NMR Spectroscopy of supramolecular chemistry on protein surfaces.

As one of the few analytical methods that offer atomic resolution, NMR spectroscopy is a valuable tool to study the interaction of proteins with their interaction partners, both biomolecules and synthetic ligands. In recent years, the focus in chemistry has kept expanding from targeting small binding pockets in proteins to recognizing patches on protein surfaces, mostly via supramolecular chemistry, with the goal to modulate protein-protein interactions. Here we present NMR methods that have been applied to characterize these molecular interactions and discuss the challenges of this endeavor.

The other side of the corona: nanoparticles inhibit the protease taspase1 in a size-dependent manner.

When nanoparticles enter a physiological environment, they rapidly adsorb biomolecules, in particular cellular proteins. This biological coating, the so-called nanoparticle protein corona, undoubtedly affects the biological identity and potential cytotoxicity of the nanomaterial. To elucidate a possible impact on the adsorbed biomolecules, we focused on an important group of players in cellular homeostasis, namely proteolytic enzymes. We could demonstrate that amorphous silica nanoparticles are not only able to bind to the oncologically relevant threonine protease Taspase1 as revealed by microscale thermophoresis and fluorescence anisotropy measurements, but moreover inhibit its proteolytic activity in a non-competitive manner. As revealed by temperature-dependent unfolding and CD spectroscopy, binding did not alter the stability of Taspase1 or its secondary structure. Noteworthy, inhibition of protein function seems not a general feature of nanoparticles, as several control enzymes were not affected in their proteolytic activity. Our data suggests that nanoparticles bind Taspase1 as an αß-dimer in a single layer without conformational change, resulting in noncompetitive inhibition that is either allostery-like or occludes the active site. Nanoparticle-based inhibition of Taspase1 could be also achieved in cell lysates and in live cells as shown by the use of a protease-specific cellular cleavage biosensor. Collectively, we could demonstrate that nanoparticles could not only bind but also selectively inhibit cellular enzymes, which might explain observed cytotoxicity but might serve as a starting point for the development of nanoparticle-based inhibitors as therapeutics.

Structural Analysis of the 42 kDa Parvulin of Trypanosoma brucei.

Trypanosoma brucei is a unicellular eukaryotic parasite, which causes the African sleeping sickness in humans. The recently discovered trypanosomal protein Parvulin 42 (TbPar42) plays a key role in parasite cell proliferation. Homologues of this two-domain protein are exclusively found in protozoa species. TbPar42 exhibits an N-terminal forkhead associated (FHA)-domain and a peptidyl-prolyl-cis/trans-isomerase (PPIase) domain, both connected by a linker. Using NMR and X-ray analysis as well as activity assays, we report on the structures of the single domains of TbPar42, discuss their intra-molecular interplay, and give some initial hints as to potential cellular functions of the protein.

Structure and function of the human parvulins Pin1 and Par14/17.

Parvulins belong to the family of peptidyl-prolyl cis/trans isomerases (PPIases) assisting in protein folding and in regulating the function of a broad variety of proteins in all branches of life. The human representatives Pin1 and Par14/17 are directly involved in processes influencing cellular maintenance and cell fate decisions such as cell-cycle progression, metabolic pathways and ribosome biogenesis. This review on human parvulins summarizes the current knowledge of these enzymes and intends to oppose the well-studied Pin1 to its less well-examined homolog human Par14/17 with respect to structure, catalytic and cellular function.

The RxLR Motif of the Host Targeting Effector AVR3a of Phytophthora infestans Is Cleaved before Secretion.

When plant-pathogenic oomycetes infect their hosts, they employ a large arsenal of effector proteins to establish a successful infection. Some effector proteins are secreted and are destined to be translocated and function inside host cells. The largest group of translocated proteins from oomycetes is the RxLR effectors, defined by their conserved N-terminal Arg-Xaa-Leu-Arg (RxLR) motif. However, the precise role of this motif in the host cell translocation process is unclear. Here, detailed biochemical studies of the RxLR effector AVR3a from the potato pathogen Phytophthora infestans are presented. Mass spectrometric analysis revealed that the RxLR sequence of native AVR3a is cleaved off prior to secretion by the pathogen and the N terminus of the mature effector was found likely to be acetylated. High-resolution NMR structure analysis of AVR3a indicates that the RxLR motif is well accessible to potential processing enzymes. Processing and modification of AVR3a is to some extent similar to events occurring with the export element (PEXEL) found in malaria effector proteins from Plasmodium falciparum These findings imply a role for the RxLR motif in the secretion of AVR3a by the pathogen, rather than a direct role in the host cell entry process itself.

The N-terminal Region of the Ubiquitin Regulatory X (UBX) Domain-containing Protein 1 (UBXD1) Modulates Interdomain Communication within the Valosin-containing Protein p97.

Valosin-containing protein/p97 is an ATP-driven protein segregase that cooperates with distinct protein cofactors to control various aspects of cellular homeostasis. Mutations at the interface between the regulatory N-domain and the first of two ATPase domains (D1 and D2) deregulate the ATPase activity and cause a multisystem degenerative disorder, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia/amyotrophic lateral sclerosis. Intriguingly, the mutations affect only a subset of p97-mediated pathways correlating with unbalanced cofactor interactions and most prominently compromised binding of the ubiquitin regulatory X domain-containing protein 1 (UBXD1) cofactor during endolysosomal sorting of caveolin-1. However, how the mutations impinge on the p97-cofactor interplay is unclear so far. In cell-based endosomal localization studies, we identified a critical role of the N-terminal region of UBXD1 (UBXD1-N). Biophysical studies using NMR and CD spectroscopy revealed that UBXD1-N can be classified as intrinsically disordered. NMR titration experiments confirmed a valosin-containing protein/p97 interaction motif and identified a second binding site at helices 1 and 2 of UBXD1-N as binding interfaces for p97. In reverse titration experiments, we identified two distant epitopes on the p97 N-domain that include disease-associated residues and an additional interaction between UBXD1-N and the D1D2 barrel of p97 that was confirmed by fluorescence anisotropy. Functionally, binding of UBXD1-N to p97 led to a reduction of ATPase activity and partial protection from proteolysis. These findings indicate that UBXD1-N intercalates into the p97-ND1 interface, thereby modulating interdomain communication of p97 domains and its activity with relevance for disease pathogenesis. We propose that the polyvalent binding mode characterized for UBXD1-N is a more general principle that defines a subset of p97 cofactors.

Transient domain interactions enhance the affinity of the mitotic regulator Pin1 toward phosphorylated peptide ligands.

The mitotic regulator Pin1 plays an important role in protein quality control and age-related medical conditions such as Alzheimer disease and Parkinson disease. Although its cellular role has been thoroughly investigated during the past decade, the molecular mechanisms underlying its function remain elusive. We provide evidence for interactions between the two domains of Pin1. Several residues displayed unequivocal peak splits in nuclear magnetic resonance spectra, indicative of two different conformational states in equilibrium. Pareto analysis of paramagnetic relaxation enhancement data demonstrates that the two domains approach each other upon addition of a nonpeptidic ligand. Titration experiments with phosphorylated peptides monitored by fluorescence anisotropy and chemical shift perturbation indicate that domain interactions increase Pin1's affinity toward peptide ligands. We propose this interplay of the domains and ligands to be a general mechanism for a large class of two-domain proteins.

Identification and characterization of peptides that bind the PPIase domain of Parvulin17.

Peptidyl-prolyl cis-trans isomerases (PPIases) are the enzymes that increase the rate of isomerization of the peptide bond N-terminal to the proline substrate. Par14 and its isoform Par17 belong to the Parvulin family of PPIases. Par14 can bind AT-rich double-stranded DNA and was shown to be part of the pre-ribosomal ribonucleoprotein (pre-rRNP) complexes, where it functions as an RNA processing factor that is involved in ribosome biogenesis. Its longer isoform Par17 is expressed only in cells of hominids, where it is targeted to the mitochondria. To find binding partners (peptides or proteins) for Par17, we applied the phage display technology. We panned 7-mer and 12-mer peptide libraries against Par17. The consensus sequence XHSXVHØ, where X can be any amino acid and Ø is a hydrophobic amino acid, was enriched from both libraries. We demonstrate the binding of this motif to the PPIase domain of Par17 using phage ELISA and NMR spectroscopy. We propose that residues Met90, Val91, Phe94, Gln95, Glu96, and Ala98 of Par17 are involved in substrate recognition, and that the phage display-selected motif XHSXVHØ can be recognized by Par17 PPIase domain in vivo.

Host-targeting protein 1 (SpHtp1) from the oomycete Saprolegnia parasitica translocates specifically into fish cells in a tyrosine-O-sulphate-dependent manner.

The eukaryotic oomycetes, or water molds, contain several species that are devastating pathogens of plants and animals. During infection, oomycetes translocate effector proteins into host cells, where they interfere with host-defense responses. For several oomycete effectors (i.e., the RxLR-effectors) it has been shown that their N-terminal polypeptides are important for the delivery into the host. Here we demonstrate that the putative RxLR-like effector, host-targeting protein 1 (SpHtp1), from the fish pathogen Saprolegnia parasitica translocates specifically inside host cells. We further demonstrate that cell-surface binding and uptake of this effector protein is mediated by an interaction with tyrosine-O-sulfate-modified cell-surface molecules and not via phospholipids, as has been reported for RxLR-effectors from plant pathogenic oomycetes. These results reveal an effector translocation route based on tyrosine-O-sulfate binding, which could be highly relevant for a wide range of host-microbe interactions.

Crystallographic proof for an extended hydrogen-bonding network in small prolyl isomerases.

Parvulins compose a family of small peptidyl-prolyl isomerases (PPIases) involved in protein folding and protein quality control. A number of amino acids in the catalytic cavity are highly conserved, but their precise role within the catalytic mechanism is unknown. The 0.8 Å crystal structure of the prolyl isomerase domain of parvulin Par14 shows the electron density of hydrogen atoms between the D74, H42, H123, and T118 side chains. This threonine residue has previously not been associated with catalysis, but a corresponding T152A mutant of Pin1 shows a dramatic reduction of catalytic activity without compromising protein stability. The observed catalytic tetrad is strikingly conserved in Pin1- and parvulin-type proteins and hence constitutes a common feature of small peptidyl prolyl isomerases.

A heterodimer of human 3'-phospho-adenosine-5'-phosphosulphate (PAPS) synthases is a new sulphate activating complex.

3'-Phospho-adenosine-5'-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Förster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that--in spite of its low quantum yield--is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5'-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.