Gabriella D’Auria

Structural insights into the interaction between the Cripto CFC domain and the ALK4 receptor.

The protein Cripto is the founding member of the extra‐cellular EGF–CFC growth factors, which are composed of two adjacent cysteine‐rich domains: the EGF‐like and the CFC. Members of the EGF–CFC family play key roles in embryonic development and are also implicated in tumourigenesis. Cripto is highly over‐expressed in many tumours, while it is poorly detectable in normal tissues.

Dynamical properties of cold shock protein A from Mycobacterium tuberculosis

Bacterial cold shock proteins (Csps) are over-expressed as response to cold stress. They have a role in transcriptional and translational events due to their ability to bind single stranded (ss) nucleic acids. Csps so far characterized show similar structures with a closed five stranded antiparallel β-barrel. Here we report a structural and functional study of cold shock protein A from Mycobacterium tuberculosis, MTB-CspA. Structural investigations by CD and NMR reveal that MTB-CspA is less ordered than expected and is the least thermal stable cold shock protein so far characterized. However, electrophoretic mobility shift assays show that MTB-CspA is functionally active, as it is able to bind oligonucleotides. The dynamic behavior of MTB-CspA, compared to its homolog from Bacillus subtilis, was investigated by molecular dynamics simulations at room and high temperatures. Analysis of trajectories point to specific regions on β1 and β4 strands, likely responsible for the higher structural fragility of MTB-CspA. Also, they show that the nucleic-acid binding region of MTB-CspA is the least prone to unfolding, a finding which explains the ability of MTB-CspA to exert its function.

PASTA in penicillin binding proteins and serine/threonine kinases: a recipe of structural, dynamic and binding properties

BACKGROUND Penicillin binding proteins (PBPs) and Serine Threonine kinases (STPKs) are two classes of bacterial enzymes whose involvement in a series of vital processes in bacterial growth and division is well assessed. Many PBPs and STPKs show linked an ancillary domain named PASTA, whose functional role is not completely deciphered so far. It has been proposed that PASTAs are sensor modules that by binding opportune ligands (i.e. muropeptides) activate the cognate proteins to their functions. However, based on recent data, the sensor annotation sounds true for PASTA from STPKs, and false for PASTA from PBPs. OBJECTIVE Different PASTA domains, belonging or not to different protein classes, sharing or not appreciable sequence identities, always show identical folds. This survey of the structural, binding and dynamic properties of PASTA domains pursues the reasons why identical topologies may turn in different roles. RESULTS Amino acid compositions, total charges and distribution of the hydrophobic/hydrophilic patches on the surface, significantly vary among PASTAs from STPKs and PBPs and appear to correlate with different functions. A possible criterion to discriminate between PASTA modules of STPKs or PBPs solely based on their sequences is proposed. Possibly reflecting different species as well as functional roles and evolutionary profile, our routine represents a fast even though approximate method to distinguish between PASTA belonging to different classes.

Toward a better understanding of the interaction between TGF-β family members and their ALK receptors

Transforming growth factor-beta (TGF-β) proteins are a family of structurally related extracellular proteins that trigger their signaling functions through interaction with the extracellular domains of their cognate serine/threonine kinase receptors. The specificity of TGF-β/receptor binding is complex and gives rise to multiple functional roles. Additionally, it is not completely understood at the atomic level. Here, we use the most reliable computational methods currently available to study systems involving activin-like kinase (ALK) receptors ALK4 and ALK7 and their multiple TGF-β ligands. We built models for all these proteins and their complexes for which experimental structures are not available. By analyzing the surfaces of interaction in six different TGF-β/ALK complexes we could infer which are the structural distinctive features of the ligand-receptor binding mode. Furthermore, this study allowed us to rationalize why binding of the growth factors GDF3 and Nodal to the ALK4 receptor requires the Cripto co-factor, whilst binding to the ALK7 receptor does not.

Structural and dynamic features of PASTA domains with different functional roles

Structural and dynamic features of PASTA domains with different functional roles Luisa Calvanese, Lucia Falcigno, Flavia Squeglia, Gabriella D’Auria and Rita Berisio* CIRPeB, University of Naples “Federico II”, via Mezzocannone, 16, Naples 80134, Italy; Department of Pharmacy, University of Naples “Federico II”, via Mezzocannone, 16, Naples 80134, Italy; Institute of Biostructures and Bioimaging-CNR, via Mezzocannone, 16, Naples 80134, Italy

In vitro and in vivo pro-angiogenic effects of thymosin-β4-derived peptides

Thymosin-β4 (Tβ4) is a G-actin sequestering peptide involved in regeneration and remodeling of injured tissues. In this work, we have designed and synthesized three peptide sequences containing the N-terminus (TYB4-n), the central part (TYB4-i) or the C-terminus (TYB4-c) of Tβ4. All fragments are overlapping on the main central binding actin site. After a structural characterization, we have evaluated in vitro and in vivo their pro-angiogenic effects. The results of this study have shown that: (i) each fragment reproduces the native conformation; (ii) Tβ4-derived peptides exert both in vitro and in vivo pro-angiogenic effects; (iii) their in vitro effect seem to be related to the activation of several signaling pathways and is positively modulated by the N-terminus of Tβ4.

Structural Investigation of the HIV‐1 Envelope Glycoprotein gp160 Cleavage Site, 2: Relevance of an N‐Terminal Helix

Proteolytic activation of the HIV‐1 envelope glycoprotein gp160 is selectively performed by the proprotein convertase furin at the C‐terminus of the sequence R508–E–K–R511 (site 1), in spite of the presence of another consensus sequence, Lys500–Ala–Lys–Arg503 (site 2). On the basis of the solution structural analysis of the synthetic peptide p498, spanning the gp160 sequence Pro498–Gly516, we previously suggested a possible role of an N‐terminal helix in regulating the exposure and accessibility of the gp160 physiological cleavage site, enclosed in a loop. Here we report on the activity and conformation of the 23‐residue peptide h‐REKR, designed to exhibit a large N‐terminal helix, followed by the gp160 native sequence, Arg508–Gly516. h‐REKR is digested by furin with high efficiency, comparable to the full native p498. Circular dichroism analyses, in mixtures from pure water to 98 % trifluoroethanol, outline a significant content of helical structure in the peptide conformation. The molecular model obtained from NMR data collected in trifluoroethanol/water, by means of DYANA and AMBER simulations, indeed has helical structure on a large N‐terminal segment. Such a long helix does not seem to affect the loop conformation of the C‐terminal site 1‐containing sequence, which exhibits the same proton chemical shifts already observed for the full native p498.

Structural Basis for Mutations of Human Aquaporins Associated to Genetic Diseases

Aquaporins (AQPs) are among the best structural-characterized membrane proteins, fulfilling the role of allowing water flux across cellular membranes. Thus far, 34 single amino acid polymorphisms have been reported in HUMSAVAR for human aquaporins as disease-related. They affect AQP2, AQP5 and AQP8, where they are associated with nephrogenic diabetes insipidus, keratoderma and colorectal cancer, respectively. For half of these mutations, although they are mostly experimentally characterized in their dysfunctional phenotypes, a structural characterization at a molecular level is still missing. In this work, we focus on such mutations and discuss what the structural defects are that they appear to cause. To achieve this aim, we built a 3D molecular model for each mutant and explored the effect of the mutation on all of their structural features. Based on these analyses, we could collect the structural defects of all the pathogenic mutations (here or previously analysed) under few main categories, that we found to nicely correlate with the experimental phenotypes reported for several of the analysed mutants. Some of the structural analyses we present here provide a rationale for previously experimentally observed phenotypes. Furthermore, our comprehensive overview can be used as a reference frame for the interpretation, on a structural basis, of defective phenotypes of other aquaporin pathogenic mutants.

Targeting VEGF receptors with non-neutralizing cyclopeptides for imaging applications

Pharmacological strategies aimed at preventing cancer growth are in most cases paralleled by diagnostic investigations for monitoring and prognosticating therapeutic efficacy. A relevant approach in cancer is the suppression of pathological angiogenesis, which is principally driven by vascular endothelial growth factor (VEGF) or closely related factors and by activation of specific receptors, prevailingly VEGFR1 and VEGFR2, set on the surface of endothelial cells. Monitoring the presence of these receptors in vivo is henceforth a way to predict therapy outcome. We have designed small peptides able to bind and possibly antagonize VEGF ligands by targeting VEGF receptors. Peptide systems have been designed to be small, cyclic and to host triplets of residues known to be essential for VEGF receptors recognition and we named them ‘mini-factors’. They have been structurally characterized by CD, NMR and molecular dynamics (MD) simulations. Mini-factors do bind with different specificity and affinity VEGF receptors but none blocks receptor activity. Following derivatization with suitable tracers they have been employed as molecular probes for sensing receptors on cell surface without affecting their activity as is usually observed with other binders having neutralizing activity.