Lelio Mazzarella

Crystal structure of the collagen triple helix model [(Pro-Pro-Gly)(10)](3)

The first report of the full‐length structure of the collagen‐like polypeptide [(Pro‐Pro‐Gly)10]3 is given. This structure was obtained from crystals grown in a microgravity environment, which diffracted up to 1.3 Å, using synchrotron radiation. The final model, which was refined to an Rfactor of 0.18, is the highest‐resolution description of a collagen triple helix reported to date. This structure provides clues regarding a series of aspects related to collagen triple helix structure and assembly. The strict dependence of proline puckering on the position inside the Pro‐Pro‐Gly triplets and the correlation between backbone and side chain dihedral angles support the propensity‐based mechanism of triple helix stabilization/destabilization induced by hydroxyproline. Furthermore, the analysis of [(Pro‐Pro‐Gly)10]3 packing, which is governed by electrostatic interactions, suggests that charges may act as locking features in the axial organization of triple helices in the collagen fibrils.

High-resolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity

The G-quadruplex architecture is a peculiar structure adopted by guanine-rich oligonucleotidic sequences, and, in particular, by several aptamers, including the thrombin-binding aptamer (TBA) that has the highest inhibitory activity against human α-thrombin. A crucial role in determining structure, stability and biological properties of G-quadruplexes is played by ions. In the case of TBA, K+ ions cause an enhancement of the aptamer clotting inhibitory activity. A detailed picture of the interactions of TBA with the protein and with the ions is still lacking, despite the importance of this aptamer in biomedical field for detection and inhibition of α-thrombin. Here, we fill this gap by presenting a high-resolution crystallographic structural characterization of the thrombin–TBA complex formed in the presence of Na+ or K+ and a circular dichroism study of the structural stability of the aptamer both free and complexed with α-thrombin, in the presence of the two ionic species. The results indicate that the different effects exerted by Na+ and K+ on the inhibitory activity of TBA are related to a subtle perturbation of a few key interactions at the protein–aptamer interface. The present data, in combination with those previously obtained on the complex between α-thrombin and a modified aptamer, may allow the design of new TBA variants with a pharmacological performance enhancement.

Preferred proline puckerings in cis and trans peptide groups: implications for collagen stability.

The interplay between side‐chain and main‐chain conformations is a distinctive characteristic of proline residues. Here we report the results of a statistical analysis of proline conformations using a large protein database. In particular, we found that proline residues with the preceding peptide bond in the cis state preferentially adopt a down puckering. Indeed, out of 178 cis proline residues, as many as 145 (81%) are down. By analyzing the 1–4 and 1–5 nonbonding distances between backbone atoms, we provide a structural explanation for the observed trend. The observed correlation between proline puckering and peptide bond conformation suggests a new mechanism to explain the reported shift of the cis‐trans equilibrium in proline derivatives. The implications of these results for the current models of collagen stability are also discussed.

Thrombin–aptamer recognition: a revealed ambiguity

Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human α-thrombin was solved at 2.9-Å resolution, but did not provide details of the aptamer conformation and the interactions with the protein molecule. TBA is rapidly processed by nucleases. To improve the properties of TBA, a number of modified analogs have been produced. In particular, a modified TBA containing a 5′-5′ polarity inversion site, mTBA, has higher stability and higher affinity toward thrombin with respect to TBA, although it has a lower inhibitory activity. We present the crystal structure of the thrombin–mTBA complex at 2.15-Å resolution; the resulting model eventually provides a clear picture of thrombin–aptamers interaction, and also highlights the structural bases of the different properties of TBA and mTBA. Our findings open the way for a rational design of modified aptamers with improved potency as anticoagulant drugs.

Crystal structure of a collagen-like polypeptide with repeating sequence Pro–Hyp–Gly at 1.4 Å resolution: Implications for collagen hydration

The use of polypeptide models has proved to be a valuable tool to obtain accurate information on the collagen triple helix. Here we report the high resolution crystal structure of a collagen-like polypeptide with repeating sequence Pro-Hyp-Gly. The structure has been refined to an R(factor) of 0.137 and an R(free) of 0.163 using synchrotron diffraction data extending up to 1.4 A resolution. The polypeptide triple-helical structure binds a large number of water molecules, in contrast with a previous structure determination at lower resolution. The highly hydrated nature of this polypeptide confirms a number of previous studies conducted both in solution and in the crystal state. In addition, neighboring polypeptide triple helices are directly bound in the crystal through Hyp-Hyp hydrogen-bonding interactions. This finding supports the idea that Hyp residues may be important for the assembly of the triple helices in the collagen fibrils and may stabilize the fibrils by mediating direct contacts between neighboring molecules.

Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains

FMR1 is an RNA-binding protein that is either absent or mutated in patients affected by the fragile X syndrome, the most common inherited cause of mental retardation in humans. Sequence analysis of the FMR1 protein has suggested that RNA binding is related to the presence of two K-homologous (KH) modules and an RGG box. However, no attempt has been so far made to map the RNA-binding sites along the protein sequence and to identify possible differential RNA-sequence specificity. In the present article, we describe work done to dissect FMR1 into regions with structurally and functionally distinct properties. A semirational approach was followed to identify four regions: an N-terminal stretch of 200 amino acids, the two KH regions, and a C-terminal stretch. Each region was produced as a recombinant protein, purified, and probed for its state of folding by spectroscopical techniques. Circular dichroism and NMR spectra of the N-terminus show formation of secondary structure with a strong tendency to aggregate. Of the two homologous KH motifs, only the first one is folded whereas the second remains unfolded even when it is extended both N- and C-terminally. The C-terminus is, as expected from its amino acid composition, nonglobular. Binding assays were then performed using the 4-nt homopolymers. Our results show that only the first KH domain but not the second binds to RNA, and provide the first direct evidence for RNA binding of both the N-terminal and the C-terminal regions. RNA binding for the N-terminus could not be predicted from sequence analysis because no known RNA-binding motif is identifiable in this region. Different sequence specificity was observed for the fragments: both the N-terminus of the protein and KH1 bind preferentially to poly-(rG). The C-terminal region, which contains the RGG box, is nonspecific, as it recognizes the bases with comparable affinity. We therefore conclude that FMR1 is a protein with multiple sites of interaction with RNA: sequence specificity is most likely achieved by the whole block that comprises the first approximately 400 residues, whereas the C-terminus provides a nonspecific binding surface.

Crystal structure of the alcohol dehydrogenase from the hyperthermophilic archaeon Sulfolobus solfataricus at 1.85 A resolution.

The crystal structure of a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) from an archaeon has been solved by multiwavelength anomalous diffraction, using a selenomethionine-substituted enzyme. The protein (SsADH), extracted from the hyperthermophilic organism Sulfolobus solfataricus, is a homo-tetramer with a crystallographic 222 symmetry. Despite the low level of sequence identity, the overall fold of the monomer is similar to that of the other homologous ADHs of known structure. However, a significant difference is the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft. At the bottom of this cleft, the catalytic zinc ion is coordinated tetrahedrally and lacks the zinc-bound water molecule that is usually found in ADH apoform structures. The fourth coordination position is indeed occupied by a Glu residue, as found in bacterial tetrameric ADHs. Other differences are found in the architecture of the substrate pocket whose entrance is more restricted than in other ADHs. SsADH is the first tetrameric ADH X-ray structure containing a second zinc ion playing a structural role. This latter metal ion shows a peculiar coordination, with a glutamic acid residue replacing one of the four cysteine ligands that are highly conserved throughout the structural zinc-containing dimeric ADHs.

Seminal Ribonuclease: The Importance of Diversity

Publisher Summary This chapter provides an overview of the seminal ribonuclease. Bovine seminal RNase (BS-RNase) is a diverse RNase “different” from the historic prototype RNase A—and from all other RNases of the vertebrate superfamily—for its dimeric structure, for its non-Michaelian kinetics, and for its special, noncatalytic, biological actions. BS-RNase is also a diverse RNase because it exists in a multiplicity of structural forms, and is endowed with a multiplicity of biological actions. Two quaternary conformations and three isoenzymatic subunit compositions are known for BS-RNase. BS-RNase performs a surprising array of biological actions: aspermatogenic, antitumor, immunosuppressive, and antiviral. BS-RNase may not be the only seminal RNase: an RNase has been purified from human semen and low levels of RNase activity have been detected in the semen of several mammals, including mouse, rabbit, and sheep. This chapter discusses isolation and production of seminal RNase. It explains preparation of seminal ribonuclease from natural sources. Production of recombinant BS-RNase is discussed. The chapter elaborates covalent structure, three-dimensional structure, and folding pathway of seminal RNase. The chapter also outlines the functions of seminal ribonuclease.

Unprecedented alkaloid skeleton from the Mediterranean sponge Reniera sarai: X-ray structure of an acetate derivative of sarain-A

Abstract The Mediterranean sponge Reniera sarai is a rich source of new alkaloids. Until now the study has been limited to the less polar compounds. In this paper we report the first studies on the more polar UV absorbing alkaloid fraction, which resulted a mixture of threehomologs, sarains A-C. A resolutive X-ray study on an acetate derivative of sarain-A has descovered an unprecedented alkaloid skeletonexhibiting a central cage structure, characterized by a chargedpseudobase moiety, in the middle of two cyclic alkyl chains.

Atomic resolution structures of ribonuclease A at six pH values

The diffraction pattern of protein crystals extending to atomic resolution guarantees a very accurate picture of the molecular structure and enables the study of subtle phenomena related to protein functionality. Six structures of bovine pancreatic ribonuclease at the pH* values 5.2, 5.9, 6.3, 7.1, 8.0 and 8.8 and at resolution limits in the range 1.05-1.15A have been refined. An overall description of the six structures and several aspects, mainly regarding pH-triggered conformational changes, are described here. Since subtle variations were expected, a thorough validation assessment of the six refined models was first carried out. Some stereochemical parameters, such as the N[bond]C(alpha)[bond]C angle and the pyramidalization at the carbonyl C atoms, indicate that the standard target values and their weights typically used in refinement may need revision. A detailed comparison of the six structures has provided experimental evidence on the role of Lys41 in catalysis. Furthermore, insights are given into the structural effects related to the pH-dependent binding of a sulfate anion, which mimics the phosphate group of RNA, in the active site. Finally, the results support a number of thermodynamic and kinetic experimental data concerning the role of the disulfide bridge between Cys65 and Cys72 in the folding of RNase A.