Davide Cavazzini

Dramatic modulation of electron transfer in protein complexes by crosslinking

The transfer of electrons between proteins is an essential step in biological energy production. Two protein redox partners are often artificially crosslinked to investigate the poorly understood mechanism by which they interact. To better understand the effect of crosslinking on electron transfer rates, we have constructed dimers of azurin by crosslinking the monomers. The measured electron exchange rates, combined with crystal structures of the dimers, demonstrate that the length of the linker can have a dramatic effect on the structure of the dimer and the electron transfer rate. The presence of ordered water molecules in the protein–protein interface may considerably influence the electronic coupling between redox centers.

A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: Structural determinants of redox potential

Flavodoxin II from Azotobacter vinelandii is a “long‐chain” flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three‐dimensional structure determined to a resolution of 2.25 Å by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five‐stranded parallel β‐sheet flanked on either side by α‐helices. An eight‐residue insertion, compared with other long‐chain flavodoxins, forms a short 310 helix preceding the start of the α3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent‐accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E2 value in particular.

Retinoic acid synthesis in the somatic cells of rat seminiferous tubules.

At physiological plasma concentrations, retinoic acid (RA) cannot cross the blood-testis barrier formed by Sertoli and peritubular cells, and it is thought to be mainly synthesized in situ through the oxidation of retinol. We have thus examined the in vitro RA biosynthetic capacity of cultured Sertoli and peritubular cells isolated from the seminiferous tubules of prepubertal rats, using holo-cellular retinol binding protein (CRBP) as a substrate. Although both somatic cell types contain CRBP and retinoic acid nuclear receptors, RA synthesis was only detected with Sertoli cell subcellular fractions. Most of the RA synthesizing activity of these cells is contributed by a microsomal-cytosolic system that shares many functional similarities with a RA biosynthetic pathway originally identified in rat liver. RA synthesis is maximal at a time of postnatal life (20 days) preceding meiotic cell accumulation and remains nearly constant thereafter. The unique ability of Sertoli cell subcellular fractions to support RA formation from holoCRBP, along with the observed age-dependent modulation of this activity, indicate that Sertoli cells represent the main site of intratubular RA production and that they may play a key role in controlling RA-dependent processes within the seminiferous tubule.

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge-charge interactions pre-orient cytochrome c6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.

Comparison of the refined crystal structures of wild-type (1.34 Å) flavodoxin from Desulfovibrio vulgaris and the S35C mutant (1.44 Å) at 100 K

Engineered flavodoxins in which a surface residue has been replaced by an exposed cysteine are useful modules to link multi-domain redox proteins obtained by gene fusion to electrode surfaces. In the present work, the crystal structure of the S35C mutant of Desulfovibrio vulgaris flavodoxin in the oxidized state has been determined and compared with a refined structure of the wild type (wt). The structure of wt flavodoxin (space group P4(3)2(1)2, unit-cell parameters a = 50.52, b = 50.52, c = 138.59 A) at 1.34 A resolution has been refined to R = 0.16 and R(free) = 0.18. The structure of the S35C mutant (space group P4(3)2(1)2, unit-cell parameters a = 50.55, b = 50.55, c = 138.39 A) at 1.44 A resolution has been refined to R = 0.13 and R(free) = 0.16. Data sets were collected with synchrotron radiation at 100 K. In the S35C mutant, the Cys35 thiol group points towards a hydrophobic region, whilst in the wt the Ser35 hydroxyl group points towards a more polar region. The solvent exposure of Cys35 is 43 A(2), of which 8 A(2) is for the sulfur. This is comparable to the exposure of 48 A(2) found for the wt Ser35, where that of the hydroxyl oxygen is also 8 A(2).

Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A2

Background: TbSP1 is a phospholipase A2 strongly up-regulated during the symbiotic phase of the truffle Tuber borchii. Results: An activated enzyme species composed of five α-helices is generated by self-proteolysis through an intermolecular reaction. Conclusion: TbSP1 autoproteolysis is a site-specific post-translational modification not involving the phospholipase active site. Significance: Autoproteolytic activation is described for the first time for a microbial PLA2, with possible implications for symbiosis establishment. Fungal phospholipases are members of the fungal/bacterial group XIV secreted phospholipases A2 (sPLA2s). TbSP1, the sPLA2 primarily addressed in this study, is up-regulated by nutrient deprivation and is preferentially expressed in the symbiotic stage of the ectomycorrhizal fungus Tuber borchii. A peculiar feature of this phospholipase and of its ortholog from the black truffle Tuber melanosporum is the presence of a 54-amino acid sequence of unknown functional significance, interposed between the signal peptide and the start of the conserved catalytic core of the enzyme. X-ray diffraction analysis of a recombinant TbSP1 form corresponding to the secreted protein previously identified in T. borchii mycelia revealed a structure comprising the five α-helices that form the phospholipase catalytic module but lacking the N-terminal 54 amino acids. This finding led to a series of functional studies that showed that TbSP1, as well as its T. melanosporum ortholog, is a self-processing pro-phospholipase A2, whose phospholipase activity increases up to 80-fold following autoproteolytic removal of the N-terminal peptide. Proteolytic cleavage occurs within a serine-rich, intrinsically flexible region of TbSP1, does not involve the phospholipase active site, and proceeds via an intermolecular mechanism. Autoproteolytic activation, which also takes place at the surface of nutrient-starved, sPLA2 overexpressing hyphae, may strengthen and further control the effects of phospholipase up-regulation in response to nutrient deprivation, also in the context of symbiosis establishment and mycorrhiza formation.

New insights on the protein-ligand interaction differences between the two primary cellular retinol carriers

The main retinol carriers in the cytosol are the cellular retinol-binding proteins types I and II (CRBP-I and CRBP-II), which exhibit distinct tissue distributions. They play different roles in the maintenance of vitamin A homeostasis and feature a 100-fold difference in retinol affinity whose origin has not been described in detail. NMR-based hydrogen/deuterium exchange measurements show that, while retinol binding endows both proteins with a more rigid structure, many amide protons exchange much faster in CRBP-II than in CRBP-I in both apo and holo form, despite the conserved three-dimensional fold. The remarkable difference in intrinsic stability between the two homologs appears to modulate their binding properties: the stronger retinol binder CRBP-I displays a reduced flexibility of the backbone structure with respect to CRBP-II. This difference must derive from specific evolution-based amino acid substitutions, resulting in additional stabilization of the CRBP-I scaffold: in fact, we identified a number of potential salt bridges on the protein surface as well as several key interactions inside the binding cavity. Furthermore, our NMR data demonstrate that helix αII of the characteristic helix-turn-helix motif in the ligand portal region exists in both apo and holo CRBP-II. Hence, the previously proposed model of retinol binding needs to be revised.

Redox properties and crystal structures of a Desulfovibrio vulgaris flavodoxin mutant in the monomeric and homodimeric forms.

The mutant S64C of the short-chain flavodoxin from Desulfovibrio vulgaris has been designed to introduce an accessible and reactive group on the protein surface. Crystals have been obtained of both the monomeric and homodimeric forms of the protein, with the cofactor FMN in either the oxidized or the one electron-reduced (semiquinone) state, and the structures have been determined to high resolution. The redox properties of the different species have been investigated and the variations observed with respect to wild type have been related to the structural changes induced by the mutation and S-S bridge formation.

Structure of S35C flavodoxin mutant from Desulfovibrio vulgaris in the semiquinone state.

The crystallographic structure of an engineered flavodoxin mutant from Desulfovibrio vulgaris has been analysed. Site-directed mutagenesis was used to substitute serine 35 with a cysteine to provide a possible covalent linkage. The crystal structure of the semiquinone form of this mutant is similar to the corresponding oxidation state of the wild-type flavodoxin. Analysis of the structural changes reveals the interaction between N(5)H of the flavin and the carbonyl O atom of Gly61 to be critical for modulation of the electrochemical properties of the protein.

Complexes between recombinant intracellular carriers of vitamin A and their specific ligands investigated by electrospray-mass spectrometry.

The intracellular carriers of vitamin A, cellular retinol-binding protein type I, cellular retinol-binding protein type II and cellular retinoic acid-binding protein type I are members of the intracellular lipid-binding proteins family, in which the ligand-binding cavity is located in the interior of a barrel-like structure. The dissociation constants of the specific complexes in water solutions around neutrality are very low (in the 0.1 to 10 nM range). Because of their high stability, they represent ideal systems to verify the adequacy of electrospray ionization mass spectrometry in the analysis of non-covalent protein–ligand complexes. The electrospray interface parameters were varied to detect the presence of species not present in solution but generated as artefacts during transfer of complexes from the condensed state to the gas-phase. The results clearly indicate that mass-spectrometry data reflect the situation present in solution only if the electrospray conditions are carefully selected. In particular, the values of cone voltage and temperature compatible with persistence of the complexes in the gas phase were determined for each vitamin A carrier. Lack of correlation between complex stability in solution and in the gas phase is attributable to the specific and differential effects of the two environments on protein conformation and ligand–protein interactions.