The structural properties of full-length annexin A11.

Annexin A11 (ANXA11) is a calcium-dependent phospholipid-binding protein belonging to the annexin protein family and implicated in the neurodegenerative amyotrophic lateral sclerosis. Structurally, ANXA11 contains a conserved calcium-binding C-terminal domain common to all annexins and a putative intrinsically unfolded N-terminus specific for ANXA11. Little is known about the structure and functions of this region of the protein. By analogy with annexin A1, it was suggested that residues 38 to 59 within the ANXA11 N-terminus could form a helical region that would be involved in interactions. Interestingly, this region contains residues that, when mutated, may lead to clinical manifestations. In the present study, we have studied the structural features of the full-length protein with special attention to the N-terminal region using a combination of biophysical techniques which include nuclear magnetic resonance and small angle X-ray scattering. We show that the N-terminus is intrinsically disordered and that the overall features of the protein are not markedly affected by the presence of calcium. We also analyzed the 38-59 helix hypothesis using synthetic peptides spanning both the wild-type sequence and clinically relevant mutations. We show that the peptides have a remarkable character typical of a native helix and that mutations do not alter the behaviour suggesting that they are required for interactions rather than being structurally important. Our work paves the way to a more thorough understanding of the ANXA11 functions.

Invisible leashes: The tethering VAPs from infectious diseases to neurodegeneration.

Intracellular organelles do not, as thought for a long time, act in isolation but are dynamically tethered together by entire machines responsible for interorganelle trafficking and positioning. Among the proteins responsible for tethering is the family of VAMP-associated proteins (VAPs) that appear in all eukaryotes and are localized primarily in the endoplasmic reticulum. The major functional role of VAPs is to tether the endoplasmic reticulum with different organelles and regulate lipid metabolism and transport. VAPs have gained increasing attention because of their role in human pathology where they contribute to infections by viruses and bacteria and participate in neurodegeneration. In this review, we discuss the structure, evolution, and functions of VAPs, focusing more specifically on VAP-B for its relationship with amyotrophic lateral sclerosis and other neurodegenerative diseases.

Tumor-Suppressor p53TAD1-60 Forms a Fuzzy Complex with Metastasis-Associated S100A4: Structural Insights and Dynamics by an NMR/MD Approach.

Conformationally flexible protein complexes represent a major challenge for structural and dynamical studies. We present herein a method based on a hybrid NMR/MD approach to characterize the complex formed between the disordered p53TAD1-60 and the metastasis-associated S100A4. Disorder-to-order transitions of both TAD1 and TAD2 subdomains upon interaction is detected. Still, p53TAD1-60 remains highly flexible in the bound form, with residues L26, M40, and W53 being anchored to identical hydrophobic pockets of the S100A4 monomer chains. In the resulting "fuzzy" complex, the clamp-like binding of p53TAD1-60 relies on specific hydrophobic anchors and on the existence of extended flexible segments. Our results demonstrate that structural and dynamical NMR parameters (cumulative Δδ, SSP, temperature coefficients, relaxation time, hetNOE) combined with MD simulations can be used to build a structural model even if, due to high flexibility, the classical solution structure calculation is not possible.

Quantitative, Diffusion NMR Based Analytical Tool To Distinguish Folded, Disordered, and Denatured Biomolecules.

The key questions in folding studies are the protein dimensions and the degree of folding. These properties are best characterized by the self-diffusion coefficients D determining the hydrodynamic dimensions. In our present study, we derive empirical variations of D as a function of molecular mass M that distinguish folded, intrinsically disordered, and urea-denatured biomolecules. Reliable D values are obtained from diffusion NMR measurements performed under identical conditions using a representative set of proteins/peptides with diverse amino acid sequence and length. The established relations are easy to use analytical tools for molecular mass analysis and aggregation studies as well. Deriving equations under denaturing conditions has several pitfalls, and here, we provide a simple quantitative method for estimating the debated end point of denaturation, while already the 1D 1H spectrum gives a qualitative picture of the collapsed, denatured structure. Data indicate that the intrinsically disordered proteins have a similar behavior as synthetic polymers and urea-denatured proteins.