Oliver Zerbe

Structure–Activity Studies in a Family of β-Hairpin Protein Epitope Mimetic Inhibitors of the p53–HDM2 Protein–Protein Interaction

Inhibitors of the interaction between the p53 tumor‐suppressor protein and its natural human inhibitor HDM2 are attractive as potential anticancer agents. In earlier work we explored designing β‐hairpin peptidomimetics of the α‐helical epitope on p53 that would bind tightly to the p53‐binding site on HDM2. The β‐hairpin is used as a scaffold to display energetically hot residues in an optimal array for interaction with HDM2. The initial lead β‐hairpin mimetic, with a weak inhibitory activity (IC50=125 μM), was optimized to afford cyclo‐(L‐Pro‐Phe‐Glu‐6ClTrp‐Leu‐Asp‐Trp‐Glu‐Phe‐D‐Pro) (where 6ClTrp=L‐6‐chlorotryptophan), which has an affinity almost 1000 times higher (IC50=140 nM). In this work, insights into the origins of this affinity maturation based on structure–activity studies and an X‐ray crystal structure of the inhibitor/HDM2(residues 17–125) complex at 1.4 Å resolution are described. The crystal structure confirms the β‐hairpin conformation of the bound ligand, and also reveals that a significant component of the affinity increase arises through new aromatic/aromatic stacking interactions between side chains around the hairpin and groups on the surface of HDM2.

Designed Armadillo Repeat Proteins as General Peptide-Binding Scaffolds: Consensus Design and Computational Optimization of the Hydrophobic Core

Armadillo repeat proteins are abundant eukaryotic proteins involved in several cellular processes, including signaling, transport, and cytoskeletal regulation. They are characterized by an armadillo domain, composed of tandem armadillo repeats of approximately 42 amino acids, which mediates interactions with peptides or parts of proteins in extended conformation. The conserved binding mode of the peptide in extended form, observed for different targets, makes armadillo repeat proteins attractive candidates for the generation of modular peptide-binding scaffolds. Taking advantage of the large number of repeat sequences available, a consensus-based approach combined with a force field-based optimization of the hydrophobic core was used to derive soluble, highly expressed, stable, monomeric designed proteins with improved characteristics compared to natural armadillo proteins. These sequences constitute the starting point for the generation of designed armadillo repeat protein libraries for the selection of peptide binders, exploiting their modular structure and their conserved binding mode.

The βE-Domain of Wheat Ec-1 Metallothionein: A Metal-Binding Domain with a Distinctive Structure

Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent metal ions such as Zn(II), Cu(I), and Cd(II) that are involved in metal ion homeostasis and detoxification, as well as protection against reactive oxygen species. Here we show the NMR solution structure of the beta(E)-domain of the early cysteine-labeled protein (E(c)-1) from wheat (beta(E)-E(c)-1), which represents the first three-dimensional structure of a plant MT. The beta(E)-domain comprises the 51 C-terminal residues of E(c)-1 and exhibits a distinctive unprecedented structure with two separate metal-binding centers, a mononuclear Zn(II) binding site constituted by two cysteine and two highly conserved histidine residues as found in certain zinc-finger motifs, and a cluster formed by three Zn(II) ions coordinated by nine Cys residues that resembles the cluster in the beta-domain of vertebrate MTs. Cys-metal ion connectivities were determined by exhaustive structure calculations for all 7560 possible configurations of the three-metal cluster. Backbone dynamics investigated by (15)N relaxation experiments support the results of the structure determination in that beta(E)-E(c)-1 is a rigidly folded polypeptide. To further investigate the influence of metal ion binding on the stability of the structure, we replaced Zn(II) with Cd(II) ions and examined the effects of metal ion release on incubation with a metal ion chelator.

In vitro and in vivo evaluation of a somatostatin analogue released from PLGA microspheres.

The purpose of this study was to design poly(lactide-co-glycolide) (PLGA) microspheres for the continuous delivery of the somatostatin analogue, vapreotide, over 2-4 weeks. The microspheres were produced by spray-drying and the desired characteristics, i.e. high encapsulation efficiency and controlled release over 2-4 weeks, achieved through optimizing the type of polymer, processing solvent, and co-encapsulated additive. The in vitro release was tested in fetal bovine serum preserved with 0.02% of thiomersal. Furthermore, formulations were injected intramuscularly into rats to obtain pharmacokinetic profiles. Encapsulation efficiency was between 34 and 91%, depending on the particular formulation. The initial peptide release (within 6 h) was lowest, i.e. <20%, when acetic acid was used as processing solvent and highest, i.e. 57%, with dichloromethane. The various co-encapsulated additives generally lowered the encapsulation efficiency by 15-30%. The best formulation in terms of low burst and effective drug serum levels (>1 ng/ml) over 21-28 days in rats was the one made with end-group uncapped PLGA 50:50, the solvent acetic acid and the additive polyethyleneglycol. In conclusion, the optimization of formulation parameters allowed us to produce vapreotide-loaded PLGA microspheres of suitable characteristics for therapeutic use.

Structural and biochemical studies on procaspase-8: new insights on initiator caspase activation.

Caspases are proteases with an active-site cysteine and aspartate specificity in their substrates. They are involved in apoptotic cell death and inflammation, and dysfunction of these enzymes is directly linked to a variety of diseases. Caspase-8 initiates an apoptotic pathway triggered by external stimuli. It was previously characterized in its active inhibitor bound state by crystallography. Here we present the solution structure of the monomeric unprocessed catalytic domain of the caspase-8 zymogen, procaspase-8, showing for the first time the position of the linker and flexibility of the active site forming loops. Biophysical studies of carefully designed mutants allowed disentangling dimerization and processing, and we could demonstrate lack of activity of monomeric uncleaved procaspase-8 and of a processed but dimerization-incompetent mutant. The data provide experimental support in so-far unprecedented detail, and reveal why caspase-8 (and most likely other initiator caspases) needs the dimerization platform during activation.

Engineering of metallothionein-3 neuroinhibitory activity into the inactive isoform metallothionein-1.

The third isoform of mammalian metallothioneins (MT-3), mainly expressed in brain and down-regulated in Alzheimers disease, exhibits neuroinhibitory activity in vitro and a highly flexible structure that distinguishes it from the widely expressed MT-1/-2 isoforms. Previously, we showed that two conserved prolyl residues of MT-3 are crucial for both the bioactivity and cluster dynamics of this isoform. We have now used genetic engineering to introduce these residues into mouse MT-1. The S6P,S8P MT-1 mutant is inactive in neuronal survival assays. However, the additional introduction of the unique Thr5 insert of MT-3 resulted in a bioactive MT-1 form. Temperature-dependent and saturation transfer113Cd NMR experiments performed on the113Cd-reconstituted wild-type and mutant Cd7-MT-1 forms revealed that the gain of MT-3-like neuronal inhibitory activity is paralleled by an increase in conformational flexibility and intersite metal exchange in the N-terminal Cd3-thiolate cluster. The observed correlation suggests that structure/cluster dynamics are critical for the biological activity of MT-3. We propose that the interplay between the specific Pro-induced conformational requirements and those of the metal-thiolate bonds gives rise to an alternate and highly fluctuating cluster ensemble kinetically trapped by the presence of the5TCPCP9 motif. The functional significance of such heterogeneous cluster ensemble is discussed.

Interactions of lipopolysaccharide and polymyxin studied By NMR spectroscopy

In the light of occurrence of bacterial strains with multiple resistances against most antibiotics, antimicrobial peptides that interact with the outer layer of Gram-negative bacteria, such as polymyxin (PMX), have recently received increased attention. Here we present a study of the interactions of PMX-B, -E, and -M with lipopolysaccharide (LPS) from a deep rough mutant strain of Escherichia coli. A method for efficient purification of biosynthetically produced LPS using reversed-phase high-performance liquid chromatography in combination with ternary solvent mixtures was developed. LPS was incorporated into a membrane model, dodecylphosphocholine micelles, and its interaction with polymyxins was studied by heteronuclear NMR spectroscopy. Data from chemical shift mapping using isotope-labeled LPS or labeled polymyxin, as well as from isotope-filtered nuclear Overhauser effect spectroscopy experiments, reveal the mode of interaction of LPS with polymyxins. Using molecular dynamics calculations the complex of LPS with PMX-B in the presence of dodecylphosphocholine micelles was modeled using restraints derived from chemical shift mapping data and intermolecular nuclear Overhauser effects. In the modeled complex the macrocycle of PMX is centered around the phosphate group at GlcN-B, and additional contacts from polar side chains are formed to GlcN-A and Kdo-C, whereas hydrophobic side chains penetrate the acyl-chain region.

Kinetic response of a photoperturbed allosteric protein

By covalently linking an azobenzene photoswitch across the binding groove of a PDZ domain, a conformational transition, similar to the one occurring upon ligand binding to the unmodified domain, can be initiated on a picosecond timescale by a laser pulse. The protein structures have been characterized in the two photoswitch states through NMR spectroscopy and the transition between them through ultrafast IR spectroscopy and molecular dynamics simulations. The binding groove opens on a 100-ns timescale in a highly nonexponential manner, and the molecular dynamics simulations suggest that the process is governed by the rearrangement of the water network on the protein surface. We propose this rearrangement of the water network to be another possible mechanism of allostery.

Prolyl Isomerase PIN1 Regulates DNA Double-Strand Break Repair by Counteracting DNA End Resection

The regulation of DNA double-strand break (DSB) repair by phosphorylation-dependent signaling pathways is crucial for the maintenance of genome stability; however, remarkably little is known about the molecular mechanisms by which phosphorylation controls DSB repair. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, interacts with key DSB repair factors and affects the relative contributions of homologous recombination (HR) and nonhomologous end-joining (NHEJ) to DSB repair. We find that PIN1-deficient cells display reduced NHEJ due to increased DNA end resection, whereas resection and HR are compromised in PIN1-overexpressing cells. Moreover, we identify CtIP as a substrate of PIN1 and show that DSBs become hyperresected in cells expressing a CtIP mutant refractory to PIN1 recognition. Mechanistically, we provide evidence that PIN1 impinges on CtIP stability by promoting its ubiquitylation and subsequent proteasomal degradation. Collectively, these data uncover PIN1-mediated isomerization as a regulatory mechanism coordinating DSB repair.

Macrocyclic hairpin mimetics of the cationic antimicrobial peptide protegrin I: a new family of broad-spectrum antibiotics.

The problems associated with increasing antibiotic resistance have stimulated great interest in newly discovered families of naturally occurring cationic antimicrobial peptides. These include protegrin, tachyplesin, and RTD‐1, which adopt β‐hairpin‐like structures. We report here an approach to novel peptidomimetics based on these natural products. The mimetics were designed by transplanting the cationic and hydrophobic residues onto a β‐hairpin‐inducing template, either a D‐Pro‐L‐Pro dipeptide or a xanthene derivative. The mimetics have good antimicrobial activity against Gram‐positive and Gram‐negative bacteria (minimal inhibitory concentration≈6–25 μg mL−1). Analogues with improved selectivity for microbial rather than red blood cells (1 % hemolysis at 100 μg mL−1) were identified from a small library prepared by parallel synthesis. Thus, it is possible to separate the antimicrobial and hemolytic activities in this class of mimetics. NMR studies on one mimetic revealed a largely unordered structure in water, but a transition to a regular β‐hairpin backbone conformation in the presence of dodecylphosphocholine micelles. This family of mimetics may provide a starting point for the optimization of antimicrobial agents of potential clinical value in the fight against multiple‐drug‐resistant microorganisms.