Linda Celeste Montemiglio

Investigating the Structural Plasticity of a Cytochrome P450 THREE-DIMENSIONAL STRUCTURES OF P450 EryK AND BINDING TO ITS PHYSIOLOGICAL SUBSTRATE

Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of substrate recognition. In this work, we analyzed the three-dimensional structures of a monomeric cytochrome P450 from Saccharopolyspora erythraea, commonly called EryK, and the binding kinetics to its physiological ligand, erythromycin D. Three different structures of EryK were obtained: two ligand-free forms and one in complex with its substrate. Analysis of the substrate-bound structure revealed the key structural determinants involved in substrate recognition and selectivity. Interestingly, the ligand-free structures of EryK suggested that the protein may explore an open and a closed conformation in the absence of substrate. In an effort to validate this hypothesis and to investigate the energetics between such alternative conformations, we performed stopped-flow absorbance experiments. Data demonstrated that EryK binds erythromycin D via a mechanism involving at least two steps. Contrary to previously characterized cytochrome P450s, analysis of double jump mixing experiments confirmed that this complex scenario arises from a pre-existing equilibrium between the open and closed subpopulations of EryK, rather than from an induced-fit type mechanism.

Sequence-specific Long Range Networks in PSD-95/Discs Large/ZO-1 (PDZ) Domains Tune Their Binding Selectivity

Protein-protein interactions mediated by modular protein domains are critical for cell scaffolding, differentiation, signaling, and ultimately, evolution. Given the vast number of ligands competing for binding to a limited number of domain families, it is often puzzling how specificity can be achieved. Selectivity may be modulated by intradomain allostery, whereby a remote residue is energetically connected to the functional binding site via side chain or backbone interactions. Whereas several energetic pathways, which could mediate intradomain allostery, have been predicted in modular protein domains, there is a paucity of experimental data to validate their existence and roles. Here, we have identified such functional energetic networks in one of the most common protein-protein interaction modules, the PDZ domain. We used double mutant cycles involving site-directed mutagenesis of both the PDZ domain and the peptide ligand, in conjunction with kinetics to capture the fine energetic details of the networks involved in peptide recognition. We performed the analysis on two homologous PDZ-ligand complexes and found that the energetically coupled residues differ for these two complexes. This result demonstrates that amino acid sequence rather than topology dictates the allosteric pathways. Furthermore, our data support a mechanism whereby the whole domain and not only the binding pocket is optimized for a specific ligand. Such cross-talk between binding sites and remote residues may be used to fine tune target selectivity.

Side-chain interactions form late and cooperatively in the binding reaction between disordered peptides and PDZ domains

Intrinsically disordered proteins are very common and mediate numerous protein-protein and protein-DNA interactions. While it is clear that these interactions are instrumental for the life of the mammalian cell, there is a paucity of data regarding their molecular binding mechanisms. Here we have used short peptides as a model system for intrinsically disordered proteins. Linear free energy relationships based on rate and equilibrium constants for the binding of these peptides to ordered target proteins, PDZ domains, demonstrate that native side-chain interactions form mainly after the rate-limiting barrier for binding and in a cooperative fashion. This finding suggests that these disordered peptides first form a weak encounter complex with non-native interactions. The data do not support the recent notion that the affinities of intrinsically disordered proteins toward their targets are generally governed by their association rate constants. Instead, we observed the opposite for peptide-PDZ interactions, namely, that changes in K(d) correlate with changes in k(off).

Azole Drugs Trap Cytochrome P450 Eryk in Alternative Conformational States.

EryK is a bacterial cytochrome P450 that catalyzes the last hydroxylation occurring during the biosynthetic pathway of erythromycin A in Streptomyces erythraeus. We report the crystal structures of EryK in complex with two widely used azole inhibitors: ketoconazole and clotrimazole. Both of these ligands use their imidazole moiety to coordinate the heme iron of P450s. Nevertheless, because of the different chemical and structural properties of their N1-substituent group, ketoconazole and clotrimazole trap EryK, respectively, in a closed and in an open conformation that resemble the two structures previously described for the ligand-free EryK. Indeed, ligands induce a distortion of the internal helix I that affects the accessibility of the binding pocket by regulating the kink of the external helix G via a network of interactions that involves helix F. The data presented thus constitute an example of how a cytochrome P450 may be selectively trapped in different conformational states by inhibitors.

Engineering the internal cavity of neuroglobin demonstrates the role of the haem-sliding mechanism.

Neuroglobin is a member of the globin family involved in neuroprotection; it is primarily expressed in the brain and retina of vertebrates. Neuroglobin belongs to the heterogeneous group of hexacoordinate globins that have evolved in animals, plants and bacteria, endowed with the capability of reversible intramolecular coordination, allowing the binding of small gaseous ligands (O2, NO and CO). In a unique fashion among haemoproteins, ligand-binding events in neuroglobin are dependent on the sliding of the haem itself within a preformed internal cavity, as revealed by the crystal structure of its CO-bound derivative. Point mutants of the neuroglobin internal cavity have been engineered and their functional and structural characterization shows that hindering the haem displacement leads to a decrease in CO affinity, whereas reducing the cavity volume without interfering with haem sliding has negligible functional effects.

Functional analysis and crystallographic structure of clotrimazole bound OleP, a cytochrome P450 epoxidase from Streptomyces antibioticus involved in oleandomycin biosynthesis.

BACKGROUND OleP is a cyt P450 from Streptomyces antibioticus carrying out epoxigenation of the antibiotic oleandomycin during its biosynthesis. The timing of its reaction has not been fully clarified, doubts remain regarding its substrate and catalytic mechanism. METHODS The crystal structure of OleP in complex with clotrimazole, an inhibitor of P450s used in therapy, was solved and the complex formation dynamics was characterized by equilibrium and kinetic binding studies and compared to ketoconazole, another azole differing for the N1-substituent. RESULTS Clotrimazole coordinates the heme and occupies the active site. Most of the residues interacting with clotrimazole are conserved and involved in substrate binding in MycG, the P450 epoxigenase with the highest homology with OleP. Kinetic characterization of inhibitor binding revealed OleP to follow a simple bimolecular reaction, without detectable intermediates. CONCLUSIONS Clotrimazole-bound OleP adopts an open form, held by a π-π stacking chain that fastens helices F and G and the FG loop. Affinity is affected by the interactions of the N1 substituent within the active site, given the one order of magnitude difference of the off-rate constants between clotrimazole and ketoconazole. Based on structural similarities with MycG, we propose a binding mode for both oleandomycin intermediates, that are the candidate substrates of OleP. GENERAL SIGNIFICANCE Among P450 epoxigenases OleP is the only one that introduces an epoxide on a non-activated C–C bond. The data here presented are necessary to understand the rare chemistry carried out by OleP, to engineer it and to design more selective and potent P450-targeted drugs.

Culture of skeletal muscle cells in unprecedented proximity to a gold surface

Culturing of skeletal muscle cells on conductive surfaces is required to develop electronic device-muscle junctions for tissue engineering and medical applications. We characterized from a molecular and morphological point of view myogenic cells cultured on gold and on cysteamine-coated gold, as compared to the standard plastic for cell culture. Our results show that cell proliferation and survival are comparable between cells grown on either of the gold surface or plastic. The majority of the cells cultured on gold surfaces retain the ability to respond to differentiation cues, as shown by nuclear translocation of myogenin. Following terminal differentiation, the myotubes cultured on cysteamine-coated gold resemble myotube cultures obtained on plastic for the size and orientation of the myotube bundles retaining most of myosin expression; on the contrary, the myotube cultures on gold show a clumped morphology, likely due to repulsive cell-substratum interaction resulting in aberrant differentiation. On the basis of the aforementioned evidences, the culture of muscle cells on cysteamine-coated gold represents an advance with respect to previously reported substrata. The cysteamine self-assembled monolayer coating is a simple approach to accomplish cultures of myotubes in unprecedented tight proximity to conductive surfaces.

Redirecting P450 EryK specificity by rational site-directed mutagenesis

The C-12 hydroxylase EryK is a bacterial cytochrome P450, active during one of the final tailoring steps of erythromycin A (ErA) biosynthesis. Its tight substrate specificity, restricted to the metabolic intermediate ErD, leads to the accumulation in the culture broth of a shunt metabolite, ErB, that originates from the competitive action of a methyltranferase on the substrate of EryK. Although the methylation of the mycarosyl moiety represents the only difference between the two metabolites, EryK exhibits very low conversion of ErB in ErA via a parallel pathway. Given its limited antimicrobial activity and its moderate toxicity, contamination by such a byproduct decreases the yield and purity of the antibiotic. In this study, EryK has been redesigned to make it suitable for industrial application. Taking advantage of the three-dimensional structure of the enzyme in complex with ErD, three single active-site mutants of EryK (M86A, H88E, and E89L) have been designed to allow hydroxylation of the nonphysiological substrate ErB. The binding and catalytic properties of these three variants on both ErD and ErB have been analyzed. Interestingly, we found the mutation of Met 86 to Ala to yield enzymatic activity on both ErB and ErD. The three-dimensional structure of the complex of mutated EryK with ErB revealed that the mutation allows ErB to accommodate in the active site of the enzyme and to induce its closure, thus assuring the progress of the catalytic reaction. Therefore, by single mutation the fine substrate recognition, active site closure, and locking were recovered.

Structure of the adenylation domain Thr1 involved in the biosynthesis of 4-chlorothreonine in Streptomyces sp. OH-5093: protein flexibility and molecular bases of substrate specificity.

We determined the crystal structure of Thr1, the self‐standing adenylation domain involved in the nonribosomal‐like biosynthesis of free 4‐chlorothreonine in Streptomyces sp. OH‐5093. Thr1 shows two monomers in the crystallographic asymmetric unit with different relative orientations of the C‐ and N‐terminal subdomains both in the presence of substrates and in the unliganded form. Cocrystallization with substrates, adenosine 5′‐triphosphate and l‐threonine, yielded one monomer containing the two substrates and the other in complex with l‐threonine adenylate, locked in a postadenylation state. Steady‐state kinetics showed that Thr1 activates l‐Thr and its stereoisomers, as well as d‐Ala, l‐ and d‐Ser, albeit with lower efficiency. Modeling of these substrates in the active site highlighted the molecular bases of substrate discrimination. This work provides the first crystal structure of a threonine‐activating adenylation enzyme, a contribution to the studies on conformational rearrangement in adenylation domains and on substrate recognition in nonribosomal biosynthesis.

Subcellular localization of the five members of the human steroid 5α-reductase family

In humans the steroid 5α-reductase (SRD5A) family comprises five integral membrane enzymes that carry out reduction of a double bond in lipidic substrates: Δ4-3-keto steroids, polyprenol and trans-enoyl CoA. The best-characterized reaction is the conversion of testosterone into the more potent dihydrotestosterone carried out by SRD5A1-2. Some controversy exists on their possible nuclear or endoplasmic reticulum localization. We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5α-reductase family as both N- and C-terminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates.