Summary MinD recruits MinE to the membrane leading to a coupled oscillation required for spatial regulation of the cytokinetic Z ring in E. coli. How these proteins interact, however, is not clear since the MinD binding regions of MinE are sequestered within a 6-stranded β-sheet and masked by N-terminal helices. Here, minE mutations are isolated that restore interaction to some MinD and MinE mutants. These mutations alter the MinE structure releasing the MinD binding regions and N-terminal helices that bind MinD and the membrane, respectively. Crystallization of MinD-MinE complexes reveals a 4-stranded β-sheet MinE dimer with the released β strands (MinD binding regions) converted to α-helices bound to MinD dimers. These results suggest a 6 stranded, β-sheet dimer of MinE ‘senses’ MinD and switches to a 4-stranded β-sheet dimer that binds MinD and contributes to membrane binding. Also, the results indicate how MinE persists at the MinD-membrane surface.

The induced fit and conformational selection/population shift models are two extreme cases of a continuum aimed at understanding the mechanism by which the final key-lock or active enzyme conformation is achieved upon formation of the correctly ligated enzyme. Structures of complexes representing the Michaelis and enolate intermediate complexes of the reaction catalyzed by phosphoenolpyruvate carboxykinase provide direct structural evidence for the encounter complex that is intrinsic to the induced fit model and not required by the conformational selection model. In addition, the structural data demonstrate that the conformational selection model is not sufficient to explain the correlation between dynamics and catalysis in phosphoenolpyruvate carboxykinase and other enzymes in which the transition between the uninduced and the induced conformations occludes the active site from the solvent. The structural data are consistent with a model in that the energy input from substrate association results in changes in the free energy landscape for the protein, allowing for structural transitions along an induced fit pathway.enzyme dynamics ͉ population shift ͉ phosphoenolpyruvate carboxykinase I t has been 50 years since the original presentation of the induced fit hypothesis by Daniel Koshland (1) that built upon Emil Fisher's key-lock principle (2) and introduced the idea that the inherent dynamic properties of enzymes play an essential role in the processes of molecular recognition and catalysis. Over the last decade, with the advent of new instrumentation and novel experimental approaches there has been a resurgence in investigations focused upon understanding the specific ways in that these dynamic properties influence the ability of enzymes to achieve enormous levels of selectivity and catalytic power (refs. 3-6 and references therein).Presently there are two primary models used to explain the mechanism by which an enzyme can move toward adopting the final key-lock state ( Fig. 1): (i) the induced fit model and (ii) the conformational selection/population shift model (7-14). As defined originally by Koshland (1), the induced fit model states that the achievement of the final key-lock state, that is, the precise orientation of catalytic groups and residues necessary for catalysis, occurs only after changes in protein structure that are induced by the binding of a substrate. This pathway is represented by the equilibrium constants K 1 and K 2 in Fig. 1. Intrinsic to this model is the ability of the enzyme to form an ''encounter complex'' † (Fig. 1B). In this complex the enzyme is liganded identically to the eventual catalytically competent key-lock state; however, the conformational change leading to the active key-lock state has not yet occurred. In contrast, the conformational selection model states that the enzyme exists in multiple conformational states, of which the substrate has a high affinity for the active or key-lock state. In this model the ligand is suggested to bind to this lowly populated high-energy ...

The isoform of pyruvate kinase from brain and muscle of mammals (M(1)-PYK) is allosterically inhibited by phenylalanine. Initial observations in this model allosteric system indicate that Ala binds competitively with Phe, but elicits a minimal allosteric response. Thus, the allosteric ligand of this system must have requirements for eliciting an allosteric response in addition to the requirements for binding. Phe analogues have been used to dissect what chemical properties of Phe are responsible for eliciting the allosteric response. We first demonstrate that the l-2-aminopropanaldehyde substructure of the amino acid ligand is primarily responsible for binding to M(1)-PYK. Since the allosteric response to Ala is minimal and linear addition of methyl groups beyond the beta-carbon increase the magnitude of the allosteric response, we conclude that moieties beyond the beta-carbon are primarily responsible for allostery. Instead of an all-or-none mechanism of allostery, these findings support the idea that the bulk of the hydrophobic side chain, but not the aromatic nature, is the primary determinant of the magnitude of the observed allosteric inhibition. The use of these results to direct structural studies has resulted in a 1.65 A structure of M(1)-PYK with Ala bound. The coordination of Ala in the allosteric amino acid binding site confirms the binding role of the l-2-aminopropanaldehyde substructure of the ligand. Collectively, this study confirms that a ligand can have chemical regions specific for eliciting the allosteric signal in addition to the chemical regions necessary for binding.

SummaryThe three Min proteins spatially regulate Z ring positioning in Escherichia coli and are dynamically associated with the membrane. MinD binds to vesicles in the presence of ATP and can recruit MinC or MinE. Biochemical and genetic evidence indicate the binding sites for these two proteins on MinD overlap. Here we solved the structure of a hydrolytic-deficient mutant of MinD truncated for the C-terminal amphipathic helix involved in binding to the membrane. The structure solved in the presence of ATP is a dimer and reveals the face of MinD abutting the membrane. Using a combination of random and extensive sitedirected mutagenesis additional residues important for MinE and MinC binding were identified. The location of these residues on the MinD structure confirms that the binding sites overlap and reveals that the binding sites are at the dimer interface and exposed to the cytosol. The location of the binding sites at the dimer interface offers a simple explanation for the ATP dependence of MinC and MinE binding to MinD.

This paper reports the first structure of a member of the Kex2/furin family of eukaryotic pro-protein processing proteases, which cleave sites consisting of pairs or clusters of basic residues. Reported is the 2.4 A resolution crystal structure of the two-domain protein ssKex2 in complex with an Ac-Ala-Lys-boroArg inhibitor (R = 20.9%, R(free) = 24.5%). The Kex2 proteolytic domain is similar in its global fold to the subtilisin-like superfamily of degradative proteases. Analysis of the complex provides a structural basis for the extreme selectivity of this enzyme family that has evolved from a nonspecific subtilisin-like ancestor. The P-domain of ssKex2 has a novel jelly roll like fold consisting of nine beta strands and may potentially be involved, along with the buried Ca(2+) ion, in creating the highly determined binding site for P(1) arginine.