A-I). X-ray crystallographic and biochemical data have shown that ␣ 1 -AT can adopt a number of ...

Analysis of the crystal structures of the free enzyme and of the binary complexes with NAD(+) and glycerol show that the active site of GlyDH lies in the cleft between the enzyme's two domains, with the catalytic zinc ion playing a role in stabilizing an alkoxide intermediate. In addition, the specificity of this enzyme for a range of diols can be understood, as both hydroxyls of the glycerol form ligands to the enzyme-bound Zn(2+) ion at the active site. The structure further reveals a previously unsuspected similarity to dehydroquinate synthase, an enzyme whose more complex chemistry shares a common chemical step with that catalyzed by glycerol dehydrogenase, providing a striking example of divergent evolution. Finally, the structure suggests that the NAD(+) binding domain of GlyDH may be related to that of the classical Rossmann fold by switching the sequence order of the two mononucleotide binding folds that make up this domain.