Structural characterization suggests models for monomeric and dimeric forms of full-length ezrin

Ezrin is a member of the ERM (ezrin–radixin–moesin) family of proteins that have been conserved through metazoan evolution. These proteins have dormant and active forms, where the latter links the actin cytoskeleton to membranes. ERM proteins have three domains: an N-terminal FERM [band Four-point-one (4.1) ERM] domain comprising three subdomains (F1, F2, and F3); a helical domain; and a C-terminal actin-binding domain. In the dormant form, FERM and C-terminal domains form a stable complex. We have determined crystal structures of the active FERM domain and the dormant FERM:C-terminal domain complex of human ezrin. We observe a bistable array of phenylalanine residues in the core of subdomain F3 that is mobile in the active form and locked in the dormant form. As subdomain F3 is pivotal in binding membrane proteins and phospholipids, these transitions may facilitate activation and signaling. Full-length ezrin forms stable monomers and dimers. We used small-angle X-ray scattering to determine the solution structures of these species. As expected, the monomer shows a globular domain with a protruding helical coiled coil. The dimer shows an elongated dumbbell structure that is twice as long as the monomer. By aligning ERM sequences spanning metazoan evolution, we show that the central helical region is conserved, preserving the heptad repeat. Using this, we have built a dimer model where each monomer forms half of an elongated antiparallel coiled coil with domain-swapped FERM:C-terminal domain complexes at each end. The model suggests that ERM dimers may bind to actin in a parallel fashion

Stabilising Peptoid Helices Using Non‐Chiral Fluoroalkyl Monomers

Stability towards protease degradation combined with modular synthesis has made peptoids of considerable interest in the fields of chemical biology, medicine, and biomaterials. Given their tertiary amide backbone, peptoids lack the capacity to hydrogen‐bond, and as such, controlling secondary structure can be challenging. The incorporation of bulky, charged, or chiral aromatic monomers can be used to control conformation but such building blocks limit applications in many areas. Through NMR and X‐ray analysis we demonstrate that non‐chiral neutral fluoroalkyl monomers can be used to influence the Kcis/trans equilibria of peptoid amide bonds in model systems. The cis‐isomer preference displayed is highly unprecedented given that neither chirality nor charge is used to control the peptoid amide conformation. The application of our fluoroalkyl monomers in the design of a series of linear peptoid oligomers that exhibit stable helical structures is also reported

Crystal Structure of Serratia fonticola Sfh-I: Activation of the Nucleophile in Mono-Zinc Metallo-beta-Lactamases

Metallo-β-lactamases (MBLs) or class B β-lactamases are zinc-dependent enzymes capable of inactivating almost all classes of β-lactam antibiotics. To date, no MBL inhibitors are available for clinical use. Of the three MBL subclasses, B2 enzymes, unlike those from subclasses B1 and B3, are fully active with one zinc ion bound and possess a narrow spectrum of activity, hydrolyzing carbapenem substrates almost exclusively. These remain the least studied MBLs. Sfh-I, originally identified from the aquatic bacterium Serratia fonticola UTAD54, is a divergent member of this group. Previous B2 MBL structures, available only for the CphA enzyme from Aeromonas hydrophila, all contain small molecules bound in their active sites. In consequence, the mechanism by which these enzymes activate the water nucleophile required for β-lactam hydrolysis remains to be unambiguously established. Here we report crystal structures of Sfh-I as a complex with glycerol and in the unliganded form, revealing for the first time the disposition of water molecules in the B2 MBL active site. Our data indicate that the hydrolytic water molecule is activated by His118 rather than by Asp120 and/or zinc. Consistent with this proposal, we show that the environment of His118 in B2 MBLs is distinct from that of the B1 and B3 enzymes, where this residue acts as a zinc ligand, and offer a structure-based mechanism for β-lactam hydrolysis by these enzymes.This work was supported by the Center for Environmental and Marine Studies, University of Aveiro, and by the Fundação para a Ciência e a Tecnologia, Portugal (PhD grant BD/30490/2006 to F.F.). We thank the staff of Diamond Light Source beamline IO4 for their assistance with data collection, M. N. Lisa and Prof. A. J. Vila (University of Rosario, Argentina) for sharing results in advance of publication, and Dr. Nick Burton, Dr. Rebecca Conners, Dr. Martin Challand, Natalia Lisa, and Alejandro Vila for their critical reading of the manuscript.publishe

Structural insights into quinolone antibiotic resistance mediated by pentapeptide repeat proteins: conserved surface loops direct the activity of a Qnr protein from a Gram-negative bacterium.

Quinolones inhibit bacterial type II DNA topoisomerases (e.g. DNA gyrase) and are among the most important antibiotics in current use. However, their efficacy is now being threatened by various plasmid-mediated resistance determinants. Of these, the pentapeptide repeat-containing (PRP) Qnr proteins are believed to act as DNA mimics and are particularly prevalent in Gram-negative bacteria. Predicted Qnr-like proteins are also present in numerous environmental bacteria. Here, we demonstrate that one such, Aeromonas hydrophila AhQnr, is soluble, stable, and relieves quinolone inhibition of Escherichia coli DNA gyrase, thus providing an appropriate model system for Gram-negative Qnr proteins. The AhQnr crystal structure, the first for any Gram-negative Qnr, reveals two prominent loops (1 and 2) that project from the PRP structure. Deletion mutagenesis demonstrates that both contribute to protection of E. coli DNA gyrase from quinolones. Sequence comparisons indicate that these are likely to be present across the full range of Gram-negative Qnr proteins. On this basis we present a model for the AhQnr:DNA gyrase interaction where loop1 interacts with the gyrase A ‘tower’ and loop2 with the gyrase B TOPRIM domains. We propose this to be a general mechanism directing the interactions of Qnr proteins with DNA gyrase in Gram-negative bacteria

Log D versus HPLC derived hydrophobicity: The development of predictive tools to aid in the rational design of bioactive peptoids

Hydrophobicity has proven to be an extremely useful parameter in small molecule drug discovery programmes given that it can be used as a predictive tool to enable rational design. For larger molecules, including peptoids, where folding is possible, the situation is more complicated and the average hydrophobicity (as determined by RP-HPLC retention time) may not always provide an effective predictive tool for rational design. Herein, we report the first ever application of partitioning experiments to determine the log D values for a series of peptoids. By comparing log D and average hydrophobicities we highlight the potential advantage of employing the former as a predictive tool in the rational design of biologically active peptoids. This article is protected by copyright

Time-dependent motor properties of multipedal molecular spiders

Molecular spiders are synthetic biomolecular walkers that use the asymmetry resulting from cleavage of their tracks to bias the direction of their stepping motion. Using Monte Carlo simulations that implement the Gillespie algorithm, we investigate the dependence of the biased motion of molecular spiders, along with binding time and processivity, on tunable experimental parameters, such as number of legs, span between the legs, and unbinding rate of a leg from a substrate site. We find that an increase in the number of legs increases the spiders' processivity and binding time but not their mean velocity. However, we can increase the mean velocity of spiders with simultaneous tuning of the span and the unbinding rate of a spider leg from a substrate site. To study the efficiency of molecular spiders, we introduce a time-dependent expression for the thermodynamic efficiency of a molecular motor, allowing us to account for the behavior of spider populations as a function of time. Based on this definition, we find that spiders exhibit transient motor function over time scales of many hours and have a maximum efficiency on the order of 1%, weak compared to other types of molecular motors

Biochemical Characterization of Sfh-I, a Subclass B2 Metallo-beta-Lactamase from Serratia fonticola UTAD54

The subclass B2 metallo-β-lactamase (MBL) Sfh-I from Serratia fonticola UTAD54 was cloned and overexpressed in Escherichia coli. The recombinant protein binds one equivalent of zinc, as shown by mass spectrometry, and preferentially hydrolyzes carbapenem substrates. However, compared to other B2 MBLs, Sfh-I also shows limited hydrolytic activity against some additional substrates and is not inhibited by a second equivalent of zinc. These data confirm Sfh-I to be a subclass B2 metallo-β-lactamase with some distinctive properties

Motor properties from persistence: a linear molecular walker lacking spatial and temporal asymmetry

The stepping direction of linear molecular motors is usually defined by a spatial asymmetry of the motor, its track, or both. Here we present a model for a molecular walker that undergoes biased directional motion along a symmetric track in the presence of a temporally symmetric chemical cycle. Instead of using asymmetry, directionality is achieved by persistence. At small load force the walker can take on average thousands of steps in a given direction until it stochastically reverses direction. We discuss a specific experimental implementation of a synthetic motor based on this design and find, using Langevin and Monte Carlo simulations, that a realistic walker can work against load forces on the order of picoNewtons with an efficiency of ~18%, comparable to that of kinesin. In principle, the walker can be turned into a permanent motor by externally monitoring the walker's momentary direction of motion, and using feedback to adjust the direction of a load force. We calculate the thermodynamic cost of using feedback to enhance motor performance in terms of the Shannon entropy, and find that it reduces the efficiency of a realistic motor only marginally. We discuss the implications for natural protein motor performance in the context of the strong performance of this design based only on a thermal ratchet