Phosphorylation drives a dynamic switch in serine/arginine-rich proteins.

Serine/arginine-rich (SR) proteins are important players in RNA metabolism and are extensively phosphorylated at serine residues in RS repeats. Here, we show that phosphorylation switches the RS domain of the serine/arginine-rich splicing factor 1 from a fully disordered state to a partially rigidified arch-like structure. Nuclear magnetic resonance spectroscopy in combination with molecular dynamics simulations revealed that the conformational switch is restricted to RS repeats, critically depends on the phosphate charge state and strongly decreases the conformational entropy of RS domains. The dynamic switch also occurs in the 100 kDa SR-related protein hPrp28, for which phosphorylation at the RS repeat is required for spliceosome assembly. Thus, a phosphorylation-induced dynamic switch is common to the class of serine/arginine-rich proteins and provides a molecular basis for the functional redundancy of serine/arginine-rich proteins and the profound influence of RS domain phosphorylation on protein-protein and protein-RNA interactions.

Structural characterization of polyglutamine fibrils by solid-state NMR spectroscopy.

Protein aggregation via polyglutamine stretches occurs in a number of severe neurodegenerative diseases such as Huntington's disease. We have investigated fibrillar aggregates of polyglutamine peptides below, at, and above the toxicity limit of around 37 glutamine residues using solid-state NMR and electron microscopy. Experimental data are consistent with a dry fibril core of at least 70-80 Å in width for all constructs. Solid-state NMR dipolar correlation experiments reveal a largely ß-strand character of all samples and point to tight interdigitation of hydrogen-bonded glutamine side chains from different sheets. Two approximately equally frequent populations of glutamine residues with distinct sets of chemical shifts are found, consistent with local backbone dihedral angles compensating for ß-strand twist or with two distinct sets of side-chain conformations. Peptides comprising 15 glutamine residues are present as single extended ß-strands. Data obtained for longer constructs are most compatible with a superpleated arrangement with individual molecules contributing ß-strands to more than one sheet and an antiparallel assembly of strands within ß-sheets.

Peptide ligands selected with CD4-induced epitopes on native dualtropic HIV-1 envelope proteins mimic extracellular coreceptor domains and bind to HIV-1 gp120 independently of coreceptor usage.

During HIV-1 entry, binding of the viral envelope glycoprotein gp120 to the cellular CD4 receptor triggers conformational changes resulting in exposure of new epitopes, the highly conserved CD4-induced (CD4i) epitopes that are essential for subsequent binding to chemokine receptor CCR5 or CXCR4. Due to their functional conservation, CD4i epitopes represent attractive viral targets for HIV-1 entry inhibition. The aim of the present study was to select peptide ligands for CD4i epitopes on native dualtropic (R5X4) HIV-1 envelope (Env) glycoproteins by phage display. Using CD4-activated retroviral particles carrying Env from the R5X4 HIV-1 89.6 strain as the target, we performed screenings of random peptide phage libraries under stringent selection conditions. Selected peptides showed partial identity with amino acids in the extracellular domains of CCR5/CXCR4, including motifs rich in tyrosines and aspartates at the N terminus known to be important for gp120 binding. A synthetic peptide derivative (XD3) corresponding to the most frequently selected phages was optimized for Env binding on peptide arrays. Interestingly, the optimized peptide could bind specifically to gp120 derived from HIV-1 strains with different coreceptor usage, competed with binding of CD4i-specific monoclonal antibody (MAb) 17b, and interfered with entry of both a CCR5 (R5)-tropic and a CXCR4 (X4)-tropic Env pseudotyped virus. This peptide ligand therefore points at unique properties of CD4i epitopes shared by gp120 with different coreceptor usage and could thus serve to provide new insight into the conserved structural details essential for coreceptor binding for further drug development.