Antonello Merlino

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.

An overview of biological macromolecule crystallization.

The elucidation of the three dimensional structure of biological macromolecules has provided an important contribution to our current understanding of many basic mechanisms involved in life processes. This enormous impact largely results from the ability of X-ray crystallography to provide accurate structural details at atomic resolution that are a prerequisite for a deeper insight on the way in which bio-macromolecules interact with each other to build up supramolecular nano-machines capable of performing specialized biological functions. With the advent of high-energy synchrotron sources and the development of sophisticated software to solve X-ray and neutron crystal structures of large molecules, the crystallization step has become even more the bottleneck of a successful structure determination. This review introduces the general aspects of protein crystallization, summarizes conventional and innovative crystallization methods and focuses on the new strategies utilized to improve the success rate of experiments and increase crystal diffraction quality.

Reversible Substrate-Induced Domain Motions in Ribonuclease A

Despite the increasing number of successful determinations of complex protein structures the understanding of their dynamics properties is still rather limited. Using X‐ray crystallography, we demonstrate that ribonuclease A (RNase A) undergoes significant domain motions upon ligand binding. In particular, when cytidine 2′‐monophosphate binds to RNase A, the structure of the enzyme becomes more compact. Interestingly, our data also show that these structural alterations are fully reversible in the crystal state. These findings provide structural bases for the dynamic behavior of RNase A in the binding of the substrate shown by Petsko and coworkers (Rasmussen et al. Nature 1992;357:423–424 ). These subtle domain motions may assume functional relevance for more complex system and may play a significant role in the cooperativity of oligomeric enzymes. Proteins 2002;46:97–104.

Molecular bases of protein halotolerance

Halophilic proteins are stable and function at high salt concentration. Understanding how these molecules maintain their fold stable and avoid aggregation under harsh conditions is of great interest for biotechnological applications. This mini-review describes what is known about the molecular determinants of protein halotolerance. Comparisons between the sequences of halophilic/non-halophilic homologous protein pairs indicated that Asp and Glu are significantly more frequent, while Lys, Ile and Leu are less frequent in halophilic proteins. Homologous halophilic and non-halophilic proteins have similar overall structure, secondary structure content, and number of residues involved in the formation of H-bonds. On the other hand, on the halophilic protein surface, a decrease of nonpolar residues and an increase of charged residues are observed. Particularly, halophilic adaptation correlates with an increase of Asp and Glu, compensated by a decrease of basic residues, mainly Lys, on protein surface. A thermodynamic model, that provides a reliable explanation of the salt effect on the conformational stability of globular proteins, is presented.

The mode of action of anticancer gold-based drugs: a structural perspective

The interactions between a few representative gold-based drugs and hen egg white lysozyme were studied by X-ray crystallography. High resolution crystal structures solved for three metallodrug-protein adducts provide valuable insight into the molecular mechanism of these promising metal compounds and the inherent protein metalation processes.

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.

Cisplatin Binding to Proteins: Molecular Structure of the Ribonuclease A Adduct

The crystal structure of the main adduct formed in the reaction between cisplatin and bovine pancreatic ribonuclease is reported here. Notably, in both of the protein molecules present in the asymmetric unit, platinum(II) binding takes place exclusively at the level of Met29. In one of the two molecules, the Gln28 side chain completes the platinum coordination sphere, anchoring the cisplatin fragment to the protein in a bidentate fashion. These results contain interesting implications for understanding the biological chemistry of this important drug.

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 β‐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 β‐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 β‐sheet motions, which are essential for substrate binding and release. Proteins 2003.

Structural and dynamic effects of α-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°C around neutral pH. An engineered form of Sso7d truncated at leucine 54 (L54Δ) is significantly less stable, with a denaturation temperature of 53°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) L54Δ 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 L54Δ suggests that the unzipping of the N‐terminal and C‐terminal β‐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. Proteins 2004.

A new RNase sheds light on the RNase/angiogenin subfamily from zebrafish.

Recently, extracellular RNases of the RNase A superfamily, with the characteristic CKxxNTF sequence signature, have been identified in fish. This has led to the recognition that these RNases are present in the whole vertebrate subphylum. In fact, they comprise the only enzyme family unique to vertebrates. Four RNases from zebrafish (Danio rerio) have been previously reported and have a very low RNase activity; some of these are endowed, like human angiogenin, with powerful angiogenic and bactericidal activities. In the present paper, we report the three-dimensional structure, the thermodynamic behaviour and the biological properties of a novel zebrafish RNase, ZF-RNase-5. The investigation of its structural and functional properties, extended to all other subfamily members, provides an inclusive description of the whole zebrafish RNase subfamily.