Biomedical subjects

Lelio Mazzarella

Publications and source records attributed to Lelio Mazzarella.

At least 19 recordsLinked to original sources

PubMed2006

High resolution crystal structure of deoxy hemoglobin from Trematomus bernacchii at different pH values: the role of histidine residues in modulating the strength of the root effect.

The Root effect is a widespread property in fish hemoglobins (Hbs) that produces a drastic reduction of cooperativity and oxygen-binding ability at acidic pH. Here, we report the high-resolution structure of the deoxy form of Hb isolated from the Antarctic fish Trematomus bernacchii (HbTb) crystallized at pH 6.2 and 8.4. The structure at acidic pH has been previously determined at a moderate resolution (Ito et al., J Mol Biol 1995;250:648-658). Our results provide a clear picture of the events occurring upon the pH increase from 6.2 to 8.4, observed within a practically unchanged crystal environment. In particular, at pH 8.4, the interaspartic hydrogen bond at the alpha(1)beta(2) interface is partially broken, suggesting a pK(a) close to 8.4 for Asp95alpha. In addition, a detailed survey of the histidine modifications, caused by the change in pH, also indicates that at least three hot regions of the molecule are modified (Ebeta helix, Cbeta-tail, CDalpha corner) and can be considered to be involved at various levels in the release of the Root protons. Most importantly, at the CDalpha corner, the break of the salt bridge Asp48alpha-His55alpha allows us to describe a detailed mechanism that transmits the modification from the CDalpha corner far to the alpha heme. More generally, the results shed light on the role played by the histidine residues in modulating the strength of the Root effect and also support the emerging idea that the structural determinants, at least for a part of the Root effect, are specific of each Hb endowed with this property.

Amino Acid Motifs↗

PubMed2006

Minimal structural requirements for root effect: crystal structure of the cathodic hemoglobin isolated from the antarctic fish Trematomus newnesi.

The cathodic hemoglobin component of the Antarctic fish Trematomus newnesi (HbCTn) is a Root-effect protein. The interpretation of its functional properties in relation to its sequence is puzzling. Indeed, HbCTn sequence is characterized by an extremely low histidyl content, and in particular by the lack of His146beta and His69beta, which are believed to be important in Bohr and Root effects, respectively. Furthermore, previous analyses suggested that the local environment of Asp95alpha, Asp99beta, and Asp101beta should not be appropriate for the formation of Asp-Asp interactions, which are important for the Root effect. Here, we report the high-resolution crystal structure of the deoxy form of HbCTn. Our data provide a structural interpretation for the very low oxygen affinity of the protein and insights into the structural determinants of the Root effect protein. The structure demonstrates that the presence of Ile41alpha and Ser97alpha at the alpha1beta2 interface does not prevent the formation of the inter-Asp interactions in HbCTn, as previous studies had suggested. The present data indicate that the hydrogen bond formed between Asp95alpha and Asp101beta, which is stabilized by Asp99beta, is per se sufficient to generate the Root effect, and it is the minimal structural requirement needed for the design of Root-effect Hbs.

Animals↗

PubMed2005

The importance of dynamic effects on the enzyme activity: X-ray structure and molecular dynamics of onconase mutants.

Onconase (ONC), a member of the RNase A superfamily extracted from oocytes of Rana pipiens, is an effective cancer killer. It is currently used in treatment of various forms of cancer. ONC antitumor properties depend on its ribonucleolytic activity that is low in comparison with other members of the superfamily. The most damaging side effect from Onconase treatment is renal toxicity, which seems to be caused by the unusual stability of the enzyme. Therefore, mutants with reduced thermal stability and/or increased catalytic activity may have significant implications for human cancer chemotherapy. In this context, we have determined the crystal structures of two Onconase mutants (M23L-ONC and C87S,des103-104-ONC) and performed molecular dynamic simulations of ONC and C87S,des103-104-ONC with the aim of explaining on structural grounds the modifications of the activity and thermal stability of the mutants. The results also provide the molecular bases to explain the lower catalytic activity of Onconase compared with RNase A and the unusually high thermal stability of the amphibian enzyme.

Animals↗

PubMed2005

The role of the hinge loop in domain swapping. The special case of bovine seminal ribonuclease.

Bovine seminal ribonuclease (BS-RNase) is a covalent homodimeric enzyme homologous to pancreatic ribonuclease (RNase A), endowed with a number of special biological functions. It is isolated as an equilibrium mixture of swapped (MxM) and unswapped (M=M) dimers. The interchanged N termini are hinged on the main bodies through the peptide 16-22, which changes conformation in the two isomers. At variance with other proteins, domain swapping in BS-RNase involves two dimers having a similar and highly constrained quaternary association, mainly dictated by two interchain disulfide bonds. This provides the opportunity to study the intrinsic ability to swap as a function of the hinge sequence, without additional effects arising from dissociation or quaternary structure modifications. Two variants, having Pro19 or the whole sequence of the hinge replaced by the corresponding residues of RNase A, show equilibrium and kinetic parameters of the swapping similar to those of the parent protein. In comparison, the x-ray structures of MxM indicate, within a substantial constancy of the quaternary association, a greater mobility of the hinge residues. The relative insensitivity of the swapping tendency to the substitutions in the hinge region, and in particular to the replacement of Pro19 by Ala, contrasts with the results obtained for other swapped proteins and can be rationalized in terms of the unique features of the seminal enzyme. Moreover, the results indirectly lend credit to the hypothesis that the major role of Pro19 resides in directing the assembly of the non-covalent dimer, the species produced by selective reduction of the interchain disulfides and considered responsible for the special biological functions of BS-RNase.

Animals↗

PubMed2004

Open interface and large quaternary structure movements in 3D domain swapped proteins: insights from molecular dynamics simulations of the C-terminal swapped dimer of ribonuclease A.

Bovine pancreatic ribonuclease (RNase A) forms two three-dimensional (3D) domain swapped dimers. Crystallographic investigations have revealed that these dimers display completely different quaternary structures: one dimer (N-dimer), which presents the swapping of the N-terminal helix, is characterized by a compact structure, whereas the other (C-dimer), which is stabilized by the exchange of the C-terminal end, shows a rather loose assembly of the two subunits. The dynamic properties of monomeric RNase A and of the N-dimer have been extensively characterized. Here, we report a molecular dynamics investigation carried out on the C-dimer. This computational experiment indicates that the quaternary structure of the C-dimer undergoes large fluctuations. These motions do not perturb the proper folding of the two subunits, which retain the dynamic properties of RNase A and the N-dimer. Indeed, the individual subunits of the C-dimer display the breathing motion of the beta-sheet structure, which is important for the enzymatic activity of pancreatic-like ribonucleases. In contrast to what has been observed for the N-dimer, the breathing motion of the two subunits of the C-dimer is not coupled. This finding suggests that the intersubunit communications in a 3D domain swapped dimer strongly rely on the extent of the interchain interface. Furthermore, the observation that the C-dimer is endowed with a high intrinsic flexibility holds interesting implications for the specific properties of 3D domain swapped dimers. Indeed, a survey of the quaternary structures of the other 3D domain swapped dimers shows that large variations are often observed when the structural determinations are conducted in different experimental conditions. The 3D domain swapping phenomenon coupled with the high flexibility of the quaternary structure may be relevant for protein-protein recognition, and in particular for the pathological aggregations.

Binding Sites↗

PubMed2004

Structural and dynamic effects of alpha-helix deletion in Sso7d: implications for protein thermal stability.

Sso7d is a 62-residue protein from the hyperthemophilic archaeon Sulfolobus solfataricus with a denaturation temperature close to 100 degrees C around neutral pH. An engineered form of Sso7d truncated at leucine 54 (L54Delta) is significantly less stable, with a denaturation temperature of 53 degrees C. Molecular dynamics (MD) studies of Sso7d and its truncated form at two different temperatures have been performed. The results of the MD simulations at 300 K indicate that: (1) the flexibility of Sso7d chain at 300 K agrees with that detected from X-ray and NMR structural studies; (2) L54Delta remains stable in the native folded conformation and possesses an overall dynamic behavior similar to that of the parent protein. MD simulations performed at 500 K, 10 ns long, indicate that, while Sso7d is in-silico resistant to high temperature, the truncated variant partially unfolds, revealing the early phases of the thermal unfolding pathway of the protein. Analysis of the trajectories of L54Delta suggests that the unzipping of the N-terminal and C-terminal beta-strands should be the first event of the unfolding pathway, and points out the regions more resistant to thermal unfolding. These findings allow one to understand the role played by specific interactions connecting the two ends of the chain for the high thermal stability of Sso7d, and support recent hypotheses on its folding mechanism emerged from site-directed mutagenesis studies.

Amino Acids↗

PubMed2004

Structure and stability of the non-covalent swapped dimer of bovine seminal ribonuclease: an enzyme tailored to evade ribonuclease protein inhibitor.

A growing number of pancreatic-type ribonucleases (RNases) present cytotoxic activity against malignant cells. The cytoxicity of these enzymes is related to their resistance to the ribonuclease protein inhibitor (RI). In particular, bovine seminal ribonuclease (BS-RNase) is toxic to tumor cells both in vitro and in vivo. BS-RNase is a covalent dimer with two intersubunit disulfide bridges between Cys(31) of one chain and Cys(32) of the second and vice versa. The native enzyme is an equilibrium mixture of two isomers, MxM and M=M. In the former the two subunits swap their N-terminal helices. The cytotoxic action is a peculiar property of MxM. In the reducing environment of cytosol, M=M dissociates into monomers, which are strongly inhibited by RI, whereas MxM remains as a non-covalent dimer (NCD), which evades RI. We have solved the crystal structure of NCD, carboxyamidomethylated at residues Cys(31) and Cys(32) (NCD-CAM), in a complex with 2'-deoxycitidylyl(3'-5')-2'-deoxyadenosine. The molecule reveals a quaternary structural organization much closer to MxM than to other N-terminal-swapped non-covalent dimeric forms of RNases. Model building of the complexes between these non-covalent dimers and RI reveals that NCD-CAM is the only dimer equipped with a quaternary organization capable of interfering seriously with the binding of the inhibitor. Moreover, a detailed comparative structural analysis of the dimers has highlighted the residues, which are mostly important in driving the quaternary structure toward that found in NCD-CAM.

Animals↗

PubMed2004

Population shift vs induced fit: the case of bovine seminal ribonuclease swapping dimer.

Bovine seminal ribonuclease (BS-RNase) is a unique member of the pancreatic-like ribonuclease superfamily. This enzyme exists as two conformational isomers with distinctive biological properties. The structure of the major isomer is characterized by the swapping of the N-terminal segment (MxM BS-RNase). In this article, the crystal structures of the ligand-free MxM BS-RNase and its complex with 2'-deoxycitidylyl(3',5')-2'-deoxyadenosine derived from isomorphous crystals have been refined. Interestingly, the comparison between this novel ligand-free form and the previously published sulfate-bound structure reveals significant differences. In particular, the ligand-free MxM BS-RNase is closer to the structure of MxM BS-RNase productive complexes than to the sulfate-bound form. These results reveal that MxM BS-RNase presents a remarkable flexibility, despite the structural constraints of the interchain disulfide bridges and the swapping of the N-terminal helices. These findings have important implications to the ligand binding mechanism of MxM BS-RNase. Indeed, a population shift rather than a substrate-induced conformational transition may occur in the MxM BS-RNase ligand binding process.

Animals↗

PubMed2004

Dynamic properties of the N-terminal swapped dimer of ribonuclease A.

Bovine pancreatic ribonuclease (RNase A) forms two 3-dimensional domain-swapped dimers with different quaternary structures. One dimer is characterized by the swapping of the C-terminal region (C-Dimer) and presents a rather loose structure. The other dimer (N-Dimer) exhibits a very compact structure with exchange of the N-terminal helix. Here we report the results of a molecular dynamics/essential dynamics (MD/ED) study carried out on the N-Dimer. This investigation, which represents the first MD/ED analysis on a three-dimensional domain-swapped enzyme, provides information on the dynamic properties of the active site residues as well as on the global motions of the dimer subunits. In particular, the analysis of the flexibility of the active site residues agrees well with recent crystallographic and site-directed mutagenesis studies on monomeric RNase A, thus indicating that domain swapping does not affect the dynamics of the active sites. A slight but significant rearrangement of N-Dimer quaternary structure, favored by the formation of additional hydrogen bonds at subunit interface, has been observed during the MD simulation. The analysis of collective movements reveals that each subunit of the dimer retains the functional breathing motion observed for RNase A. Interestingly, the breathing motion of the two subunits is dynamically coupled, as they open and close in phase. These correlated motions indicate the presence of active site intercommunications in this dimer. On these bases, we propose a speculative mechanism that may explain negative cooperativity in systems preserving structural symmetry during the allosteric transitions.

Animals↗

PubMed2004

The oxidation process of Antarctic fish hemoglobins.

Analysis of the molecular properties of proteins extracted from organisms living under extreme conditions often highlights peculiar features. We investigated by UV-visible spectroscopy and X-ray crystallography the oxidation process, promoted by air or ferricyanide, of five hemoglobins extracted from Antarctic fishes (Notothenioidei). Spectroscopic analysis revealed that these hemoglobins share a common oxidation pathway, which shows striking differences from the oxidation processes of hemoglobins from other vertebrates. Indeed, simple exposure of these hemoglobins to air leads to the formation of a significant amount of the low-spin hexacoordinated form, denoted hemichrome. This hemichrome form, which is detected under a variety of experimental conditions, can be reversibly transformed to either carbomonoxy or deoxygenated forms with reducing agents. Interestingly, the spectra of the fully oxidized species, obtained by treating the protein with ferricyanide, show the simultaneous presence of peaks corresponding to different hexacoordinated states, the aquomet and the hemichrome. In order to assign the heme region state of the alpha and beta chains, the air-oxidized and ferricyanide-oxidized forms of Trematomus bernacchii hemoglobin were crystallized. Crystallographic analysis revealed that these forms correspond to an alpha(aquomet)-beta(bishistidyl-hemichrome) state. This demonstrates that the alpha and beta chains of Antarctic fish hemoglobins follow very different oxidation pathways. As found for Trematomus newnesi hemoglobin in a partial hemichrome state [Riccio, A., Vitagliano, L., di Prisco, G., Zagari, A. & Mazzarella, L. (2002) Proc. Natl Acad. Sci. USA99, 9801-9806], the quaternary structures of these alpha(aquomet)-beta(bishistidyl-hemichrome) forms are intermediate between the physiological R and T hemoglobin states. Together, these structures provide information on the general features of this intermediate state.

Animals↗

PubMed2003

Crystal structure of a ternary complex of the alcohol dehydrogenase from Sulfolobus solfataricus.

The crystal structure of a ternary complex of the alcohol dehydrogenase from the archaeon Sulfolobus solfataricus (SsADH) has been determined at 2.3 A. The asymmetric unit contains a dimer with a NADH and a 2-ethoxyethanol molecule bound to each subunit. The comparison with the apo structure of the enzyme reveals that this medium chain ADH undergoes a substantial conformational change in the apo-holo transition, accompanied by loop movements at the domain interface. The extent of domain closure is similar to that observed for the classical horse liver ADH, although some differences are found which can be related to the different oligomeric states of the enzymes. Compared to its apo form, the SsADH ternary complex shows a change in the ligation state of the active site zinc ion which is no longer bound to Glu69, providing additional evidence of the dynamic role played by the conserved glutamate residue in ADHs. In addition, the structure presented here allows the identification of the substrate site and hence of the residues that are important in the binding of both the substrate and the coenzyme.

Alcohol Dehydrogenase↗

PubMed2003

Subtle functional collective motions in pancreatic-like ribonucleases: from ribonuclease A to angiogenin.

The analysis of the dynamic behavior of enzymes is fundamental to structural biology. A direct relationship between protein flexibility and biological function has been shown for bovine pancreatic ribonuclease (RNase A) (Rasmussen et al., Nature 1992;357:423-424). More recently, crystallographic studies have shown that functional motions in RNase A involve the enzyme beta-sheet regions that move concertedly on substrate binding and release (Vitagliano et al., Proteins 2002;46:97-104). These motions have been shown to correspond to intrinsic dynamic properties of the native enzyme by molecular dynamics (MD) simulations. To unveil the occurrence of these collective motions in other members of pancreatic-like superfamily, we carried out MD simulations on human angiogenin (Ang). Essential dynamics (ED) analyses performed on the trajectories reveal that Ang exhibits collective motions similar to RNase A, despite the limited sequence identity (33%) of the two proteins. Furthermore, we show that these collective motions are also present in ensembles of experimentally determined structures of both Ang and RNase A. Finally, these subtle concerted beta-sheet motions were also observed for other two members of the pancreatic-like superfamily by comparing the ligand-bound and ligand-free structures of these enzymes. Taken together, these findings suggest that pancreatic-like ribonucleases share an evolutionary conserved dynamic behavior consisting of subtle beta-sheet motions, which are essential for substrate binding and release.

Animals↗

PubMed2003

The unswapped chain of bovine seminal ribonuclease: Crystal structure of the free and liganded monomeric derivative.

Bovine seminal ribonuclease, a homodimeric enzyme joined covalently by two interchain disulphide bonds, is an equilibrium mixture of two conformational isomers, MxM and M=M. The major form, MxM, whose crystal structure has been previously determined at 1.9 A resolution, presents the swapping of the N-terminal segments (residues 1-15) and composite active sites formed by residues of different chains. The three-dimensional domain swapping does not occur in the M=M form. The different fold of each N-terminal tail is directed by the hinge loop (residue 16-22) connecting the swapping domain to the body of the protein. Reduction and alkylation of interchain disulphide bridges produce a monomeric derivative and a noncovalent swapped dimer, which are both active. The free and nucleotide-bound forms of the monomer have been crystallized at an alkaline pH and refined at 1.45 and 1.65 A resolution, respectively. In both cases, the N-terminal fragment is folded on the main body of the protein to produce an intact active site and a chain architecture very similar to that of bovine pancreatic ribonuclease. In this new fold of the seminal chain, the hinge loop is disordered. Despite the difference between the tertiary structure of the monomer and that of the chains in the MxM form, the active sites of the two enzymes are virtually indistinguishable. Furthermore, the structure of the liganded enzyme represents the first example of a ribonuclease complex studied at an alkaline pH and provides new information on the binding of a nucleotide when the catalytic histidines are deprotonated.

Animals↗

PubMed2003

Structural study of a single-point mutant of Sulfolobus solfataricus alcohol dehydrogenase with enhanced activity.

Alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) is the only enzyme from Archaea among the structurally studied members of the medium-chain ADH family described so far. Here, we present the three-dimensional structure of the apo form of the mutant N249Y which exhibits increased catalytic activity when compared to the wild-type enzyme. The substitution, located in the coenzyme binding domain, decreases the affinity for NAD(H) cofactor. The rearrangement of segments 248-250 and 270-275, induced by the mutation, suggests an explanation for the lower coenzyme affinity. This study also highlights the role in SsADH catalysis of the flexible loops located at the interface between the catalytic and the coenzyme domains.

Alcohol Dehydrogenase↗

PubMed2002

Global and local motions in ribonuclease A: a molecular dynamics study.

The understanding of protein dynamics is one of the major goals of structural biology. A direct link between protein dynamics and function has been provided by x-ray studies performed on ribonuclease A (RNase A) (B. F. Rasmussen et al., Nature, 1992, Vol. 357, pp. 423-424; L. Vitagliano et al., Proteins: Structure, Function, and Genetics, 2002, Vol. 46, pp. 97-104). Here we report a 3 ns molecular dynamics simulation of RNase A in water aimed at characterizing the dynamical behavior of the enzyme. The analysis of local and global motions provides interesting insight on the dynamics/function relationship of RNase A. In agreement with previous crystallographic reports, the present study confirms that the RNase A active site is constituted by rigid (His12, Asn44, Thr45) and flexible (Lys41, Asp83, His119, Asp121) residues. The analysis of the global motions, performed using essential dynamics, shows that the two beta-sheet regions of RNase A move coherently in opposite directions, thus modifying solvent accessibility of the active site, and that the mixed alpha/3(10)-helix (residues 50-60) behaves as a mechanical hinge during the breathing motion of the protein. These data demonstrate that this motion, essential for RNase A substrate binding and release, is an intrinsic dynamical property of the ligand-free enzyme.

Animals↗

PubMed2002

Crystallization of the collagen-like polypeptide (PPG)10 aboard the International Space Station. 1. Video observation.

Single chains of the collagen model polypeptide with sequence (Pro-Pro-Gly)(10), hereafter referred to as (PPG)(10), aggregate to form rod-shaped triple helices. Crystals of (PPG)(10) were grown in the Advanced Protein Crystallization Facility (APCF) both onboard the International Space Station (ISS) and on Earth. The experiments allow the direct comparison of four different crystallization environments for the first time: solution in microgravity ((g), agarose gel in (g, solution on earth, and gel on earth. Both on board and on ground, the crystal growth was monitored by a CCD video camera. The image analysis provided information on the spatial distribution of the crystals, their movement and their growth rate. The analysis of the distribution of crystals reveals that the crystallization process occurs as it does in batch conditions. Slow motions have been observed onboard the ISS. Different to Space-Shuttle experiment, the crystals onboard the ISS moved coherently and followed parallel trajectories. Growth rate and induction time are very similar both in gel and in solution, suggesting that the crystal growth rate is controlled by the kinetics at the interface under the used experimental conditions. These results provide the first data in the crystallogenesis of (PPG)(10), which is a representative member of non-globular, rod-like proteins.

Collagen↗

PubMed2002

Crystallization of the collagen-like polypeptide (PPG)10 aboard the International Space Station. 2. Comparison of crystal quality by X-ray diffraction.

Crystals of the collagen-like polypeptide (PPG)(10) were obtained within the Advanced Protein Crystallization Facility on board the International Space Station, during the STS-105/STS-108 mission. The duration of this mission was such to ensure that the crystallization process had reached its end. Crystals were grown both in the presence and in the absence of agarose gel, to compare the quality of the crystals obtained from these different environments. As a result, crystals grown in the absence of agarose on Earth as well as in microgravity showed X-ray diffraction up to 1.15 A. The intensity/sigma ratio was slightly higher for microgravity grown crystals. Crystals grown in agarose gel, both in microgravity and on ground, showed a comparable diffraction power, with a resolution limit of 1.45 A.

Collagen↗

PubMed2002

3,6-Bis(dimethylamino)-10-(10-iododecyl)acridinium iodide.

In the title compound, C(27)H(39)IN(3)(+).I(-), the acridinium system shows the usual approximate mirror symmetry about the central C.N line, and the corresponding bond lengths and angles in the two halves agree within experimental error. The alkyl chain at the ring N atom is initially perpendicular to the ring plane and then bends sharply at the fourth C atom. Pairs of centrosymmetrically related cations overlap two of their rings and the dimethylamino groups are also partly involved in the overlap. Each I(-) ion is involved in short-range interactions with two cations. These interactions give rise to a 14-membered cyclic structure, which involves pairs of cations and anions across an inversion centre.

Journal Article↗