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Human CA150, a transcriptional activator, binds to and is co-deposited with huntingtin during Huntington's disease. The second WW domain of CA150 is a three-stranded β-sheet that folds in vitro in microseconds and forms amyloid fibers under physiological conditions. We found from exhaustive alanine scanning studies that fibrillation of this WW domain begins from its denatured conformations, and we identified a subset of residues critical for fibril formation. We used high-resolution magic-angle-spinning NMR studies on site-specific isotopically labeled fibrils to identify abundant long-range interactions between side chains. The distribution of critical residues identified by the alanine scanning and NMR spectroscopy, along with the electron microscopy data, revealed the protofilament repeat unit: a 26-residue nonnative β-hairpin. The structure we report has similarities to the hairpin formed by the A β (1–40) protofilament, yet also contains closely packed side-chains in a “steric zipper” arrangement found in the cross-β spine formed from small peptides from the Sup35 prion protein. Fibrillation of unrelated amyloidogenic sequences shows the common feature of zippered repeat units that act as templates for fiber elongation.

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Conventional cooperative protein folding invokes discrete ensembles of native and denatured state structures in separate freeenergy wells. Unimodal noncooperative (''downhill'') folding, however, proposes an ensemble of states occupying a single free-energy well for proteins folding at >4 ؋ 10 4 s ؊1 at 298 K. It is difficult to falsify unimodal mechanisms for such fast folding proteins by standard equilibrium experiments because both cooperative and unimodal mechanisms can present the same timeaveraged structural, spectroscopic, and thermodynamic properties when the time scale used for observation is longer than for equilibration. However, kinetics can provide the necessary evidence. Chevron plots with strongly sloping linear refolding arms are very difficult to explain by downhill folding and are a signature for cooperative folding via a transition state ensemble. The folding kinetics of the peripheral subunit binding domain POB and its mutants fit to strongly sloping chevrons at observed rate constants of >6 ؋ 10 4 s ؊1 in denaturant solution, extrapolating to 2 ؋ 10 5 s ؊1 in water. Protein A, which folds at 10 5 s ؊1 at 298 K, also has a well-defined chevron. Single-molecule fluorescence energy transfer experiments on labeled Protein A in the presence of denaturant demonstrated directly bimodal distributions of native and denatured states.A currently controversial subject in protein folding is unimodal ''downhill'' versus classical cooperative folding. It is generally accepted that proteins fold on a free-energy landscape in which there are ensembles of states separated by free-energy barriers (1). Accordingly, there are cooperative transitions between those ensembles of states, as, for example, the native N and denatured D ensembles, which have a bimodal distribution of properties. However, when there is an extreme energetic bias toward the native state, the protein may fold ''downhill,'' without an energy barrier (1). This conventional (''chemical'') view of folding has been challenged by Muñoz and colleagues (2, 3), who claim that for a protein NapBBL, a truncated and naphthylalanine-labeled derivative of the BBL peripheral subunit binding domain (PSBD) from Escherichia coli, in particular, and for all proteins that fold faster than 40,000 s Ϫ1 at 298 K (4), the D and N states are not separated by an energy barrier, but slowly merge into each other with changing conditions; Muñoz and colleagues (2) call this mechanism ''downhill'' folding ( Fig. 1). We use the term noncooperative or unimodal (5) for this downhill folding. Various criteria derived from equilibrium experiments have been proposed to be signatures of unimodal folding (2, 6). Downhill folding is proposed to be important, in particular as a means for the PSBDs to adjust their sizes as ''molecular rheostats,'' and in general because it is suggested (2, 4, 7) that it opens up the exciting prospect of examining the whole pathway of folding from equilibrium spectroscopic observations.Until recently, the proteins that were studied folded slowly com...

Urinary tract infections (UTIs), predominantly caused by uropathogenic Escherichia coli (UPEC), belong to the most prevalent infectious diseases worldwide. The attachment of UPEC to host cells is mediated by FimH, a mannose-binding adhesin at the tip of bacterial type 1 pili. To date, UTIs are mainly treated with antibiotics, leading to the ubiquitous problem of increasing resistance against most of the currently available antimicrobials. Therefore, new treatment strategies are urgently needed. Here, we describe the development of an orally available FimH antagonist. Starting from the carboxylate substituted biphenyl α-d-mannoside 9, affinity and the relevant pharmacokinetic parameters (solubility, permeability, renal excretion) were substantially improved by a bioisosteric approach. With 3'-chloro-4'-(α-d-mannopyranosyloxy)biphenyl-4-carbonitrile (10j) a FimH antagonist with an optimal in vitro PK/PD profile was identified. Orally applied, 10j was effective in a mouse model of UTI by reducing the bacterial load in the bladder by about 1000-fold.