Biancamaria Farina

Deciphering the zinc coordination properties of the prokaryotic zinc finger domain: The solution structure characterization of Ros87 H42A functional mutant

The zinc coordination sphere in prokaryotic zinc finger domain is extremely versatile and influences the stability and the folding property of the domain. Of a particular interest is the fourth zinc coordinating position, which is frequently occupied by two successive histidines, both able to coordinate the metal ion. To clarify their structural and functional role we report the NMR solution structure and the dynamics behavior of Ros87 H42A, which is a functional mutant of Ros87, the DNA binding domain of the Ros protein containing a prokaryotic Cys2His2 zinc finger domain. The structural analysis indicates that reducing the spacer among the two coordinating histidines from 4 (among His37 and His42) amino acids to 3 (among His37 and His41) increases the helicity of the first α-helix. At the same time, the second helix appears more mobile in the μs-ms timescale and the hydrophobic core is reduced. These data explain the high frequency of three-residue His spacers in the eukaryotic zinc finger domain and their absence in the prokaryotic counterpart. Furthermore, the structural comparison shows that the second coordination position is more sensitive to H42A mutation with respect to the first and the third position, providing the rationale of the high variability of the second and the fourth zinc coordinating position in Ros homologs, which adopt different metal coordination but preserve similar tertiary structures and DNA binding activities. Finally, H/D exchange measurements and NMR thermal unfolding analysis indicate that this mutant likely unfolds via a different mechanism with respect to the wild-type.

Structural and functional studies of Stf76 from the Sulfolobus islandicus plasmid–virus pSSVx: a novel peculiar member of the winged helix–turn–helix transcription factor family

The hybrid plasmid–virus pSSVx from Sulfolobus islandicus presents an open reading frame encoding a 76 amino acid protein, namely Stf76, that does not show significant sequence homology with any protein with known 3D structure. The recombinant protein recognizes specifically two DNA-binding sites located in its own promoter, thus suggesting an auto-regulated role of its expression. Circular dichroism, spectrofluorimetric, light scattering and isothermal titration calorimetry experiments indicated a 2:1 molar ratio (protein:DNA) upon binding to the DNA target containing a single site. Furthermore, the solution structure of Stf76, determined by nuclear magnetic resonance (NMR) using chemical shift Rosetta software, has shown that the protein assumes a winged helix–turn–helix fold. NMR chemical shift perturbation analysis has been performed for the identification of the residues responsible for DNA interaction. In addition, a model of the Stf76–DNA complex has been built using as template a structurally related homolog.

Molecular strategies to replace the structural metal site in the prokaryotic zinc finger domain

The specific arrangement of secondary elements in a local motif often totally relies on the formation of coordination bonds between metal ions and protein ligands. This is typified by the ~30 amino acid eukaryotic zinc finger motif in which a β-sheet and an α-helix are clustered around a zinc ion by various combinations of four ligands. The prokaryotic zinc finger domain (found in the Ros protein from Agrobacterium tumefaciens) is different from the eukaryotic counterpart as it consists of 58 amino acids arranged in a βββαα topology stabilized by a 15-residue hydrophobic core. Also, this domain tetrahedrally coordinates zinc and unfolds in the absence of the metal ion. The characterization of proteins belonging to the Ros homologs family has however shown that the prokaryotic zinc finger domain can overcome the metal requirement to achieve the same fold and DNA-binding activity. In the present work, two zinc-lacking Ros homologs (Ml4 and Ml5 proteins) have been thoroughly characterized using bioinformatics, biochemical and NMR techniques. We show how in these proteins a network of hydrogen bonds and hydrophobic interactions surrogate the zinc coordination role in the achievement of the same functional fold.

NMR backbone dynamics studies of human PED⁄PEA-15 outline protein functional sites

PED/PEA‐15 (phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes) is a ubiquitously expressed protein and a key regulator of cell growth and glucose metabolism. PED/PEA‐15 mediates both homotypic and heterotypic interactions and is constituted by an N‐terminal canonical death effector domain and a C‐terminal tail. In the present study, the backbone dynamics of PED/PEA‐15 via 15N R1 and R2 and steady‐state [1H]‐15N NOE measurements is reported. The dynamic parameters were analyzed using both Lipari–Szabo model‐free formalism and a reduced spectral density mapping approach. The results obtained define a polar and charged surface of the death effector domain characterized by internal motions in the micro‐ to millisecond timescale, which is crucial for the multiple heterotypic functional protein–protein interactions in which PED/PEA‐15 is involved. The present study contributes to a better understanding of the molecular basis of the PED/PEA‐15 functional interactions and provides a more detailed surface for the design and development of PED/PEA‐15 binders.

The (unusual) aspartic acid in the metal coordination sphere of the prokaryotic zinc finger domain.

The possibility of choices of protein ligands and coordination geometries leads to diverse Zn(II) binding sites in zinc-proteins, allowing a range of important biological roles. The prokaryotic Cys2His2 zinc finger domain (originally found in the Ros protein from Agrobacterium tumefaciens) tetrahedrally coordinates zinc through two cysteine and two histidine residues and it does not adopt a correct fold in the absence of the metal ion. Ros is the first structurally characterized member of a family of bacterial proteins that presents several amino acid changes in the positions occupied in Ros by the zinc coordinating residues. In particular, the second position is very often occupied by an aspartic acid although the coordination of structural zinc by an aspartate in eukaryotic zinc fingers is very unusual. Here, by appropriately mutating the protein Ros, we characterize the aspartate role within the coordination sphere of this family of proteins demonstrating how the presence of this residue only slightly perturbs the functional structure of the prokaryotic zinc finger domain while it greatly influences its thermodynamic properties.

Molecular basis of the PED/PEA15 interaction with the C-terminal fragment of phospholipase D1 revealed by NMR spectroscopy.

PED/PEA15 is a small protein involved in many protein-protein interactions that modulates the function of a number of key cellular effectors involved in major cell functions, including apoptosis, proliferation and glucose metabolism. In particular, PED/PEA15 interacts with the phospholipase D (PLD) isoforms 1 and 2 increasing protein kinase C-α isoform activity and affects both insulin-stimulated glucose transport and glucose-stimulated insulin secretion. The C-terminal portion (residues 712-1074) of PLD1, named D4, is still able to interact with PED/PEA15. In this study we characterized, by means of NMR spectroscopy, the molecular interaction of PED/PEA15 with D4α, a smaller region of D4, encompassing residues 712-818, shown to have the same affinity for PED/PEA15 and to induce the same effects as D4 in PED/PEA15-overexpressing cells. Chemical shift perturbation (CSP) studies allowed to define D4α binding site of PED/PEA15 and to identify a smaller region likely affected by an allosteric effect. Moreover, ELISA-like experiments showed that three 20-mer overlapping synthetic peptides, covering the 762-801 region of D4α, strongly inhibit PED/PEA15-D4α interaction through their binding to PED/PEA15 with KDs in low micromolar range. Finally, molecular details of the interaction of PED/PEA15 with one of the three peptides have been revealed by CSP and saturation transfer difference (STD) analyses.

Targeting metalloproteins by fragment-based lead discovery.

It has been estimated that nearly one‐third of functional proteins contain a metal ion. These constitute a wide variety of possible drug targets including metalloproteinases, dehydrogenases, oxidoreductases, hydrolases, deacetylases, or many others in which the metal ion is either of catalytic or of structural nature. Despite the predominant role of a metal ion in so many classes of drug targets, current high‐throughput screening techniques do not usually produce viable hits against these proteins, likely due to the lack of proper metal‐binding pharmacophores in the current screening libraries. Herein, we describe a novel fragment‐based drug discovery approach using a metal‐targeting fragment library that is based on a variety of distinct classes of metal‐binding groups designed to reliably anchor the fragments at the target’s metal ions. We show that the approach can effectively identify novel, potent and selective agents that can be readily developed into metalloprotein‐targeted therapeutics.

Synthesis, conformational analysis and immunological activity of β3Phe‐substituted Cyclolinopeptide A analogues

CLA, a natural, highly hydrophobic cyclic nonapeptide with sequence c(Pro1‐Pro2‐Phe3‐Phe4‐Leu5‐Ile6‐Ile7‐Leu8‐Val9‐), isolated from linseed oil, was found to possess a strong immunosuppressive activity comparable, in low doses, with that of CsA, with a mechanism that depends on the inhibition of the interleukin‐1 and interleukin‐2 action. Structural analysis of CLA and its related compounds has underlined that the presence of the tetrapeptide Pro‐Pro‐Phe‐Phe sequence, the Pro‐Pro cis amide bond, and the ‘edge‐to‐face’ interaction are possible important features for the immunosuppressive activity of CLA. To evaluate the role and significance of ‘edge‐to‐face’ interaction in the process of molecular recognition by receptors, we have synthesised three linear precursors and three cyclic analogues of CLA, in which one or both Phe residues have been replaced by β3Phe residues. A conformational analysis by NMR in CD3CN/H2O mixture has been carried out on the CLA analogue, in which Phe3 has been replaced by a βPhe, to study the influence of the mutation on the three‐dimensional structure. All linear and cyclic CLA analogues containing βPhe have been tested in the humoral and cellular immune response in vivo assays in mice. The peptide activities have been compared with CsA, as a reference drug. Copyright

A Combined NMR and Computational Approach to Determine the RGDechi-hCit-αv β3 Integrin Recognition Mode in Isolated Cell Membranes.

The critical role of integrins in tumor progression and metastasis has stimulated intense efforts to identify pharmacological agents that can modulate integrin function. In recent years, αv β3 and αv β5 integrin antagonists were demonstrated to be effective in blocking tumor progression. RGDechi-hCit, a chimeric peptide containing a cyclic RGD motif linked to an echistatin C-terminal fragment, is able to recognize selectively αv β3 integrin both in vitro and in vivo. High-resolution molecular details of the selective αv β3 recognition of the peptide are certainly required, nonetheless RGDechi-hCit internalization limited the use of classical in cell NMR experiments. To overcome such limitations, we used WM266 isolated cellular membranes to accomplish a detailed NMR interaction study that, combined with a computational analysis, provides significant structural insights into αv β3 molecular recognition by RGDechi-hCit. Remarkably, on the basis of the identified molecular determinants, we design a RGDechi-hCit mutant that is selective for αv β5 integrin.

Targeting Zinc Finger Domains with Small Molecules: Solution Structure and Binding Studies of the RanBP2-Type Zinc Finger of RBM5.

The RNA binding motif protein 5 (RBM5), also known as Luca15 or H37, is a component of prespliceosomal complexes that regulates the alternative splicing of several mRNAs, such as Fas and caspase‐2. The RBM5 gene is located at the 2p21.3 chromosomal region, which is strongly associated with lung cancer and many other cancers. Both increased and decreased levels of RBM5 can play a role in tumor progression. In particular, downregulation of rbm5 is involved in lung cancer and other cancers upon Ras activation, and, also, represents a molecular signature associated with metastasis in various solid tumors. On the other hand, upregulation of RBM5 occurs in breast and ovarian cancer. Moreover, RBM5 was also found to be involved in the early stage of the HIV‐1 viral cycle, representing a potential target for the treatment of the HIV‐1 infection. While the molecular basis for RNA recognition and ubiquitin interaction has been structurally characterized, small molecules binding this zinc finger (ZF) domain that might contribute to characterizing their activity and to the development of potential therapeutic agents have not yet been reported. Using an NMR screening of a fragment library we identified several binders and the complex of the most promising one, compound 1, with the RBM5 ZF1 was structurally characterized in solution. Interestingly, the binding mechanism reveals that 1 occupies the RNA binding pocket and is therefore able to compete with the RNA to bind RBM5 RanBP2‐type ZF domain, as indicated by NMR studies.