Claudia Muhle-Goll

Hydrophobic Matching Controls the Tilt and Stability of the Dimeric Platelet-derived Growth Factor Receptor (PDGFR) β Transmembrane Segment

Background: Dimerization regulates activation of PDGF receptor in signal transduction. Results: The transmembrane segment of PDGFR forms a left-handed helical dimer, which becomes more tilted and less stable in model membranes with decreasing lipid acyl chain lengths. Conclusion: The membrane thickness controls the ability of the transmembrane segments to dimerize. Significance: Receptor dimerization and activation in vivo may require relocation to thick lipid rafts. The platelet-derived growth factor receptor β is a member of the cell surface receptor tyrosine kinase family and dimerizes upon activation. We determined the structure of the transmembrane segment in dodecylphosphocholine micelles by liquid-state NMR and found that it forms a stable left-handed helical dimer. Solid-state NMR and oriented circular dichroism were used to measure the tilt angle of the helical segments in macroscopically aligned model membranes with different acyl chain lengths. Both methods showed that decreasing bilayer thickness (DEPC-POPC-DMPC) led to an increase in the helix tilt angle from 10° to 30° with respect to the bilayer normal. At the same time, reconstitution of the comparatively long hydrophobic segment became less effective, eventually resulting in complete protein aggregation in the short-chain lipid DLPC. Unrestrained molecular dynamics simulations of the dimer were carried out in explicit lipid bilayers (DEPC, POPC, DMPC, sphingomyelin), confirming the observed dependence of the helix tilt angle on bilayer thickness. Notably, molecular dynamics revealed that the left-handed dimer gets tilted en bloc, whereas conformational transitions to alternative (e.g. right-handed dimeric) states were not supported. The experimental data along with the simulation results demonstrate a pronounced interplay between the platelet-directed growth factor receptor β transmembrane segment and the bilayer thickness. The effect of hydrophobic mismatch might play a key role in the redistribution and activation of the receptor within different lipid microdomains of the plasma membrane in vivo.

Short cationic antimicrobial peptides interact with ATP.

ABSTRACT The mode of action of short, nonhelical antimicrobial peptides is still not well understood. Here we show that these peptides interact with ATP and directly inhibit the actions of certain ATP-dependent enzymes, such as firefly luciferase, DnaK, and DNA polymerase. α-Helical and planar or circular antimicrobial peptides did not show such interaction with ATP.

Analysis of the specific interactions between the lectin domain of malectin and diglucosides

The endoplasmic reticulum malectin is a highly conserved protein in the animal kingdom that has no counterpart so far in lower organisms. We recently determined the structure of its conserved domain and found a highly selective binding to Glc(2)Man(9)GlcNAc(2), an intermediate of N-glycosylation. In our quest for putative ligands during the initial characterization of the protein, we noticed that the malectin domain is highly specific for diglucosides but quite tolerant towards the linkage of the glucosidic bond. To understand the molecular requirements for the observed promiscuity of the malectin domain, here we analyze the binding to a range of diglucosides through comparison of the protein chemical shift perturbation patterns and the saturation transfer difference spectra of the ligands including two maltose-mimicking drugs. A comparison of the maltose-bound structure of the malectin domain with the complex of the native ligand nigerose reveals why malectin is able to tolerate such a diversity of ligands.

Coregulator Control of Androgen Receptor Action by a Novel Nuclear Receptor-binding Motif

Background: The interaction surface of coactivators and the androgen receptor (AR) is an important target for prostate cancer therapeutics. Results: A new interface formed by binding of the sequence (GARRPR) and the allosteric pocket (BF-3) of the AR has been identified. Conclusion: GARRPR binding modulates AR activity. Significance: The GARRPR/BF-3 interaction is a novel regulatory hub for AR activity. The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR.

ON THE SIGNIFICANCE OF TOC-GTPASE HOMODIMERS *

Precursor protein translocation across the outer chloroplast membrane depends on the action of the Toc complex, containing GTPases as recognizing receptor components. The G domains of the GTPases are known to dimerize. In the dimeric conformation an arginine contacts the phosphate moieties of bound nucleotide in trans. Kinetic studies suggested that the arginine in itself does not act as an arginine finger of a reciprocal GTPase-activating protein (GAP). Here we investigate the specific function of the residue in two GTPase homologues. Arginine to alanine replacement variants have significantly reduced affinities for dimerization compared with wild-type GTPases. The amino acid exchange does not impact on the overall fold and nucleotide binding, as seen in the monomeric x-ray crystallographic structure of the Arabidopsis Toc33 arginine-alanine replacement variant at 2.0Å. We probed the catalytic center with the transition state analogue GDP/AlFx using NMR and analytical ultracentrifugation. AlFx binding depends on the arginine, suggesting the residue can play a role in catalysis despite the non-GAP nature of the homodimer. Two non-exclusive functional models are discussed: 1) the coGAP hypothesis, in which an additional factor activates the GTPase in homodimeric form; and 2) the switch hypothesis, in which a protein, presumably the large Toc159 GTPase, exchanges with one of the homodimeric subunits, leading to activation.

Autoinduced Catalysis and Inverse Equilibrium Isotope Effect in the Frustrated Lewis Pair Catalyzed Hydrogenation of Imines

The frustrated Lewis pair (FLP)-catalyzed hydrogenation and deuteration of N-benzylidene-tert-butylamine (2) was kinetically investigated by using the three boranes B(C6F5)3 (1), B(2,4,6-F3-C6H2)3 (4), and B(2,6-F2-C6H3)3 (5) and the free activation energies for the H2 activation by FLP were determined. Reactions catalyzed by the weaker Lewis acids 4 and 5 displayed autoinductive catalysis arising from a higher free activation energy (2 kcal mol(-1)) for the H2 activation by the imine compared to the amine. Surprisingly, the imine reduction using D2 proceeded with higher rates. This phenomenon is unprecedented for FLP and resulted from a primary inverse equilibrium isotope effect.

Influence of Freezing and Storage Procedure on Human Urine Samples in NMR-Based Metabolomics

It is consensus in the metabolomics community that standardized protocols should be followed for sample handling, storage and analysis, as it is of utmost importance to maintain constant measurement conditions to identify subtle biological differences. The aim of this work, therefore, was to systematically investigate the influence of freezing procedures and storage temperatures and their effect on NMR spectra as a potentially disturbing aspect for NMR-based metabolomics studies. Urine samples were collected from two healthy volunteers, centrifuged and divided into aliquots. Urine aliquots were frozen either at −20 °C, on dry ice, at −80 °C or in liquid nitrogen and then stored at −20 °C, −80 °C or in liquid nitrogen vapor phase for 1–5 weeks before NMR analysis. Results show spectral changes depending on the freezing procedure, with samples frozen on dry ice showing the largest deviations. The effect was found to be based on pH differences, which were caused by variations in CO2 concentrations introduced by the freezing procedure. Thus, we recommend that urine samples should be frozen at −20 °C and transferred to lower storage temperatures within one week and that freezing procedures should be part of the publication protocol.

Structural basis for a distinct catalytic mechanism in Trypanosoma brucei tryparedoxin peroxidase.

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three cysteine homologues (Px I-III) of classical selenocysteine-containing glutathione peroxidases. The enzymes obtain their reducing equivalents from the unique trypanothione (bis(glutathionyl)spermidine)/tryparedoxin system. During catalysis, these tryparedoxin peroxidases cycle between an oxidized form with an intramolecular disulfide bond between Cys47 and Cys95 and the reduced peroxidase with both residues in the thiol state. Here we report on the three-dimensional structures of oxidized T. brucei Px III at 1.4Å resolution obtained by x-ray crystallography and of both the oxidized and the reduced protein determined by NMR spectroscopy. Px III is a monomeric protein unlike the homologous poplar thioredoxin peroxidase (TxP). The structures of oxidized and reduced Px III are essentially identical in contrast to what was recently found for TxP. In Px III, Cys47, Gln82, and Trp137 do not form the catalytic triad observed in the selenoenzymes, and related proteins and the latter two residues are unaffected by the redox state of the protein. The mutational analysis of three conserved lysine residues in the vicinity of the catalytic cysteines revealed that exchange of Lys107 against glutamate abrogates the reduction of hydrogen peroxide, whereas Lys97 and Lys99 play a crucial role in the interaction with tryparedoxin.

Structural Role of the Conserved Cysteines in the Dimerization of the Viral Transmembrane Oncoprotein E5

The E5 oncoprotein is the major transforming protein of bovine papillomavirus type 1. This 44-residue transmembrane protein can interact with the platelet-derived growth factor receptor β, leading to ligand-independent activation and cell transformation. For productive interaction, E5 needs to dimerize via a C-terminal pair of cysteines, though a recent study suggested that its truncated transmembrane segment can dimerize on its own. To analyze the structure of the full protein in a membrane environment and elucidate the role of the Cys-Ser-Cys motif, we produced recombinantly the wild-type protein and four cysteine mutants. Comparison by circular dichroism in detergent micelles and lipid vesicular dispersion and by NMR in trifluoroethanol demonstrates that the absence of one or both cysteines does not influence the highly α-helical secondary structure, nor does it impair the ability of E5 to dimerize, observations that are further supported by sodium dodecylsulfate polyacrylamide gel electrophoresis. We also observed assemblies of higher order. Oriented circular dichroism in lipid bilayers shows that E5 is aligned as a transmembrane helix with a slight tilt angle, and that this membrane alignment is also independent of any cysteines. We conclude that the Cys-containing motif represents a disordered region of the protein that serves as an extra covalent connection for stabilization.

Functional and structural characterization of a novel member of the natriuretic family of peptides from the venom of Pseudocerastes persicus.

A novel peptide, PNP ( seudocerastes persicus atriuretic eptide), was isolated from the venom of the Iranian viper P. persicus. Amino acid sequencing revealed that the 37‐residue peptide belongs to the family of natriuretic peptides. The physiological effects of intra‐venously PNP infused into anesthetized rats on urine flow, sodium excretion and blood pressure were comparable to those of atrial natriuretic peptide (ANP). In PC12 cells that were treated with either PNP, ANP, or C‐type natriuretic peptide, PNP induced a similar cGMP response as ANP. Since PC12 cells only express the natriuretic peptide receptor (NPR)‐A receptor we conclude that PNP binds to the NPR‐A receptor. The solution conformation of PNP was characterized using 1H nuclear magnetic resonance spectroscopy and indicates a high degree of conformational flexibility.