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The molecular mechanism of how human serum transferrin (hTF) recognizes cisplatin at the atomic level is still unclear. Here, we report the molecular structure of the adduct formed upon the reaction of hTF with cisplatin. Pt binds the side chain of Met256 (at the N-lobe), without altering the protein overall conformation.

AbstractHuman α-thrombin (thrombin) is a multifunctional enzyme that plays a pivotal role in the coagulation pathway. Thrombin activity can be effectively modulated by G-quadruplex-based oligonucleotide aptamers that specifically interact with the two positively charged regions (exosites I and II) on the protein surface. Although insightful atomic-level snapshots of the recognition between thrombin and aptamers have been recently achieved through crystallographic analyses, some dynamic aspects of this interaction have not been fully characterized. We here report molecular dynamics simulations of thrombin in different association states: ligand-free and binary/ternary complexes with the aptamers TBA (at exosite I) and HD22_27mer (at exosite II). The simulations carried out on the binary and ternary complexes formed by thrombin with these aptamers provide a dynamic view of the interactions that stabilize them in a crystal-free environment. Interestingly, the analysis of the dynamics of the exosites in different thrombin binding states clearly indicates that the HD22_27mer binding at the exosite II favours conformations of exosite I that are prone to the TBA binding. Similar effects are observed upon the binding of TBA to the exosite I. These observations provide an atomic-level picture of the exosite inter-communication in thrombin and explain the experimentally detected cooperativity of the TBA/HD22_27mer binding.

The Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) produces a cold-active iron superoxide dismutase (SOD). PhSOD is a homodimeric enzyme, that displays a high catalytic activity even at low temperature. Using hanging-drop vapour-diffusion technique, PhSOD has been successfully crystallized in two different crystal forms. Both crystal forms are monoclinic with space group P21 and diffract to 2.1 Å resolution. Form I has unit-cell parameters a45.49Å b103.63Å c50.37Å 108.2° and contains a homodimer in the asymmetric unit. Form II has unit-cell parameters a50.48Å b103.78Å c90.25Å 103.8° and an asymmetric unit containing two PhSOD homodimers. Structure determination has been achieved using molecular replacement. The crystallographic study of this cold-adapted enzyme could contribute to the understanding of the molecular mechanisms of cold-adaptation and of the high catalytic efficiency at low temperature.

Crystals of the deamidated form of bovine pancreatic ribonuclease which contains an isoaspartyl residue in position 67 diffract to 0.87 A˚ at 100 K. We have refined the crystallographic model using anisotropic displacement parameters for all atoms to a conventional crystallographic residual R=0.101 for all observed reflections in the resolution range 61.0–0.87 A˚. The ratio observations/parameters is 7.2 for the final model. This structure represents one of the highest resolution protein structures to date and interestingly, it is the only example containing more than one molecule in the asymmetric unit with a resolution better than 1.0 A˚. The non-crystallographic symmetry has been used as a validation check of the geometrical parameters and it has allowed an estimate for an upper limit of errors associated with this high resolution model. In the present structure it was possible to obtain a more accurate picture of the active site whose electron density was not clearly interpretable in the previous 1.9 A˚ resolution structure. In particular, the P1 site is alternatively occupied either by a sulphate anion or by a water molecule network. Most of hydrogen atoms were visible in the electron density maps, including those involved in Cα—Hα···O interactions. Analysis of protein-solvent interactions has revealed the occurrence of an extensive cluster of water molecules, predominantly arranged in pentagonal fused rings and surrounding hydrophobic moiety of side-chains. Finally, in spite of the limited sample of residues, we have detected a clear dependence of backbone N—Cα—C angle on residue conformation. This correlation can be fruitfully used as a valuable tool in protein structure validation.

Bovine seminal ribonuclease (BS-RNase) is a peculiar member of the pancreatic-like ribonuclease superfamily endowed with unique biological functions. It has been shown that native BS-RNase is a mixture of two distinct dimeric forms. The most abundant form is characterised by the swapping of the N-terminal helix. Kinetic studies have shown that this dimer is allosterically regulated, whereas the minor component, in which no swapping occurs, exhibits typical Michaelian kinetics. In order to correlate the catalytic properties with the structural features of BS-RNase, we have determined the crystal structure of the BS-RNase swapping dimer complexed with uridylyl(2′-5′)guanosine. The structure of the complex was refined to an Rvalue of 0.189 at 1.9 Å resolution. Surprisingly, the enzyme binds four dinucleotide molecules, all in a non-productive way. In the two active sites, the guanine base is located in the subsite that is specific for pyrimidines. This unusual binding has been observed also in complexes of RNase A with guanine-containing nucleotides (retro-binding). One of the two additional dinucleotide molecules bound to the enzyme is located on the surface of the protein in a pocket generated by crystal packing; the second was found in a cavity at the interface between the two subunits of the swapping dimer. There are indications that the interface site plays a role in the allosteric regulation exhibited by BS-RNase. This finding suggests that domain swapping may not merely be a mechanism that proteins adopt for the transition from a monomeric to oligomeric state but can be used to achieve modulations in catalytic function.

In the ribonuclease superfamily, dimericity is a unique feature of bovine seminal RNase (BS‐RNase). In about two‐thirds of native BS‐RNase molecules, the two subunits interchange their N‐terminal tails, thus generating domain‐swapped dimers (MxM), which mostly responsible for enzyme biological activities and allostericity. Higher molecular weight BS‐RNase oligomers can also be prepared [Libonati, M. (1969) Ital. J. Biochem. 18, 407–417.]. This paper reports on BS‐RNase tetrameric derivatives which were isolated and enzymatically characterized. The data collected and the analysis of the crystal packing of MxM dimers suggested a structural model for tetramer assembly, in which the four subunits are enchained by multiple domain‐swapping events.

An NAD+-dependent alcohol dehydrogenase from the extreme thermophilic archaebacterium Sulfolobus solfataricushas been crystallized in the holo-enzyme and apo-enzyme forms. Crystals of the holo-enzyme grow from 2-methyl-2,4-pentanediol at pH 8·4 with the addition of NADH and at pH 7·0 with the addition of NADH and dimethyl sulphoxide. Crystals of the apo-enzyme grow at pH 6·3 from a mixture of polyethylene glycol 4000 and propan-2-ol. The holo-enzyme crystallizes in C2 with a dimer in the asymmetric unit, however the crystals are twinned and unsuitable for data collection. The apo-enzyme crystallizes in I4122 (a= 126·82 Å, b= 118·95 Å) with a monomer in the asymmetric unit, and the single crystals diffract to 2·8 Å.