Tommaso Eliseo

NMR Dynamic Studies Suggest that Allosteric Activation Regulates Ligand Binding in Chicken Liver Bile Acid-binding Protein

Apo chicken liver bile acid-binding protein has been structurally characterized by NMR. The dynamic behavior of the protein in its apo- and holo-forms, complexed with chenodeoxycholate, has been determined via 15N relaxation and steady state heteronuclear 15N(1H) nuclear Overhauser effect measurements. The dynamic parameters were obtained at two pH values (5.6 and 7.0) for the apoprotein and at pH 7.0 for the holoprotein, using the model free approach. Relaxation studies, performed at three different magnetic fields, revealed a substantial conformational flexibility on the microsecond to millisecond time scales, mainly localized in the C-terminal face of the β-barrel. The observed dynamics are primarily caused by the protonation/deprotonation of a buried histidine residue, His98, located on this flexible face. A network of polar buried side chains, defining a spine going from the E to J strand, is likely to provide the long range connectivity needed to communicate motion from His98 to the EF loop region. NMR data are accompanied by molecular dynamics simulations, suggesting that His98 protonation equilibrium is the triggering event for the modulation of a functionally important motion, i.e. the opening/closing at the protein open end, whereas ligand binding stabilizes one of the preexisting conformations (the open form). The results presented here, complemented with an analysis of proteins belonging to the intracellular lipid-binding protein family, are consistent with a model of allosteric activation governing the binding mechanism. The functional role of this mechanism is thoroughly discussed within the framework of the mechanism for the enterohepatic circulation of bile acids.

ATP Acts as a Regulatory Effector in Modulating Structural Transitions of Cytochrome c: Implications for Apoptotic Activity†

The binding of lipids (free fatty acids as well as acidic phospholipids) to cytochrome c (cyt c) induces conformational changes and partial unfolding of the protein, strongly influencing cyt c oxidase/peroxidase activity. ATP is unique among the nucleotides in being able to turn non-native states of cyt c back to the native conformation. The peroxidase activity acquired by lipid-bound cyt c turns out to be very critical in the early stages of apoptosis. Nucleotide specificity is observed for apoptosome formation and caspase activation, the cleavage occurring only in the presence of dATP or ATP. In this study, we demonstrate the connection between peroxidase activity and oleic acid-induced conformational transitions of cyt c and show how ATP is capable of modulating such interplay. By NMR measurement, we have demonstrated that ATP interacts with a site (S1) formed by K88, R91, and E62 and such interaction was weakened by mutation of E62, suggesting the selective role in the interaction played by the base moiety. Interestingly, the interactions of ATP and GTP with cyt c are significantly different at low nucleotide concentrations, with GTP being less effective in perturbing the S1 site and in eliciting apoptotic activity. To gain insights into the structural features of cyt c required for its pro-apoptotic activity and to demonstrate a regulatory role for ATP (compared to the effect of GTP), we have performed experiments on cell lysates by using cyt c proteins mutated on amino acid residues that, as suggested by NMR measurements, belong to S1. Thus, we provide evidence that ATP acts as an allosteric effector, regulating structural transitions among different conformations and different oxidation states of cyt c, which are endowed with apoptotic activity or not. On this basis, we suggest a previously unrecognized role for ATP binding to cyt c at low millimolar concentrations in the cytosol, beyond the known regulatory role during the oxidative phosphorylation in mitochondria.

Indirect DNA Readout on the Protein Side: Coupling between Histidine Protonation, Global Structural Cooperativity, Dynamics, and DNA Binding of the Human Papillomavirus Type 16 E2C Domain

DNA sequence recognition by the homodimeric C-terminal domain of the human papillomavirus type 16 E2 protein (E2C) is known to involve both direct readout and DNA-dependent indirect readout mechanisms, while protein-dependent indirect readout has been deduced but not directly observed. We have investigated coupling between specific DNA binding and the dynamics of the unusual E2C fold, using pH as an external variable. Nuclear magnetic resonance and isothermal titration calorimetry show that pH titration of His318 in the complex interface and His288 in the core of the domain is coupled to both binding and the dynamics of the beta-barrel core of E2C, with a tradeoff between dimer stability and function. Specific DNA binding is, in turn, coupled to the slow dynamics and amide hydrogen exchange in the entire beta-barrel, reaching residues far apart from the DNA recognition elements but not affecting the two helices of each monomer. The changes are largest in the dimerization interface, suggesting that the E2C beta-barrel acts as a hinge that regulates the relative position of the DNA recognition helices. In conclusion, the cooperative dynamics of the human papillomavirus type 16 E2C beta-barrel is coupled to sequence recognition in a protein-dependent indirect readout mechanism. The patterns of residue substitution in genital papillomaviruses support the importance of the protonation states of His288 and His318 and suggest that protein-dependent indirect readout and histidine pH titration may regulate DNA binding in the cell.

Thymosin α1 inserts N terminus into model membranes assuming a helical conformation

Objective: Thymosin α1 (Tα1) is a peptide hormone whose therapeutic application has been approved in several diseases, but the description of a precise receptor for its therapeutic action still remains elusive and some knowledge of the mechanism of interaction with the cell membrane still needs to be clarified. This work is aimed at studying the folding and interaction of Tα1, which is completely unstructured in water solution, with model membranes. Methods: The folding and interaction of Tα1 with sodium dodecyl sulfate micelles was monitored by NMR and CD spectroscopy techniques. Results: Tα1 assumes a helical conformation in the presence of sodium dodecyl sulfate micelles, showing a helical fold with a structural break around residues 9 and 14. These results were confirmed by circular dichroism and NMR spectroscopy. Moreover, by paramagnetic NMR relaxation it was found that Tα1 is inserted in the hydrophobic region of the micelles by the residues 1 – 5 of the N-terminal end. This result clarifies the modality of insertion that was not obtained in previous NMR studies in trifluoroethanol. Conclusions: These findings suggest that Tα1 folds on the membrane and, when inserted, may be able to interact with nearby proteins and/or receptors acting as an effector and causing a biological signaling cascade.

MD Simulations of Papillomavirus DNA-E2 Protein Complexes Hints at a Protein Structural Code for DNA Deformation

The structural dynamics of the DNA binding domains of the human papillomavirus strain 16 and the bovine papillomavirus strain 1, complexed with their DNA targets, has been investigated by modeling, molecular dynamics simulations, and nuclear magnetic resonance analysis. The simulations underline different dynamical features of the protein scaffolds and a different mechanical interaction of the two proteins with DNA. The two protein structures, although very similar, show differences in the relative mobility of secondary structure elements. Protein structural analyses, principal component analysis, and geometrical and energetic DNA analyses indicate that the two transcription factors utilize a different strategy in DNA recognition and deformation. Results show that the protein indirect DNA readout is not only addressable to the DNA molecule flexibility but it is finely tuned by the mechanical and dynamical properties of the protein scaffold involved in the interaction.

Molecular dynamics of the DNA-binding domain of the papillomavirus E2 transcriptional regulator uncover differential properties for DNA target accommodation.

Papillomaviruses are small DNA tumor viruses that infect mammalian hosts, with consequences from benign to cancerous lesions. The Early protein 2 is the master regulator for the virus life cycle, participating in gene transcription, DNA replication, and viral episome migration. All of these functions rely on primary target recognition by its dimeric DNA‐binding domain. In this work, we performed molecular dynamics simulations in order to gain insights into the structural dynamics of the DNA‐binding domains of two prototypic strains, human papillomavirus strain 16 and the bovine papillomavirus strain 1. The simulations underline different dynamic features in the two proteins. The human papillomavirus strain 16 domain displays a higher flexibility of the β2–β3 connecting loop in comparison with the bovine papillomavirus strain 1 domain, with a consequent effect on the DNA‐binding helices, and thus on the modulation of DNA recognition. A compact β‐barrel is found in human papillomavirus strain 16, whereas the bovine papillomavirus strain 1 protein is characterized by a loose β‐barrel with a large number of cavities filled by water, which provides great flexibility. The rigidity of the human papillomavirus strain 16 β‐barrel prevents protein deformation, and, as a consequence, deformable spacers are the preferred targets in complex formation. In contrast, in bovine papillomavirus strain 1, a more deformable β‐barrel confers greater adaptability to the protein, allowing the binding of less flexible DNA regions. The flexibility data are confirmed by the experimental NMR S2 values, which are reproduced well by calculation. This feature may provide the protein with an ability to discriminate between spacer sequences. Clearly, the deformability required for the formation of the Early protein 2 C‐terminal DNA‐binding domain–DNA complexes of various types is based not only on the rigidity of the base sequences in the DNA spacers, but also on the intrinsic deformability properties of each domain.

Structure of the lipodepsipeptide syringomycin e in phospholipids and sodium dodecylsulphate micelle studied by circular dichroism, NMR spectroscopy and molecular dynamics

Syringomycin E (SRE) is a member of a family of lipodepsipeptides that characterize the secondary metabolism of the plant-associated bacteria Pseudomonas syringae pv. syringae. It displays phytotoxic, antifungal and haemolytic activities, due to the membrane interaction and ion channel formation. To gain an insight into the conformation of SRE in the membrane environment, we studied the conformation of SRE bound to SDS micelle, a suitable model for the membrane-bound SRE. In fact, highly similar circular dichroism (CD) spectra were obtained for SRE bound to sodium dodecylsulphate (SDS) and to a phospholipid bilayer, indicating the conformational equivalence of SRE in these two media, at difference with the CD spectrum of SRE in water solution. The structure of SDS-bound SRE was determined by NMR spectroscopy combined with molecular dynamics calculations in octane environment. The results of this study highlight the influence of the interaction with lipids in determining the three-dimensional structure of SRE and provide the basis for further investigations on structural determinants of syringomycin E-membrane interaction.

Structural characterization of the Hepatitis C Virus NS3 protease from genotype 3a: The basis of the genotype 1b vs. 3a inhibitor potency shift

The first structural characterization of the genotype 3a Hepatitis C Virus NS3 protease is reported, providing insight into the differential susceptibility of 1b and 3a proteases to certain inhibitors. Interaction of the 3a NS3 protease with a P2-P4 macrocyclic and a linear phenethylamide inhibitor was investigated. In addition, the effect of the NS4A cofactor binding on the conformation of the protease was analyzed. Complexation of NS3 with the phenethylamide inhibitor significantly stabilizes the protease but binding does not involve residues 168 and 123, two key amino acids underlying the different inhibition of genotype 1b vs. 3a proteases by P2-P4 macrocycles. Therefore, we studied the dynamic behavior of these two residues in the phenethylamide complex, serving as a model of the situation in the apo 3a protein, in order to explore the structural basis of the inhibition potency shift between the proteases of the genotypes 1b and 3a.

NMR-based homology model for the solution structure of the C-terminal globular domain of EMILIN1

EMILIN1 is a glycoprotein of elastic tissues that has been recently linked to the pathogenesis of hypertension. The protein is formed by different independently folded structural domains whose role has been partially elucidated. In this paper the solution structure, inferred from NMR-based homology modelling of the C-terminal trimeric globular C1q domain (gC1q) of EMILIN1, is reported. The high molecular weight and the homotrimeric structure of the protein required the combined use of highly deuterated 15N, 13C-labelled samples and TROSY experiments. Starting from a homology model, the protein structure was refined using heteronuclear residual dipolar couplings, chemical shift patterns, NOEs and H-exchange data. Analysis of the gC1q domain structure of EMILIN1 shows that each protomer of the trimer adopts a nine-stranded β sandwich folding topology which is related to the conformation observed for other proteins of the family. Distinguishing features, however, include a missing edge-strand and an unstructured 19-residue loop. Although the current data do not allow this loop to be precisely defined, the available evidence is consistent with a flexible segment that protrudes from each subunit of the globular trimeric assembly and plays a key role in inter-molecular interactions between the EMILIN1 gC1q homotrimer and its integrin receptor α4β1.

A new member of the ribbon-helix-helix transcription factor superfamily from the plant pathogen Xanthomonas axonopodis pv. citri

XACb0070 is an uncharacterized protein coded by the two large plasmids isolated from Xanthomonas axonopodis pv. citri, the agent of citrus canker and responsible for important economical losses in citrus world production. XACb0070 presents sequence homology only with other hypothetical proteins belonging to plant pathogens, none of which have their structure determined. The NMR-derived solution structure reveals this protein is a homodimer in which each monomer presents two domains with different structural and dynamic properties: a folded N-terminal domain with beta alpha alpha topology which mediates dimerization and a long disordered C-terminal tail. The folded domain shows high structural similarity to the ribbon-helix-helix transcriptional repressors, a family of DNA-binding proteins of conserved 3D fold but low sequence homology: indeed XACb0070 binds DNA. Primary sequence and fold comparison of XACb0070 with other proteins of the ribbon-helix-helix family together with examination of the genes in the vicinity of xacb0070 suggest the protein might be the component of a toxin-antitoxin system.