Many peptides and proteins with large sequences and structural differences self-assemble into disease-causing amyloids that share very similar biochemical and biophysical characteristics, which may contribute to their cross-interaction. Here, we demonstrate how the self-assembled, cyclic d,l-α-peptide CP-2, which has similar structural and functional properties to those of amyloids, acts as a generic inhibitor of the Parkinson's disease associated α-synuclein (α-syn) aggregation to toxic oligomers by an "off-pathway" mechanism. We show that CP-2 interacts with the N-terminal and the non-amyloid-β component region of α-syn, which are responsible for α-syn's membrane intercalation and self-assembly, thus changing the overall conformation of α-syn. CP-2 also remodels α-syn fibrils to nontoxic amorphous species and permeates cells through endosomes/lysosomes to reduce the accumulation and toxicity of intracellular α-syn in neuronal cells overexpressing α-syn. Our studies suggest that targeting the common structural conformation of amyloids may be a promising approach for developing new therapeutics for amyloidogenic diseases.

Wiskott-Aldrich syndrome protein (WASp) is exclusively expressed in hematopoietic cells and responsible for actin-dependent processes, including cellular activation, migration, and invasiveness. The C-terminal domain of WASp-Interacting Protein (WIP) binds to WASp and regulates its activity by shielding it from degradation in a phosphorylation dependent manner as we previously demonstrated. Mutations in the WAS-encoding gene lead to the primary immunodeficiencies Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). Here, we shed a first structural light upon this function of WIP using nuclear magnetic resonance (NMR) and in vivo molecular imaging. Coexpression of fragments WASp(20-158) and WIP(442-492) allowed the purification and structural characterization of a natively folded complex, determined to form a characteristic pleckstrin homology domain with a mixed α/β-fold and central two-winged β-sheet. The WIP-derived peptide, unstructured in its free form, wraps around and interacts with WASp through short structural elements. Förster resonance energy transfer (FRET) and biochemical experiments demonstrated that, of these elements, WIP residues 454-456 are the major contributor to WASp affinity, and the previously overlooked residues 449-451 were found to have the largest effect upon WASp ubiquitylation and, presumably, degradation. Results obtained from this complementary combination of technologies link WIP-WASp affinity to protection from degradation. Our findings about the nature of WIP·WASp complex formation are relevant for ongoing efforts to understand hematopoietic cell behavior, paving the way for new therapeutic approaches to WAS and XLT.

Intrinsically disordered proteins (IDPs) are multi-conformational polypeptides that lack a single stable three-dimensional structure. It has become increasingly clear that the versatile IDPs play key roles in a multitude of biological processes, and, given their flexible nature, NMR is a leading method to investigate IDP behavior on the molecular level. Here we present an IDP-tailored J-modulated experiment designed to monitor changes in the conformational ensemble characteristic of IDPs by accurately measuring backbone one- and two-bond J(N,Cα) couplings. This concept was realized using a unidirectional (H)NCO C-detected experiment suitable for poor spectral dispersion and optimized for maximum coverage of amino acid types. To demonstrate the utility of this approach we applied it to the disordered actin-binding N-terminal domain of WASp interacting protein (WIP), a ubiquitous key modulator of cytoskeletal changes in a range of biological systems. One- and two-bond J(N,Cα) couplings were acquired for WIP residues 2-65 at various temperatures, and in denaturing and crowding environments. Under native conditions fitted J-couplings identified in the WIP conformational ensemble a propensity for extended conformation at residues 16-23 and 45-60, and a helical tendency at residues 28-42. These findings are consistent with a previous study of the based upon chemical shift and RDC data and confirm that the WIP conformational ensemble is biased towards the structure assumed by this fragment in its actin-bound form. The effects of environmental changes upon this ensemble were readily apparent in the J-coupling data, which reflected a significant decrease in structural propensity at higher temperatures, in the presence of 8 M urea, and under the influence of a bacterial cell lysate. The latter suggests that crowding can cause protein unfolding through protein-protein interactions that stabilize the unfolded state. We conclude that J-couplings are a useful measureable in characterizing structural ensembles in IDPs, and that the proposed experiment provides a practical method for accurately performing such measurements, once again emphasizing the power of NMR in studying IDP behavior.

intracellular nucleotide binding sites. Fluorescence spectra of ANAP labelled Kir6.2 subunits were acquired after exposure to increasing concentrations of TNP-ATP. Binding of TNP-ATP was measured as quenching of the ANAP fluorescence at 470 nm and could be competed off with addition of unlabelled ATP. Both Kir6.2-I182ANAP and F183ANAP co-expressed with SUR1 bound TNP-ATP in the low mM range in the absence of Mg 2þ , comparable to the apparent affinity for inhibition of wild-type Kir6.2/SUR1 by TNP-ATP. Similar apparent affinities were obtained from C-terminally truncated or GFP-tagged Kir6.2-F183 constructs expressed without SUR1. A mutation (G334D) in the ATP binding site of Kir6.2 that does not affect intrinsic K ATP gating greatly decreased the apparent nucleotide affinity. Similar effects on TNP-ATP binding were observed with the introduction of the C166S gating mutation (which increases open probability), suggesting that conformational changes at the pore of Kir6.2 can influence nucleotide binding.

changes and sequence-specific local chain motions of 4E-BP2 upon phosphorylation and upon binding to eIF4E. 1H -15N heteronuclear single quantum correlation (HSQC) spectra demonstrate that cysteine mutations of 4E-BP2 do not perturb its phosphorylated folded structure. Multiparameter smFRET analysis reveals changes in the conformational ensemble upon phosphorylation, denaturation and binding to elF4E. Nanosecond scale dynamics in 4E-BP2 were observed by sitespecific FCS, and were tentatively assigned to formation of transient intrachain contacts. Our data suggests that multi-site phosphorylation of the protein slows down the proximal chain motions and also modulates the kinetics of distal regions. Segmental rotational correlation times and wobbling cone angles extracted for different sites along the chain provide a rigidity map of this IDP and can be used to evaluate its binding mode to eIF4E. 2746-Pos Board B123LC8 Motif Recognition: Insights from the LC8-Chica Complex LC8 is a dimeric hub protein involved in a large number of interactions central to cell function. It binds short linear motifs -usually containing a Thr-Gln-Thr (TQT) triplet -in intrinsically disordered regions of its binding partners, some of which have several LC8 recognition motifs in tandem. Hallmarks of the motif are a high variability of sequence and of LC8 binding affinity. Human Chica, a spindle adaptor protein, contains a 70-residue segment with four putative recognition motifs but only three reported LC8 binding sites. Here, NMRderived secondary chemical shifts and relaxation properties show that the Chica LC8 binding domain is essentially disordered with a dynamically restricted segment in one linker between motifs. Calorimetry of LC8 binding to synthetic motif-mimicking peptides shows that the first motif dominates LC8 recruitment. Crystal structures of LC8 bound to each of two motif peptides show highly ordered and invariant TQT-LC8 interactions and more flexible and conformationally variable non-TQT-LC8 interactions. These data add a new and complex LC8 binding partner to the LC8 knowledgebase and provide new insights into the determinants of motif specificity. We propose that LC8 recognition is based on inflexible interactions between LC8 and TQT residues that act as an anchor, coupled with inherent flexibility of interactions between LC8 and non-TQT residues. The 'anchored flexibility' model combines highly rigid as well as highly flexible regions of the same binding groove on the surface of LC8. Intrinsically disordered proteins (IDPs) are multi-conformational polypeptides that lack a stable three-dimensional structure, forcing structural biology to reconsider the structure-function paradigm. The versatile IDPs play key roles in a multitude of biological processes, and, given their inherently flexible nature, nuclear magnetic resonance (NMR) is a leading method for investigating IDP behavior on the molecular level. Our research has focused on the intrinsically disordered WASp Interacting Protein (WIP) from human T cells, whose C-...