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Biomedical subjects

Gabriele Varani

Publications and source records attributed to Gabriele Varani.

At least 37 records · Page 2Linked to original sources
PubMed2005

Protein and RNA dynamics play key roles in determining the specific recognition of GU-rich polyadenylation regulatory elements by human Cstf-64 protein.

The N-terminal domain of the 64 kDa subunit of the cleavage stimulation factor (CstF-64) recognizes GU-rich elements within the 3'-untranslated region of eukaryotic mRNAs. This interaction is essential for mRNA 3' end processing and transcription termination, and its strength affects the efficiency of utilization of different polyadenylation sites. The structure of the RNA-binding N-terminal domain of CstF-64 showed how the N-terminal RNA recognition motif of CstF-64 recognizes GU-rich RNAs. However, it is still perplexing how this protein can bind selectively to RNAs that are rich in G and U residues regardless of their detailed sequence composition, yet discriminate effectively against non-GU-RNAs. We investigated by NMR the dynamics of the CstF-64 RNA-binding domain, both free and bound to two GU-rich RNA sequences that represent polyadenylation regulatory elements. While the free protein displays the motional properties typical of a well-folded protein domain and is uniformly rigid, the protein-RNA interface acquires significant mobility on the micro- to millisecond time-scale once GU-rich RNAs binds to it. These motional features, we propose, are intrinsic to the functional requirement to bind all GU-rich sequences and yet to discriminate against non-GU-rich RNAs. This behavior may be a general mechanism by which some RNA-binding proteins are able to bind to classes of sequences, as opposed to a well-defined sequence or consensus.

Cleavage Stimulation Factor↗
PubMed2005

The structure of an enzyme-activating fragment of human telomerase RNA.

The ribonucleoprotein enzyme telomerase ensures the stability and fidelity of linear chromosome ends by elongating the telomeric DNA that is lost during each round of DNA replication. All telomerases contain a catalytic protein component homologous to viral reverse transcriptases (TERT) and an RNA (TR) that provides the template sequence, acts as the scaffold for ribonucleoprotein assembly, and activates the enzyme for catalysis. Vertebrate telomerase RNAs contain three highly conserved structural and functional domains: the template domain, the "CR4-CR5" or "activation" domain essential for activation of the enzymatic activity, and a 3'-terminal "box H/ACA"-homology domain responsible for ribonucleprotein assembly and maturation. Here we report the NMR structure of a functionally essential RNA structural element derived from the human telomerase RNA CR4-CR5 domain. This RNA, referred to as hTR J6, forms a stable hairpin interrupted by a single nucleotide bulge and an asymmetric internal loop. Previous work on telomerase has shown that deletion of the hTR J6 asymmetric internal loop results in an RNA incapable of binding the enzymatic protein component of the RNP and therefore an inactive RNP without telomerase activity. We demonstrate here that the J6 internal loop introduces a twist in the RNA structure that may position the entire domain into the catalytic site of the enzyme.

Catalytic Domain↗
PubMed2005

Protein families and RNA recognition.

This minireview series examines the structural principles underlying the biological function of RNA-binding proteins. The structural work of the last decade has elucidated the structures of essentially all the major RNA-binding protein families; it has also demonstrated how RNA recognition takes place. The ribosome structures have further integrated this knowledge into principles for the assembly of complex ribonucleoproteins. Structural and biochemical work has revealed unexpectedly that several RNA-binding proteins bind to other proteins in addition to RNA or instead of RNA. This tremendous increase in the structural knowledge has expanded not only our understanding of the RNA recognition principle, but has also provided new insight into the biological function of these proteins and has helped to design better experiments to understand their biological roles.

Animals↗
PubMed2004

A new hydrogen-bonding potential for the design of protein-RNA interactions predicts specific contacts and discriminates decoys.

RNA-binding proteins play many essential roles in the regulation of gene expression in the cell. Despite the significant increase in the number of structures for RNA-protein complexes in the last few years, the molecular basis of specificity remains unclear even for the best-studied protein families. We have developed a distance and orientation-dependent hydrogen-bonding potential based on the statistical analysis of hydrogen-bonding geometries that are observed in high-resolution crystal structures of protein-DNA and protein-RNA complexes. We observe very strong geometrical preferences that reflect significant energetic constraints on the relative placement of hydrogen-bonding atom pairs at protein-nucleic acid interfaces. A scoring function based on the hydrogen-bonding potential discriminates native protein-RNA structures from incorrectly docked decoys with remarkable predictive power. By incorporating the new hydrogen-bonding potential into a physical model of protein-RNA interfaces with full atom representation, we were able to recover native amino acids at protein-RNA interfaces.

Amino Acid Sequence↗
PubMed2004

Validation of automated docking programs for docking and database screening against RNA drug targets.

The increasing awareness of the essential role of RNA in controlling viral replication and in bacterial protein synthesis emphasizes the potential of ribonucleoproteins as targets for developing new antibacterial and antiviral drugs. RNA forms well defined three-dimensional structures with clefts and binding pockets reminiscent of the active sites of proteins. Furthermore, it precedes proteins in the translation pathway; inhibiting the function of a single RNA molecule would result in inhibition of multiple proteins. Thus, small molecules that bind RNA specifically would combine the advantages of antisense and RNAi strategies with the much more favorable medicinal chemistry of small-molecule therapeutics. The discovery of small-molecule inhibitors of RNA with attractive pharmacological potential would be facilitated if we had available effective computational tools of structure-based drug design. Here, we systematically test automated docking tools developed for proteins using existing three-dimensional structures of RNA-small molecule complexes. The results show that the native structures can generally be reproduced to within 2.5 angstroms more than 50-60% of the time. For more than half of the test complexes, the native ligand ranked among the top 10% compounds in a database-scoring test. Through this work, we provide parameters for the validated application of automated docking tools to the discovery of new inhibitors of RNA function.

Algorithms↗
PubMed2004

A new alpha-helical extension promotes RNA binding by the dsRBD of Rnt1p RNAse III.

Rnt1 endoribonuclease, the yeast homolog of RNAse III, plays an important role in the maturation of a diverse set of RNAs. The enzymatic activity requires a conserved catalytic domain, while RNA binding requires the double-stranded RNA-binding domain (dsRBD) at the C-terminus of the protein. While bacterial RNAse III enzymes cleave double-stranded RNA, Rnt1p specifically cleaves RNAs that possess short irregular stem-loops containing 12-14 base pairs interrupted by internal loops and bulges and capped by conserved AGNN tetraloops. Consistent with this substrate specificity, the isolated Rnt1p dsRBD and the 30-40 amino acids that follow bind to AGNN-containing stem-loops preferentially in vitro. In order to understand how Rnt1p recognizes its cognate processing sites, we have defined its minimal RNA-binding domain and determined its structure by solution NMR spectroscopy and X-ray crystallography. We observe a new carboxy-terminal helix following a canonical dsRBD structure. Removal of this helix reduces binding to Rnt1p substrates. The results suggest that this helix allows the Rnt1p dsRBD to bind to short RNA stem-loops by modulating the conformation of helix alpha1, a key RNA-recognition element of the dsRBD.

Amino Acid Sequence↗
PubMed2004

Structural mimicry of retroviral tat proteins by constrained beta-hairpin peptidomimetics: ligands with high affinity and selectivity for viral TAR RNA regulatory elements.

An approach is described to the design of beta-hairpin peptidomimetic ligands for bovine immunodeficiency virus (BIV) Tat protein, which inhibit binding to its transactivator response element (TAR) RNA. A library of peptidomimetics was derived by grafting onto a hairpin-inducing d-Pro-l-Pro template sequences related to the RNA recognition element in Tat. One hairpin mimetic was identified that binds tightly (K(d) approximately 150 nM) to BIV TAR, and another that binds also to HIV-1 TAR RNA (K(d) approximately 1-2 microM). (In the same assay, the wild-type BIV Tat(65-81) peptide binds to BIV TAR with K(d) approximately 50 nM.) The high-affinity BIV-Tat mimetic was shown to adopt a stable beta-hairpin conformation in free solution by NMR methods. Amino acid substitutions in this mimetic were shown to impact on the hairpin structure and to disrupt binding to the RNA. This family of conformationally constrained peptidomimetics affords insights into the structural requirements for binding to TAR RNA and provides a basis for the design of new ligands with increased inhibitory activity and specificity to both BIV and HIV TAR RNAs.

Amino Acid Sequence↗
PubMed2004

Rational design of inhibitors of HIV-1 TAR RNA through the stabilisation of electrostatic "hot spots".

The targeting of RNA for the design of novel anti-viral compounds has until now proceeded largely without incorporating direct input from structure-based design methodology, partly because of lack of structural data, and complications arising from substrate flexibility. We propose a paradigm to explain the physical mechanism for ligand-induced refolding of trans-activation response element (TAR RNA) from human immunodeficiency virus 1 (HIV-1). Based upon Poisson-Boltzmann analysis of the TAR structure, as bound by a peptide derived from the transcriptional activator protein, Tat, our hypothesis shows that two specific electrostatic interactions are necessary to stabilise the conformation. This result contradicts the belief that a single argininamide residue is responsible for stabilising the TAR fold, as well as the conventional wisdom that electrostatic interactions with RNA are non-specific or dominated by phosphates. We test this hypothesis by using NMR and computational methods to model the interaction of a series of novel inhibitors of the in vitro RNA-binding activities for a peptide derived from Tat. A subset of inhibitors, including the bis-guanidine compound rbt203 and its analogues, induce a conformation in TAR similar to that brought about by the protein. Comparison of the interactions of two of these ligands with the RNA and structure-activity relationships observed within the compound series, confirm the importance of the two specific electrostatic interactions in the stabilisation of the Tat-bound RNA conformation. This work illustrates how the use of medicinal chemistry and structural analysis can provide a rational basis for prediction of ligand-induced conformational change, a necessary step towards the application of structure-based methods in the design of novel RNA or protein-binding drugs.

Anti-HIV Agents↗
PubMed2004

NMR studies of protein-nucleic acid interactions.

Protein-DNA and protein-RNA complexes play key functional roles in every living organism. Therefore, the elucidation of their structure and dynamics is an important goal of structural and molecular biology. Nuclear magnetic resonance (NMR) studies of protein and nucleic acid complexes have common features with studies of protein-protein complexes: the interaction surfaces between the molecules must be carefully delineated, the relative orientation of the two species needs to be accurately and precisely determined, and close intermolecular contacts defined by nuclear Overhauser effects (NOEs) must be obtained. However, differences in NMR properties (e.g., chemical shifts) and biosynthetic pathways for sample productions generate important differences. Chemical shift differences between the protein and nucleic acid resonances can aid the NMR structure determination process; however, the relatively limited dispersion of the RNA ribose resonances makes the process of assigning intermolecular NOEs more difficult. The analysis of the resulting structures requires computational tools unique to nucleic acid interactions. This chapter summarizes the most important elements of the structure determination by NMR of protein-nucleic acid complexes and their analysis. The main emphasis is on recent developments (e.g., residual dipolar couplings and new Web-based analysis tools) that have facilitated NMR studies of these complexes and expanded the type of biological problems to which NMR techniques of structural elucidation can now be applied.

DNA↗
PubMed2003

Design of a novel globular protein fold with atomic-level accuracy.

A major challenge of computational protein design is the creation of novel proteins with arbitrarily chosen three-dimensional structures. Here, we used a general computational strategy that iterates between sequence design and structure prediction to design a 93-residue alpha/beta protein called Top7 with a novel sequence and topology. Top7 was found experimentally to be folded and extremely stable, and the x-ray crystal structure of Top7 is similar (root mean square deviation equals 1.2 angstroms) to the design model. The ability to design a new protein fold makes possible the exploration of the large regions of the protein universe not yet observed in nature.

Algorithms↗
PubMed2003

Recognition of GU-rich polyadenylation regulatory elements by human CstF-64 protein.

Vertebrate polyadenylation sites are identified by the AAUAAA signal and by GU-rich sequences downstream of the cleavage site. These are recognized by a heterotrimeric protein complex (CstF) through its 64 kDa subunit (CstF-64); the strength of this interaction affects the efficiency of poly(A) site utilization. We present the structure of the RNA-binding domain of CstF-64 containing an RNA recognition motif (RRM) augmented by N- and C-terminal helices. The C-terminal helix unfolds upon RNA binding and extends into the hinge domain where interactions with factors responsible for assembly of the polyadenylation complex occur. We propose that this conformational change initiates assembly. Consecutive Us are required for a strong CstF-GU interaction and we show how UU dinucleotides are recognized. Contacts outside the UU pocket fine tune the protein-RNA interaction and provide different affinities for distinct GU-rich elements. The protein-RNA interface remains mobile, most likely a requirement to bind many GU-rich sequences and yet discriminate against other RNAs. The structural distinction between sequences that form stable and unstable complexes provides an operational distinction between weakly and strongly processed poly(A) sites.

Amino Acid Sequence↗
PubMed2003

Solution structure of the influenza A virus cRNA promoter: implications for differential recognition of viral promoter structures by RNA-dependent RNA polymerase.

Influenza A virus replication requires the interaction of viral RNA-dependent RNA polymerase (RdRp) with promoters in both the RNA genome (vRNA) and the full-length complementary RNA (cRNA) which serve as templates for the generation of new vRNAs. Although RdRp binds both promoters effectively, it must also discriminate between them because they serve different functional roles in the viral life cycle. Even though the inherent asymmetry between two RNA promoters is considered as a cause of the differential recognition by the RdRp, the structural basis for the ability of the RdRp to recognize the RNA promoters and discriminate effectively between them remains unsolved. Here we report the structure of the cRNA promoter of influenza A virus as determined by heteronuclear magnetic resonance spectroscopy. The terminal region is extremely unstable and does not have a rigid structure. The major groove of the internal loop is widened by the displacement of a novel A*(UU) motif toward the minor groove. These internal loop residues show distinguishable dynamic characters, with differing motional timescales for each residue. Comparison of the cRNA promoter structure with that of the vRNA promoter reveals common structural and dynamic elements in the internal loop, but also differences that provide insight into how the viral RdRp differentially recognizes the cRNA and vRNA promoters.

Base Sequence↗
PubMed2003

The solution structure of an essential stem-loop of human telomerase RNA.

The ribonucleoprotein enzyme telomerase maintains chromosome ends in most eukaryotes and is critical for a cell's genetic stability and its proliferative viability. All telomerases contain a catalytic protein component homologous to viral reverse transcriptases (TERT) and an RNA (TR) that provides the template sequence as well as a scaffold for ribonucleoprotein assembly. Vertebrate telomerase RNAs have three essential domains: the template, activation and stability domains. Here we report the NMR structure of an essential RNA element derived from the human telomerase RNA activation domain. The sequence forms a stem-loop structure stabilized by a GU wobble pair formed by two of the five unpaired residues capping a short double helical region. The remaining three loop residues are in a well-defined conformation and form phosphate-base stacking interactions reminiscent of other RNA loop structures. Mutations of these unpaired nucleotides abolish enzymatic activity. The structure rationalizes a number of biochemical observations, and allows us to propose how the loop may function in the telomerase catalytic cycle. The pre-formed structure of the loop exposes the bases of these three essential nucleotides and positions them to interact with other RNA sequences within TR, with the reverse transcriptase or with the newly synthesized telomeric DNA strand. The functional role of this stem-loop appears to be conserved in even distantly related organisms such as yeast and ciliates.

Humans↗
PubMed2002

Structure and stability of wild-type and mutant RNA internal loops from the SL-1 domain of the HIV-1 packaging signal.

The packaging signal (Psi) of the human immunodeficiency virus type 1 (HIV-1) enables encapsidation of the full-length genomic RNA against a background of a vast excess of cellular mRNAs. The core HIV-1 Psi is approximately 109 nucleotides and contains sequences critical for viral genomic dimerisation and splicing, in addition to the packaging signal. It consists of a series of stem-loops (termed SL-1 to SL-4), which can be arranged in a cloverleaf secondary structure. Using a combination of NMR spectroscopy, UV melting experiments, molecular modeling and phylogenetic analyses, we have explored the structure of two conserved internal loops proximal to the palindromic sequence of SL-1. Internal loop A, composed of six purines, forms a flexible structure that is strikingly similar to the Rev responsive element motif when bound to Rev protein. This result suggests that it may function as a protein-binding site. The absolutely conserved four-purine internal loop B is instead conformationally and thermodynamically unstable, and exhibits multiple conformations in solution. By introducing a double AGG to GGA mutation within this loop, its conformation is stabilised to form a new intra-molecular G:A:G base-triplet. The structure of the GGA mutant explains the relative instability of the wild-type loop. In a manner analogous to SL-3, we propose that conformational flexibility at this site may facilitate melting of the structure during Gag protein capture or genomic RNA dimerisation.

Base Pairing↗
PubMed2002

Molecular dynamics simulation of the RNA complex of a double-stranded RNA-binding domain reveals dynamic features of the intermolecular interface and its hydration.

The interaction between double-stranded RNA (dsRNA) and the third double-stranded domain (dsRBD) from Drosophila Staufen protein represents a paradigm to understand how the dsRBD protein family, one of the most common RNA-binding protein units, binds dsRNA. The nuclear magnetic resonance (NMR) structure of this complex and the x-ray structure of another family member revealed the stereochemical basis for recognition, but also raised new questions. Although the crystallographic studies revealed a highly ordered interface containing numerous water-mediated contacts, NMR suggested extensive residual motion at the interface. To address how interfacial motion contributes to molecular recognition in the dsRBD-dsRNA system, we conducted a 2-ns molecular dynamics simulation of the complex derived from Staufen protein and of the separate protein and RNA components. The results support the observation that a high degree of conformational flexibility is retained upon complex formation and that this involves interfacial residues that are critical for dsRBD-dsRNA binding. The structural origin of this residual flexibility is revealed by the analysis of the trajectory of motion. Individual basic side chains switch continuously from one RNA polar group to another with a residence time seldom exceeding 100 ps, while retaining favorable interaction with RNA throughout much of the simulation. Short-lived water molecules mediate some of these interactions for a large fraction of the trajectory studied here. This result indicates that water molecules are not statically associated with the interface, but continuously exchange with the bulk solvent on a 1-10-ps time scale. This work provides new insight into dsRBD-dsRNA recognition and builds upon a growing body of evidence, suggesting that short-lived dynamic interactions play important roles in protein-nucleic acid interactions.

Binding Sites↗