Gabriella D'Auria

Structural and binding properties of the PASTA domain of PonA2, a key penicillin binding protein from Mycobacterium tuberculosis

PonA2 is one of the two class A penicillin binding proteins of Mycobacterium tuberculosis, the etiologic agent of tuberculosis. It plays a complex role in mycobacterial physiology and is spotted as a promising target for inhibitors. PonA2 is involved in adaptation of M. tuberculosis to dormancy, an ability which has been attributed to the presence in its sequence of a C‐terminal PASTA domain. Since PASTA modules are typically considered as β‐lactam antibiotic binding domains, we determined the solution structure of the PASTA domain from PonA2 and analyzed its binding properties versus a plethora of potential binders, including the β‐lactam antibiotics, two typical muropeptide mimics, and polymeric peptidoglycan. We show that, despite a high structural similarity with other PASTA domains, the PASTA domain of PonA2 displays different binding properties, as it is not able to bind muropeptides, or β‐lactams, or polymeric peptidoglycan. These results indicate that the role of PASTA domains cannot be generalized, as their specific binding properties strongly depend on surface residues, which are widely variable.

Solid State and Solution Conformation of [Ala7]‐Phalloidin: A Synthetic Phallotoxin Analogue

Phallotoxins are toxic compounds produced by poisonous mushroom Amanita phalloides and belong to the class of bicyclic peptides with a transannular thioether bridge. Their intoxication mechanism in the liver involves a specific binding of the toxins to F-actin that, consequently, prevents the depolymerization equilibrium with G-actin. Even though the conformational features of phallotoxins have been worked out in solution, the exact mechanism of interaction with F-actin is still unknown. In this study a toxic phalloidin synthetic derivative, bicyclo(Ala1-D-Thr2-Cys3-cis-4-hydroxy-Pro4-Ala5-2-mercapto-Trp6-Ala7)(S-3-->6) has been synthesized. A substitution at position 7. with an Ala residue replaces the 4,5-dihydroxy-Leu present in the natural phalloidin. This analogue has formed crystals suitable for X-ray analysis, and represents the first case for such a class of compounds. The solid-state structure as well as the solution conformation have been evaluated. NMR techniques have been used to extract interproton distances as restraints in subsequent molecular dynamics calculations. Finally, a direct comparison between structures in solution and in the solid state is presented.

Design and structure of a novel Neurokinin A receptor antagonist cyclo(-Met1-Asp2-Trp3-Phe4-Dap5-Leu6-)cyclo(2β-5β)

We report here the rational design, synthesis and structural characterization in the solid state and in solution of the most potent and selective peptide-based Neurokinin A (NKA) antagonist, thus far described. We predicted the bioactive conformation of the known NKA antagonist cyclo(-Met1-Gln2-Trp3-Phe4-Gly5-Leu6-) by comparison with the known structures of other cyclohexapeptides. On this basis we designed a highly constrained peptide molecule corresponding to a bicyclic hexapeptide containing two rings of 14 atoms, namely cyclo(-Met1-Asp2-Trp3-Phe4-Dap5-Leu6-)cyclo(2β-5β). It was synthesized efficiently, using a combined solution and solid phase strategy. We fully characterized this molecule in the solid state by X-ray diffraction and we show that it adopts an almost identical conformation in acetonitrile solution by NMR spectroscopy. This structure fully confirms our hypothetical model. Its structure and conformational rigidity in solution explain the high potency and selectivity and the resistance to proteolytic degradation. Therefore the structural requirements for NKA antagonistic activity are clarified.

Structural investigations on the Nodal-Cripto binding: a theoretical and experimental approach.

Nodal, a member of the transforming growth factor-β superfamily, is a potent embryonic morphogen also implicated in tumor progression. Up to date structural information on the interaction of Nodal with its molecular partners are unknown. To deepen our understanding about mechanisms underlying both embryonic development and Nodal/Cripto-dependent tumor progression, we present here a molecular model of activin receptor-like kinase 4/Cripto/Nodal complex built by homology modeling as well as docking tests aimed at identifying potential binding epitopes. Starting from this model, we have predicted a large interaction surface on Nodal, which encompasses residues 43-69 and includes the prehelix loop and the H3 helix. This hypothesis has been subsequently assessed by surface plasmon resonance binding assays between the full-length Cripto and synthetic peptides reproducing the selected Nodal regions. In addition, the binding affinity between the full-length Nodal and Cripto proteins has been evaluated for the first time.

NMR conformational studies on a synthetic peptide reproducing the [1‐20] processing domain of the pro‐ocytocin‐neurophysin precursor

The combined use of several nuclear magnetic resonance and restrained molecular dynamics techniques allowed the formulation of a molecular model for the preferred solution conformation of a synthetic peptide reproducing the [1-20] processing domain of the pro-ocytocin-neurophysin precursor. In the model, the conformation of the 20-membered tocin ring, with the two Cys1 and Cys6 residues bridged by a disulphide bond, is very close to that observed for isolated ocytocin in the solid state; in addition, a type II beta-turn is postulated for the 7-10 segment of the acyclic tail containing the Lys11-Arg12 processing site, and connecting ocytocin to the neurophysin domain, while the C-terminal 13-20 segment of the molecule is believed to assume a helical structure. This particular structural organization could be important in participating as the favorable conformation for optimal substrate-enzyme active site recognition and processing by specific endoproteases.

Self-assembling properties of ionic-complementary peptides.

Self‐complementary synthetic peptides, composed by 8 and 16 residues, were analyzed by CD, NMR and small angle neutron scattering (SANS) techniques in order to investigate the relevance of charge and hydrophobic interactions in determining their self‐assembling properties. All the sequences are potentially able to form fibrils and membranes as they share, with the prototype EAK16, a strictly alternating arrangement of polar and nonpolar residues. We find that 16‐mer peptides show higher self‐assembling propensities than the 8‐mer analogs and that the aggregation processes are favored by salts and neutral pH. Peptide hydrophobic character appears as the most relevant factor in determining self‐assembling. Solution conformational analysis, diffusion and SANS measurements all together show that the sequences with a higher self‐assemble propensity are distributed, in mild conditions, between light and heavy forms. For some of the systems, the light form is mostly constituted by monomers in a random conformation, while the heavy one is constituted by β‐aggregates. In our study we also verified that sequences designed to adopt extended conformation, when dissolved in alcohol‐water mixtures, can easily fold in helix structures. In that media, the prototype of the series appears distributed between helical monomers and β‐aggregates. It is worth noticing that the structural conversion from helical monomer to β‐aggregates, mimics β‐amyloid peptide aggregation mechanisms. Copyright

500 MHz NMR characterization of synthetic bombesin and related peptides in DMSO-d6 by two-dimensional techniques.

The proton NMR characterization of bombesin has been carried out at 500 MHz in DMSO‐d6 using two‐dimensional homo‐ and 1H‐13C hetero‐correlated techniques. All resonances in the NMR spectra have been assigned and several coupling constants have been measured. The backbone J αCH‐NH coupling constants have constant values that vary between 7.8 and 8.2 Hz and indicate an unfolded structure in DMSO‐d6. Discrepancies with data recently obtained at 300 MHz [(1987) Eur. J. Biochem. 168, 193–199] are discussed.

Structural investigation of the HIV-1 envelope glycoprotein gp160 cleavage site

The selective proteolytic activation of the HIV-1 envelope glycoprotein gp160 by furin and other precursor convertases (PCs) occurs at the carboxyl side of the sequence Arg508-Glu-Lys-Arg511 (site 1), in spite of the presence of another consensus sequence: Lys500-Ala-Lys-Arg503 (site 2). We report on the solution structural analysis of a 19-residue synthetic peptide, p498, which spans the two gp160-processing sites 1 and 2, and is properly digested by furin at site 1. A molecular model is obtained for p498, by means of molecular dynamics simulations, from NMR data collected in trifluoroethanol/water. The peptide N-terminal side presents a 9-residue helical segment, enclosing the processing site 2; the C-terminal segment can be described as a loop exposing the processing site 1. A hypothesis for the docking of p498 onto the catalytic domain of human furin, modeled by homology and fitting previous site-directed mutagenesis studies, is also presented. p498 site 1 is shown to have easy access to the furin catalytic site, unlike the nonphysiological site 2. Finally, on the basis of available data, we suggest a possible structural motif required for the gp160-PCs recognition.

Mapping key interactions in the dimerization process of HBHA from Mycobacterium tuberculosis, insights into bacterial agglutination

HBHA and HBHA bind by blue native page (View interaction)

Conformational features and binding affinities to Cripto, ALK7 and ALK4 of Nodal synthetic fragments

Nodal, a member of the TGF‐β superfamily, is a potent embryonic morphogen also implicated in tumor progression. As for other TGF‐βs, it triggers the signaling functions through the interaction with the extracellular domains of type I and type II serine/threonine kinase receptors and with the co‐receptor Cripto. Recently, we reported the molecular models of Nodal in complex with its type I receptors (ALK4 and ALK7) as well as with Cripto, as obtained by homology modeling and docking simulations. From such models, potential binding epitopes have been identified. To validate such hypotheses, a series of mutated Nodal fragments have been synthesized. These peptide analogs encompass residues 44–67 of the Nodal protein, corresponding to the pre‐helix loop and the H3 helix, and reproduce the wild‐type sequence or bear some modifications to evaluate the hot‐spot role of modified residues in the receptor binding.