Jean Cavarelli

The active site of yeast aspartyl-tRNA synthetase: structural and functional aspects of the aminoacylation reaction.

The crystal structures of the various complexes formed by yeast aspartyl-tRNA synthetase (AspRS) and its substrates provide snapshots of the active site corresponding to different steps of the aminoacylation reaction. Native crystals of the binary complex tRNA-AspRS were soaked in solutions containing the two other substrates, ATP (or its analog AMPPcP) and aspartic acid. When all substrates are present in the crystal, this leads to the formation of the aspartyl-adenylate and/or the aspartyl-tRNA. A class II-specific pathway for the aminoacylation reaction is proposed which explains the known functional differences between the two classes while preserving a common framework. Extended signature sequences characteristic of class II aaRS (motifs 2 and 3) constitute the basic functional unit. The ATP molecule adopts a bent conformation, stabilized by the invariant Arg531 of motif 3 and a magnesium ion coordinated to the pyrophosphate group and to two class-invariant acidic residues. The aspartic acid substrate is positioned by a class II invariant acidic residue, Asp342, interacting with the amino group and by amino acids conserved in the aspartyl synthetase family. The amino acids in contact with the substrates have been probed by site-directed mutagenesis for their functional implication.

tRNA aminoacylation by arginyl‐tRNA synthetase: induced conformations during substrates binding

The 2.2 Å crystal structure of a ternary complex formed by yeast arginyl‐tRNA synthetase and its cognate tRNAArg in the presence of the L‐arginine substrate highlights new atomic features used for specific substrate recognition. This first example of an active complex formed by a class Ia aminoacyl‐tRNA synthetase and its natural cognate tRNA illustrates additional strategies used for specific tRNA selection. The enzyme specifically recognizes the D‐loop and the anticodon of the tRNA, and the mutually induced fit produces a conformation of the anticodon loop never seen before. Moreover, the anticodon binding triggers conformational changes in the catalytic center of the protein. The comparison with the 2.9 Å structure of a binary complex formed by yeast arginyl‐tRNA synthetase and tRNAArg reveals that L‐arginine binding controls the correct positioning of the CCA end of the tRNAArg. Important structural changes induced by substrate binding are observed in the enzyme. Several key residues of the active site play multiple roles in the catalytic pathway and thus highlight the structural dynamics of the aminoacylation reaction.

Functional insights from structures of coactivator-associated arginine methyltransferase 1 domains.

Coactivator‐associated arginine methyltransferase 1 (CARM1), a protein arginine methyltransferase recruited by several transcription factors, methylates a large variety of proteins and plays a critical role in gene expression. We report, in this paper, four crystal structures of isolated modules of CARM1. The 1.7 Å crystal structure of the N‐terminal domain of CARM1 reveals an unexpected PH domain, a scaffold frequently found to regulate protein–protein interactions in a large variety of biological processes. Three crystal structures of the CARM1 catalytic module, two free and one cofactor‐bound forms (refined at 2.55 Å, 2.4 Å and 2.2 Å, respectively) reveal large structural modifications including disorder to order transition, helix to strand transition and active site modifications. The N‐terminal and the C‐terminal end of CARM1 catalytic module contain molecular switches that may inspire how CARM1 regulates its biological activities by protein–protein interactions.

L‐Arginine recognition by yeast arginyl‐tRNA synthetase

The crystal structure of arginyl‐tRNA synthetase (ArgRS) from Saccharomyces cerevisiae, a class I aminoacyl‐tRNA synthetase (aaRS), with L‐Arginine bound to the active site has been solved at 2.75 Å resolution and refined to a crystallographic R‐factor of 19.7%. ArgRS is composed predominantly of α‐helices and can be divided into five domains, including the class I‐specific active site. The N‐terminal domain shows striking similarity to some completely unrelated proteins and defines a module which should participate in specific tRNA recognition. The C‐terminal domain, which is the putative anticodon‐binding module, displays an all‐α‐helix fold highly similar to that of Escherichia coli methionyl‐tRNA synthetase. While ArgRS requires tRNAArg for the first step of the aminoacylation reaction, the results show that its presence is not a prerequisite for L‐Arginine binding. All H‐bond‐forming capability of L‐Arginine is used by the protein for the specific recognition. The guanidinium group forms two salt bridge interactions with two acidic residues, and one H‐bond with a tyrosine residue; these three residues are strictly conserved in all ArgRS sequences. This tyrosine is also conserved in other class I aaRS active sites but plays several functional roles. The ArgRS structure allows the definition of a new framework for sequence alignments and subclass definition in class I aaRSs.

Structural basis for hijacking of cellular LxxLL motifs by papillomavirus E6 oncoproteins.

Targeting HPV Papillomaviruses infect mammalian epithelial cells and induce cancers, including cervical cancer in humans. Vaccines against human papillomavirus (HPV) can prevent, but not cure, infection. A key viral oncoprotein, E6, acts by binding and inactivating many host proteins. Zanier et al. (p. 694) determined high-resolution crystal structures of bovine papillomavirus bound to a peptide from the focal adhesion protein, paxillin, and of HPV bound to a peptide from the ubiquitin ligase E6AP. The structures show that the peptide binds in a pocket formed by two zinc domains and a linker helix, which represents a promising target for therapeutics. Crystal structures show how a key oncoprotein in human papillomavirus binds host proteins. E6 viral oncoproteins are key players in epithelial tumors induced by papillomaviruses in vertebrates, including cervical cancer in humans. E6 proteins target many host proteins by specifically interacting with acidic LxxLL motifs. We solved the crystal structures of bovine (BPV1) and human (HPV16) papillomavirus E6 proteins bound to LxxLL peptides from the focal adhesion protein paxillin and the ubiquitin ligase E6AP, respectively. In both E6 proteins, two zinc domains and a linker helix form a basic-hydrophobic pocket, which captures helical LxxLL motifs in a way compatible with other interaction modes. Mutational inactivation of the LxxLL binding pocket disrupts the oncogenic activities of both E6 proteins. This work reveals the structural basis of both the multifunctionality and the oncogenicity of E6 proteins.

Structural basis for the inhibition of histone deacetylase 8 (HDAC8), a key epigenetic player in the blood fluke Schistosoma mansoni.

The treatment of schistosomiasis, a disease caused by blood flukes parasites of the Schistosoma genus, depends on the intensive use of a single drug, praziquantel, which increases the likelihood of the development of drug-resistant parasite strains and renders the search for new drugs a strategic priority. Currently, inhibitors of human epigenetic enzymes are actively investigated as novel anti-cancer drugs and have the potential to be used as new anti-parasitic agents. Here, we report that Schistosoma mansoni histone deacetylase 8 (smHDAC8), the most expressed class I HDAC isotype in this organism, is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity. The crystal structure of smHDAC8 shows that this enzyme adopts a canonical α/β HDAC fold, with specific solvent exposed loops corresponding to insertions in the schistosome HDAC8 sequence. Importantly, structures of smHDAC8 in complex with generic HDAC inhibitors revealed specific structural changes in the smHDAC8 active site that cannot be accommodated by human HDACs. Using a structure-based approach, we identified several small-molecule inhibitors that build on these specificities. These molecules exhibit an inhibitory effect on smHDAC8 but show reduced affinity for human HDACs. Crucially, we show that a newly identified smHDAC8 inhibitor has the capacity to induce apoptosis and mortality in schistosomes. Taken together, our biological and structural findings define the framework for the rational design of small-molecule inhibitors specifically interfering with schistosome epigenetic mechanisms, and further support an anti-parasitic epigenome targeting strategy to treat neglected diseases caused by eukaryotic pathogens.

The structure of Staphylococcus aureus epidermolytic toxin A, an atypic serine protease, at 1.7 A resolution.

BACKGROUND Staphylococcal epidermolytic toxins A and B (ETA and ETB) are responsible for the staphylococcal scalded skin syndrome of newborn and young infants; this condition can appear just a few hours after birth. These toxins cause the disorganization and disruption of the region between the stratum spinosum and the stratum granulosum--two of the three cellular layers constituting the epidermis. The physiological substrate of ETA is not known and, consequently, its mode of action in vivo remains an unanswered question. Determination of the structure of ETA and its comparison with other serine proteases may reveal insights into ETAs catalytic mechanism. RESULTS The crystal structure of staphylococcal ETA has been determined by multiple isomorphous replacement and refined at 1.7 A resolution with a crystallographic R factor of 0.184. The structure of ETA reveals it to be a new and unique member of the trypsin-like serine protease family. In contrast to other serine protease folds, ETA can be characterized by ETA-specific surface loops, a lack of cysteine bridges, an oxyanion hole which is not preformed, an S1 specific pocket designed for a negatively charged amino acid and an ETA-specific specific N-terminal helix which is shown to be crucial for substrate hydrolysis. CONCLUSIONS Despite very low sequence homology between ETA and other trypsin-like serine proteases, the ETA crystal structure, together with biochemical data and site-directed mutagenesis studies, strongly confirms the classification of ETA in the Glu-endopeptidase family. Direct links can be made between the protease architecture of ETA and its biological activity.

Acyl derivatives of p-aminosulfonamides and dapsone as new inhibitors of the arginine methyltransferase hPRMT1.

Arginine methylation is an epigenetic modification that receives increasing interest as it plays an important role in several diseases. This is especially true for hormone-dependent cancer, seeing that histone methylation by arginine methyltransferase I (PRMT1) is involved in the activation of sexual hormone receptors. Therefore, PRMT inhibitors are potential drugs and interesting tools for cell biology. A dapsone derivative called allantodapsone previously identified by our group served as a lead structure for inhibitor synthesis. Acylated derivatives of p-aminobenzenesulfonamides and the antilepra drug dapsone were identified as new inhibitors of PRMT1 by in vitro testing. The bis-chloroacetyl amide of dapsone selectively inhibited human PRMT1 in the low micromolar region and was selective for PRMT1 as compared to the arginine methyltransferase CARM1 and the lysine methyltransferase Set7/9. It showed anticancer activity on MCF7a and LNCaP cells and blocked androgen dependent transcription specifically in a reporter gene system. Likewise, a transcriptional block was also demonstrated in LNCaP cells using quantitative RT-PCR on the mRNA of androgen dependent genes.

Crystal structure of the V-region of Streptococcus mutans antigen I/II at 2.4 A resolution suggests a sugar preformed binding site.

Antigens I/II are large multifunctional adhesins from oral viridans streptococci that exert immunomodulatory effects on human cells and play important roles in inflammatory disorders. Among them, Streptococcus mutans plays a major role in the initiation of dental caries. The structure of the V-region (SrV+, residues 464-840) of the antigen I/II of S. mutans has been determined using the multiwavelength anomalous diffraction phasing technique with seleno-methionine-substituted recombinant protein and subsequently refined at 2.4 A resolution. The crystal structure of SrV+ revealed a lectin-like fold that displays a putative preformed carbohydrate-binding site stabilized by a metal ion. Inhibition of this binding site may confer to humans a protection against dental caries and dissemination of the bacteria to extra-oral sites involved in life-threatening inflammatory diseases. This crystal structure constitutes a first step in understanding the structure-function relationship of antigens I/II and may help in delineating new preventive or therapeutic strategies against colonization of the host by oral streptococci.

Noncanonical Tandem SH2 Enables Interaction of Elongation Factor Spt6 with RNA Polymerase II

Src homology 2 (SH2) domains are mostly found in multicellular organisms where they recognize phosphotyrosine-containing signaling proteins. Spt6, a conserved transcription factor and putative histone chaperone, contains a C-terminal SH2 domain conserved from yeast to human. In mammals, this SH2 domain recognizes phosphoserines rather than phosphotyrosines and is essential for the recruitment of Spt6 by elongating RNA polymerase II (RNAPII), enabling Spt6 to participate in the coupling of transcription elongation, chromatin modulation, and mRNA export. We have determined the structure of the entire Spt6 C-terminal region from Antonospora locustae, revealing the presence of two highly conserved tandem SH2 domains rather than a single SH2 domain. Although the first SH2 domain has a canonical organization, the second SH2 domain is highly noncanonical and appears to be unique in the SH2 family. However, both SH2 domains have phosphate-binding determinants. Our biochemical and genetic data demonstrate that the complete tandem, but not the individual SH2 domains, are necessary and sufficient for the interaction of Spt6 with RNAPII and are important for Spt6 function in vivo. Furthermore, our data suggest that binding of RNAPII to the Spt6 tandem SH2 is more extensive than the mere recognition of a doubly phosphorylated C-terminal domain peptide by the tandem SH2. Taken together, our results show that Spt6 interaction with RNAPII via a novel arrangement of canonical and noncanonical SH2 domains is crucial for Spt6 function in vivo.