Peter Schmieder

Dual epitope recognition by the VASP EVH1 domain modulates polyproline ligand specificity and binding affinity

The Ena‐VASP family of proteins act as molecular adaptors linking the cytoskeletal system to signal transduction pathways. Their N‐terminal EVH1 domains use groups of exposed aromatic residues to specifically recognize ‘FPPPP’ motifs found in the mammalian zyx in and vinculin proteins, and ActA protein of the intracellular bacterium Listeria monocytogenes. Here, evidence is provided that the affinities of these EVH1–peptide interactions are strongly dependent on the recognition of residues flanking the core FPPPP motifs. Determination of the VASP EVH1 domain solution structure, together with peptide library screening, measurement of individual Kds by fluorescence titration, and NMR chemical shift mapping, revealed a second affinity‐determining epitope present in all four ActA EVH1‐binding motifs. The epitope was shown to interact with a complementary hydrophobic site on the EVH1 surface and to increase strongly the affinity of ActA for EVH1 domains. We propose that this epitope, which is absent in the sequences of the native EVH1‐interaction partners zyxin and vinculin, may provide the pathogen with an advantage when competing for the recruitment of the host VASP and Mena proteins in the infected cell.

WW domain sequence activity relationships identified using ligand recognition propensities of 42 WW domains

WW domains mediate protein–protein interactions in a number of different cellular functions by recognizing proline‐containing peptide sequences. We determined peptide recognition propensities for 42 WW domains using NMR spectroscopy and peptide library screens. As potential ligands, we studied both model peptides and peptides based on naturally occurring sequences, including phosphorylated residues. Thirty‐two WW domains were classified into six groups according to detected ligand recognition preferences for binding the motifs PPx(Y/poY), (p/ϕ)P(p,g)PPpR, (p/ϕ)PPRgpPp, PPLPp, (p/ξ)PPPPP, and (poS/poT)P (motifs according to modified Seefeld Convention 2001). In addition to these distinct binding motifs, group‐specific WW domain consensus sequences were identified. For PPxY‐recognizing domains, phospho‐tyrosine binding was also observed. Based on the sequences of the PPx(Y/poY)‐specific group, a profile hidden Markov model was calculated and used to predict PPx(Y/poY)‐recognition activity for WW domains, which were not assayed. PPx(Y/poY)‐binding was found to be a common property of NEDD4‐like ubiquitin ligases.

High resolution 1H-detected solid-state NMR spectroscopy of protein aliphatic resonances: access to tertiary structure information.

Biological magic angle spinning (MAS) solid-state nuclear magnetic resonance spectroscopy has developed rapidly over the past two decades. For the structure determination of a protein by solid-state NMR, routinely (13)C,(13)C distance restraints as well as dihedral restraints are employed. In protonated samples, this is achieved by growing the bacterium on a medium which contains [1,3]-(13)C glycerol or [2]-(13)C glycerol to dilute the (13)C spin system. Labeling schemes, which rely on heteronuclei, are insensitive both for detection and in terms of quantification of distances, since they are relying on low-γ nuclei. Proton detection can in principle provide a gain in sensitivity by a factor of 8 and 31, compared to the (13)C or (15)N detected version of the experiment. We report here a new labeling scheme, which enables (1)H-detection of aliphatic resonances with high resolution in MAS solid-state NMR spectroscopy. We prepared microcrystals of the SH3 domain of chicken α-spectrin with 5% protonation at nonexchangeable sites and obtained line widths on the order of 25 Hz for aliphatic (1)H resonances. We show further that (13)C resolved 3D-(1)H,(1)H correlation experiments yield access to long-range proton-proton distances in the protein.

Small Molecule AKAP-Protein Kinase A (PKA) Interaction Disruptors That Activate PKA Interfere with Compartmentalized cAMP Signaling in Cardiac Myocytes

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3′-diamino-4,4′-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.

NMR structure of the Wnt modulator protein Sclerostin

Sclerostin has been identified as a negative regulator of bone growth. Initially it was considered that Sclerostin performs its regulatory function via acting as a modulator of bone morphogenetic proteins (BMPs) similar to known examples such as Noggin, Chordin, and members of the DAN family. Recent findings, however, show that Sclerostin interferes with the Wnt signaling pathway due to binding to the Wnt co-receptor LRP5 thereby modulating bone growth. As Sclerostin is exclusively produced by osteocytes located in bones, neutralization of its bone-inhibiting functions makes it a highly interesting target for an osteoanabolic therapeutic approach in diseases characterized by bone loss, such as osteoporosis. Despite the huge interest in Sclerostin inhibitors the molecular basis of its function and its interaction with components of the Wnt signaling cascade has remained unclear. Here, we present the NMR structure of murine Sclerostin providing the first insights how Sclerostin might bind to LRP5.

Probing protein-chromophore interactions in Cph1 phytochrome by mutagenesis

We have investigated mutants of phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 in order to study chromophore–protein interactions. Cph1Δ2, the 514‐residue N‐terminal sensor module produced as a recombinant His6‐tagged apoprotein in Escherichia coli, autoassembles in vitro to form a holoprotein photochemically indistinguishable from the full‐length product. We generated 12 site‐directed mutants of Cph1Δ2, focusing on conserved residues which might be involved in chromophore–protein autoassembly and photoconversion. Folding, phycocyanobilin‐binding and Pr→Pfr photoconversion were analysed using CD and UV–visible spectroscopy. MALDI‐TOF‐MS confirmed C259 as the chromophore attachment site. C259L is unable to attach the chromophore covalently but still autoassembles to form a red‐shifted photochromic holoprotein. H260Q shows UV–visible properties similar to the wild‐type at pH 7.0 but both Pr and Pfr (reversibly) bleach at pH 9.0, indicating that the imidazole side chain buffers chromophore protonation. Mutations at E189 disturbed folding but the residue is not essential for chromophore–protein autoassembly. In D207A, whereas red irradiation of the ground state leads to bleaching of the red Pr band as in the wild‐type, a Pfr‐like peak does not arise, implicating D207 as a proton donor for a deprotonated intermediate prior to Pfr. UV‐Vis spectra of both H260Q under alkaline conditions and D207A point to a particular significance of protonation in the Pfr state, possibly implying proton migration (release and re‐uptake) during Pr→Pfr photoconversion. The findings are discussed in relation to the recently published 3D structure of a bacteriophytochrome fragment [Wagner JR, Brunzelle JS, Forest KT & Vierstra RD (2005) Nature438, 325–331].

Improved resolution in proton-detected heteronuclear long-range correlation

Heteronuclear long-range correlations can provide valuable information in many circumstances of spectroscopic work. They can provide correlations to quaternary carbons, which can be useful, e.g., either in the assignment of aromatic resonances or during sequencing of a peptide

Light‐Directed Protein Binding of a Biologically Relevant β‐Sheet

b-Hairpin structures are frequently involved in protein– protein interactions that control essential processes in cells and are therefore interesting targets for interference. Hairpinforming peptides that compete with such protein interactions are valuable tools for studying biological processes. Moreover, the incorporation of a photoswitchable unit into appropriate b-hairpin-forming peptide ligands could allow protein interactions in cells to be studied by light-triggered interference. However, b-hairpin structures are rarely studied because of the limited availability and stability of suitable model peptides. This is because such a model peptide has to fulfill at least three requirements: 1) the b-hairpin has to be sufficiently stable as monomer without the tendency to selfaggregate, 2) the photoswitchable unit incorporated must stabilize the biologically active peptide conformation, and 3) disturbing the protein binding site by light-induced isomerization of the photoswitch must not result in intermolecular association or even formation of insoluble fibrils. Herein, we report the first example of a b-hairpin model peptide of a biologically important protein domain that shows considerably different binding affinities for the target protein that are dependent on the isomerization state of the embedded photoswitch. PDZ domains mediate the formation of a variety of multiprotein complexes in the cell. Besides C-terminal protein sequences, PDZ domains are also able to recognize internal peptide motifs that bind at the same binding pocket as the C-terminal ones. The best example of this type of internal ligand recognition is found in the extended PDZ domain of neuronal nitric oxide synthase (nNOS) which interacts with the PDZ domain from a-1-syntrophin or the second PDZ domain from PSD95. The formation of the PDZ/PDZ heterodimer requires the b-finger structure of nNOS (30 amino acid residues) to bind at the syntrophin PDZ domain, thus mediating the membrane association of nNOS to skeletal muscle and inducing the production of the second messenger nitric oxide (NO) for muscle contraction. Crucial for binding is the internal recognition motif -LETTFof the extended PDZ domain of nNOS located in the first strand of the hairpin peptide (Scheme 1), a stable

Hyperphosphorylation of Glucosyl C6 Carbons and Altered Structure of Glycogen in the Neurodegenerative Epilepsy Lafora Disease

Laforin or malin deficiency causes Lafora disease, characterized by altered glycogen metabolism and teenage-onset neurodegeneration with intractable and invariably fatal epilepsy. Plant starches possess small amounts of metabolically essential monophosphate esters. Glycogen contains similar phosphate amounts, which are thought to originate from a glycogen synthase error side reaction and therefore lack any specific function. Glycogen is also believed to lack monophosphates at glucosyl carbon C6, an essential phosphorylation site in plant starch metabolism. We now show that glycogen phosphorylation is not due to a glycogen synthase side reaction, that C6 is a major glycogen phosphorylation site, and that C6 monophosphates predominate near centers of glycogen molecules and positively correlate with glycogen chain lengths. Laforin or malin deficiency causes C6 hyperphosphorylation, which results in malformed long-chained glycogen that accumulates in many tissues, causing neurodegeneration in brain. Our work advances the understanding of Lafora disease pathogenesis and suggests that glycogen phosphorylation has important metabolic function.

Light-dependent dimerisation in the N-terminal sensory module of cyanobacterial phytochrome 1

Phytochromes, photoreceptors controlling important physiological processes in plants and many prokaryotes, are photochromic biliproteins. The red‐absorbing Pr ground state is converted by light into the farred‐absorbing Pfr which can be photoconverted back to Pr. In plants at least Pfr is the physiologically active signalling state. Here, we show that the N‐terminal photochromic module of Cph1 homodimerises reversibly and independently in Pr and Pfr, Pfr‐dimers being significantly more stable. Implications for the mechanism of signal transduction are discussed.