Synergy and allostery in ligand binding by HIV-1 Nef.

The Nef protein of human and simian immunodeficiency viruses boosts viral pathogenicity through its interactions with host cell proteins. By combining the polyvalency of its large unstructured regions with the binding selectivity and strength of its folded core domain, Nef can associate with many different host cell proteins, thereby disrupting their functions. For example, the combination of a linear proline-rich motif and hydrophobic core domain surface allows Nef to bind tightly and specifically to SH3 domains of Src family kinases. We investigated whether the interplay between Nef's flexible regions and its core domain could allosterically influence ligand selection. We found that the flexible regions can associate with the core domain in different ways, producing distinct conformational states that alter the way in which Nef selects for SH3 domains and exposes some of its binding motifs. The ensuing crosstalk between ligands might promote functionally coherent Nef-bound protein ensembles by synergizing certain subsets of ligands while excluding others. We also combined proteomic and bioinformatics analyses to identify human proteins that select SH3 domains in the same way as Nef. We found that only 3% of clones from a whole-human fetal library displayed Nef-like SH3 selectivity. However, in most cases, this selectivity appears to be achieved by a canonical linear interaction rather than by a Nef-like 'tertiary' interaction. Our analysis supports the contention that Nef's mode of hijacking SH3 domains is a virus-specific adaptation with no or very few cellular counterparts. Thus, the Nef tertiary binding surface is a promising virus-specific drug target.

Amyloidogenicity as a driving force for the formation of functional oligomers.

Insoluble amyloid fibrils formed by self-assembly of amyloidogenic regions of proteins have a cross-ß-structure. In this work, by using targeted molecular dynamics and rigid body simulation, we demonstrate that if a protein consists of an amyloidogenic region and a globular domain(s) and if the linker between them is short enough, such molecules cannot assemble into amyloid fibrils, instead, they form oligomers with a defined and limited number of ß-strands in the cross-ß core. We show that this blockage of the amyloid growth is due to the steric repulsion of the globular structures linked to amyloidogenic regions. Furthermore, we establish a relationship between the linker length and the number of monomers in such nanoparticles. We hypothesise that such oligomerisation can be a yet unrecognised way to form natural protein complexes involved in biological processes. Our results can also be used in protein engineering for designing soluble nanoparticles carrying different functional domains.

Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1.

The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN), a large multiprotein complex that resembles the 19S lid of the 26S proteasome, plays a central role in the regulation of the E3-cullin RING ubiquitin ligases (CRLs). The catalytic activity of the CSN complex, carried by subunit 5 (CSN5/Jab1), resides in the deneddylation of the CRLs that is the hydrolysis of the cullin-neural precursor cell expressed developmentally downregulated gene 8 (Nedd8)isopeptide bond. Whereas CSN-dependent CSN5 displays isopeptidase activity, it is intrinsically inactive in other physiologically relevant forms. Here we analyze the crystal structure of CSN5 in its catalytically inactive form to illuminate the molecular basis for its activation state. We show that CSN5 presents a catalytic domain that brings essential elements to understand its activity control. Although the CSN5 active site is catalytically competent and compatible with di-isopeptide binding, the Ins-1 segment obstructs access to its substrate-binding site, and structural rearrangements are necessary for the Nedd8-binding pocket formation. Detailed study of CSN5 by molecular dynamics unveils signs of flexibility and plasticity of the Ins-1 segment. These analyses led to the identification of a molecular trigger implicated in the active/inactive switch that is sufficient to impose on CSN5 an active isopeptidase state. We show that a single mutation in the Ins-1 segment restores biologically relevant deneddylase activity. This study presents detailed insights into CSN5 regulation. Additionally, a dynamic monomer-dimer equilibrium exists both in vitro and in vivo and may be functionally relevant.

Lensless coherent imaging of proteins and supramolecular assemblies: Efficient phase retrieval by the charge flipping algorithm.

Diffractive imaging using the intense and coherent beam of X-ray free-electron lasers opens new perspectives for structural studies of single nanoparticles and biomolecules. Simulations were carried out to generate 3D oversampled diffraction patterns of non-crystalline biological samples, ranging from peptides and proteins to megadalton complex assemblies, and to recover their molecular structure from nanometer to near-atomic resolutions. Using these simulated data, we show here that iterative reconstruction methods based on standard and variant forms of the charge flipping algorithm, can efficiently solve the phase retrieval problem and extract a unique and reliable molecular structure. Contrary to the case of conventional algorithms, where the estimation and the use of a compact support is imposed, our approach does not require any prior information about the molecular assembly, and is amenable to a wide range of biological assemblies. Importantly, the robustness of this ab initio approach is illustrated by the fact that it tolerates experimental noise and incompleteness of the intensity data at the center of the speckle pattern.

The backbone model of the Arabis mosaic virus reveals new insights into functional domains of Nepovirus capsid.

Arabis mosaic virus (ArMV) and Grapevine fanleaf virus (GFLV) are two picorna-like viruses from the genus Nepovirus, consisting in a bipartite RNA genome encapsidated into a 30 nm icosahedral viral particle formed by 60 copies of a single capsid protein (CP). They are responsible for a severe degeneration of grapevines that occurs in most vineyards worldwide. Although sharing a high level of sequence identity between their CP, ArMV is transmitted exclusively by the ectoparasitic nematode Xiphinema diversicaudatum whereas GFLV is specifically transmitted by the nematode X. index. The structural determinants involved in the transmission specificity of both viruses map solely to their respective CP. Recently, reverse genetic and crystallographic studies on GFLV revealed that a positively charged pocket in the CP B domain located at the virus surface may be responsible for vector specificity. To go further into delineating the coat protein determinants involved in transmission specificity, we determined the 6.5 Å resolution cryo-electron microscopy structure of ArMV and used homology modeling and flexible fitting approaches to build its pseudo-atomic structure. This study allowed us to resolve ArMV CP architecture and delineate connections between ArMV capsid shell and its RNA. Comparison of ArMV and GFLV CPs reveals structural differences in the B domain pocket, thus strengthening the hypothesis of a key role of this region in the viral transmission specificity and identifies new potential functional domains of Nepovirus capsid.

Structural insights into the molecular mechanisms of cauliflower mosaic virus transmission by its insect vector.

Cauliflower mosaic virus (CaMV) is transmitted from plant to plant through a seemingly simple interaction with insect vectors. This process involves an aphid receptor and two viral proteins, P2 and P3. P2 binds to both the aphid receptor and P3, itself tightly associated with the virus particle, with the ensemble forming a transmissible viral complex. Here, we describe the conformations of both unliganded CaMV P3 protein and its virion-associated form. X-ray crystallography revealed that the N-terminal domain of unliganded P3 is a tetrameric parallel coiled coil with a unique organization showing two successive four-stranded subdomains with opposite supercoiling handedness stabilized by a ring of interchain disulfide bridges. A structural model of virus-liganded P3 proteins, folding as an antiparallel coiled-coil network coating the virus surface, was derived from molecular modeling. Our results highlight the structural and biological versatility of this coiled-coil structure and provide new insights into the molecular mechanisms involved in CaMV acquisition and transmission by the insect vector.

Scent evolution in Chinese roses.

The phenolic methyl ether 3,5-dimethoxytoluene (DMT) is a major scent compound of many modern rose varieties, and its fragrance participates in the characteristic "tea scent" that gave their name to Tea and Hybrid Tea roses. Among wild roses, phenolic methyl ether (PME) biosynthesis is restricted to Chinese rose species, but the progenitors of modern roses included both European and Chinese species (e.g., Rosa chinensis cv Old Blush), so this trait was transmitted to their hybrid progeny. The last steps of the biosynthetic pathways leading to DMT involve two methylation reactions catalyzed by the highly similar orcinol O-methyltransferases (OOMT) 1 and 2. OOMT1 and OOMT2 enzymes exhibit different substrate specificities that are consistent with their operating sequentially in DMT biosynthesis. Here, we show that these different substrate specificities are mostly due to a single amino acid polymorphism in the phenolic substrate binding site of OOMTs. An analysis of the OOMT gene family in 18 species representing the diversity of the genus Rosa indicated that only Chinese roses possess both the OOMT2 and the OOMT1 genes. In addition, we provide evidence that the Chinese-rose-specific OOMT1 genes most probably evolved from an OOMT2-like gene that has homologues in the genomes of all extant roses. We propose that the emergence of the OOMT1 gene may have been a critical step in the evolution of scent production in Chinese roses.

Fertilization and early seed formation.

The double fertilization of flowering plants is a complex process, encompassing multiple steps. From its discovery more than a century ago, many useful descriptive approaches have been employed to better unveil specific steps/mechanisms. More recently, the development of an in vitro assay developed in our laboratory, has allowed a better understanding of this phenomenon. However, in vitro methods may show some limitations. The search for complementary strategies, especially with the search of mutants affected in the fertilization step allowed one to elucidate this critical and unique phenomenon in living organisms. Genes involved in pollen tube guidance or pollen discharge in synergids have been identified, as well as genes exhibiting differential expression in sperm, egg and central cells before and after fertilization. A calcium wave proved to correspond to the first cellular event seen after cytoplasmic fusion in the fertilized egg cell or zygote, which develops into a multi-cellular organism with an elaborate body plan. The development of the fertilized central cell into a nourishing tissue (endosperm) starts with the formation of the coenocyte, a multinuclear single cell unique in the plant kingdom, cellularization occurring later on. The balance of the paternal and maternal genomes, which is under the control of the FIS polycomb group complex, was found to be of the utmost importance for the successful development of the seed.

Establishment of Constraints on Amyloid Formation Imposed by Steric Exclusion of Globular Domains.

In many disease-related and functional amyloids, the amyloid-forming regions of proteins are flanked by globular domains. When located in close vicinity of the amyloid regions along the chain, the globular domains can prevent the formation of amyloids because of the steric repulsion. Experimental tests of this effect are few in number and non-systematic, and their interpretation is hampered by polymorphism of amyloid structures. In this situation, modeling approaches that use such a clear-cut criterion as the steric tension can give us highly trustworthy results. In this work, we evaluated this steric effect by using molecular modeling and dynamics. As an example, we tested hybrid proteins containing an amyloid-forming fragment of Aß peptide (17-42) linked to one or two globular domains of GFP. Searching for the shortest possible linker, we constructed models with pseudo-helical arrangements of the densely packed GFPs around the Aß amyloid core. The molecular modeling showed that linkers of 7 and more residues allow fibrillogenesis of the Aß-peptide flanked by GFP on one side and 18 and more residues when Aß-peptide is flanked by GFPs on both sides. Furthermore, we were able to establish a more general relationship between the size of the globular domains and the length of the linkers by using analytical expressions and rigid body simulations. Our results will find use in planning and interpretation of experiments, improvement of the prediction of amyloidogenic regions in proteins, and design of new functional amyloids carrying globular domains.

Female control of male gamete delivery during fertilization in Arabidopsis thaliana.

Fertilization in both animals and plants relies on the correct targeting of the male gametes to the female gametes. In flowering plants, the pollen tube carries two male gametes through the maternal reproductive tissues to the embryo sac, which contains two female gametes. The pollen tube then releases its two male gametes into a specialized receptor cell of the embryo sac, the synergid cell. The mechanisms controlling this critical step of gamete delivery are unknown. Here, data based on the new sirène (srn) mutant of Arabidopsis thaliana provide the first evidence for female control over male gamete delivery. Live imaging of fertilization shows that wild-type pollen tubes do not stop their growth and do not deliver their contents in srn embryo sacs.

Structure of the mature P3-virus particle complex of cauliflower mosaic virus revealed by cryo-electron microscopy.

The cauliflower mosaic virus (CaMV) has an icosahedral capsid composed of the viral protein P4. The viral product P3 is a multifunctional protein closely associated with the virus particle within host cells. The best-characterized function of P3 is its implication in CaMV plant-to-plant transmission by aphid vectors, involving a P3-virion complex. In this transmission process, the viral protein P2 attaches to virion-bound P3, and creates a molecular bridge between the virus and a putative receptor in the aphid's stylets. Recently, the virion-bound P3 has been suggested to participate in cell-to-cell or long-distance movement of CaMV within the host plant. Thus, as new data accumulate, the importance of the P3-virion complex during the virus life-cycle is becoming more and more evident. To provide a first insight into the knowledge of the transmission process of the virus, we determined the 3D structures of native and P3-decorated virions by cryo-electron microscopy and computer image processing. By difference mapping and biochemical analysis, we show that P3 forms a network around the capsomers and we propose a structural model for the binding of P3 to CaMV capsid in which its C terminus is anchored deeply in the inner shell of the virion, while the N-terminal extremity is facing out of the CaMV capsid, forming dimers by coiled-coil interactions. Our results combined with existing data reinforce the hypothesis that this coiled-coil N-terminal region of P3 could coordinate several functions during the virus life-cycle, such as cell-to-cell movement and aphid-transmission.

Structure of the cathelicidin motif of protegrin-3 precursor: structural insights into the activation mechanism of an antimicrobial protein.

Cathelicidins are a family of antimicrobial proteins isolated from leucocytes and epithelia cells that contribute to the innate host defense mechanisms in mammalians. Located in the C-terminal part of the holoprotein, the cathelicidin-derived antimicrobial peptide is liberated by a specific protease cleavage. Here, we report the X-ray structure of the cathelicidin motif of protegrin-3 solved by MAD phasing using the selenocysteine-labeled protein. Its overall structure represents a fold homologous to the cystatin family and adopts two native states, a monomer, and a domain-swapped dimer. This crystal structure is the first example of a structural characterization of the highly conserved cathelicidin motif and thus provides insights into the possible mechanism of activation of the antimicrobial protegrin peptide.

Selenomethionine and selenocysteine double labeling strategy for crystallographic phasing.

A protocol for the quantitative incorporation of both selenomethionine and selenocysteine into recombinant proteins overexpressed in Escherichia coli is described. This methodology is based on the use of a suitable cysteine auxotrophic strain and a minimal medium supplemented with selenium-labeled methionine and cysteine. The proteins chosen for these studies are the cathelin-like motif of protegrin-3 and a nucleoside-diphosphate kinase. Analysis of the purified proteins by electrospray mass spectrometry and X-ray crystallography revealed that both cysteine and methionine residues were isomorphously replaced by selenocysteine and selenomethionine. Moreover, selenocysteines allowed the formation of unstrained and stable diselenide bridges in place of the canonical disulfide bonds. In addition, we showed that NDP kinase contains a selenocysteine adduct on Cys122. This novel selenium double-labeling method is proposed as a general approach to increase the efficiency of the MAD technique used for phase determination in protein crystallography.

The Human Mixed Lineage Leukemia 5 (MLL5), a Sequentially and Structurally Divergent SET Domain-Containing Protein with No Intrinsic Catalytic Activity.

Mixed Lineage Leukemia 5 (MLL5) plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. Chromatin binding is ensured by its plant homeodomain (PHD) through a direct interaction with the N-terminus of histone H3 (H3). In addition, MLL5 contains a Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain, a protein module that usually displays histone lysine methyltransferase activity. We report here the crystal structure of the unliganded SET domain of human MLL5 at 2.1 Å resolution. Although it shows most of the canonical features of other SET domains, both the lack of key residues and the presence in the SET-I subdomain of an unusually large loop preclude the interaction of MLL5 SET with its cofactor and substrate. Accordingly, we show that MLL5 is devoid of any in vitro methyltransferase activity on full-length histones and histone H3 peptides. Hence, the three dimensional structure of MLL5 SET domain unveils the structural basis for its lack of methyltransferase activity and suggests a new regulatory mechanism.

Multicellular structures developing during maize microspore culture express endosperm and embryo-specific genes and show different embryogenic potentialities.

During maize pollen embryogenesis, a range of multicellular structures are formed. Using different approaches, the "nature" of these structures has been determined in terms of their embryogenic potential. In situ molecular identification techniques for gene transcripts and products, and a novel cell tracking system indicated the presence of embryogenic (embryo-like structures, ELS) and non-embryogenic (callus-like structures, CLS) structures that occurred for short periods within the cultures. Some multicellular structures with a compact appearance generated embryos. RT-PCR and fluorescence in situ hybridization (FISH) with confocal microscopy techniques using specific gene markers of the endosperm (ZmESR2, ZmAE3) and embryo (LTP2 and ZmOCL1, ZmOCL3) revealed "embryo" and "endosperm" potentialities in these various multicellular structures present in the cultures. The results presented here showed distinct and specific patterns of gene expression. Altogether, the results demonstrate the presence of different molecules on both embryonic and non-embryonic structures. Their possible roles are discussed in the context of a parallel between embryo/endosperm interactions in planta and embryonic and non-embryonic structure interrelations under in vitro conditions.

Crystallization and preliminary X-ray crystallographic analysis of human Geminin coiled-coil domain.

Initially discovered in Xenopus laevis, Geminin is a DNA replication initiation inhibitor found in higher eukaryotes. The coiled-coil domain of Human Geminin (termed GemH-37) has been crystallized by the vapor-diffusion sitting-drop method. A complete 1.74 A data set has been collected on a single orthorhombic crystal with unit cell parameters a = 25.25, b = 44.35, c = 68.58 A. Successful molecular replacement shows that GemH-37 is a dimeric parallel coiled-coil. Structural analysis is now in progress.

A W-box is required for full expression of the SA-responsive gene SFR2.

Transcripts of SFR2, a member of the S family of receptor kinase genes, accumulate rapidly in Brassica oleracea leaves in response to wounding, bacterial infection and following treatment with salicylic acid (SA). Expression of a chimeric gene consisting of the SFR2 5' flanking sequence fused to the gusA reporter gene is also induced in wounded and SA-treated Arabidopsis plants indicating that the observed response is conferred by the SFR2 promoter. We show here that, in Arabidopsis plants carrying the salicylate hydroxylase (NahG) transgene, wound induction of the SFR2 promoter-gusA reporter fusion was abolished, indicating that, as has previously been shown for the response to bacterial infection, SA is required for the response to wounding. Deletion analysis of the SFR2 promoter identified a region necessary for full expression following SA treatment. This region, which includes two putative W-boxes, is conserved in the promoter of the Arabidopsis SFR2 homologue, ARK3. Deletion of a 12 bp region containing the two W-box motifs reduced the response to SA treatment. Tandem repeats of the W-box-containing element fused upstream of a CaMV 35S minimal promoter enhanced reporter gene expression in transgenic Arabidopsis both in the absence and presence of SA. Gel-mobility shift assays showed that Arabidopsis leaf extracts contained factors that bound to a fragment of the promoter spanning the putative W-boxes and that a fragment in which these motifs were mutated was unable to compete for binding. In summary, induction of the SFR2 promoter in response to bacterial infection and wounding requires SA, and full expression of the induced gene requires the presence of a functional element containing W-box motifs in the SFR2 promoter. The involvement of two W-boxes indicates that transcription factors of the WRKY family may play a key role in mediating these responses.

NMR solution structure of Mob1, a mitotic exit network protein and its interaction with an NDR kinase peptide.

Proteins of the Mob1/phocein family are found in all eukaryotic cells. In yeast, they are activating subunits of Dbf2-related protein kinases involved in cell cycle control. Despite the wide occurrence of these proteins, their biological functions remain poorly understood. Here we report the solution structure of the Mob1 protein from Xenopus laevis solved by heteronuclear multidimensional NMR. The structure reveals a fold constituted by a central left-handed four-helix bundle, one connecting helix, two flanking helices and a long flexible loop. The clustering of two Cys and two His residues, and zinc measurement by atomic absorption spectroscopy support the existence of a zinc ion binding site. Our NMR structure is in good agreement with the recently described X-ray structure of human Mob1-A. Chemical shift perturbations observed upon addition of a peptide encompassing the basic region of the N-terminal regulatory domain of NDR kinase were used to identify and map a specific interaction between Mob1 and this kinase. The chemical shift changes indicate that the main interaction occurs on the acidic and conserved surface of Mob1. This surface was previously hypothesized to be the interaction surface according to the X-ray structure and was identified as functionally important in yeast. Our data suggest that the NDR kinase is a functional Dbf2 homologue in animal cells and contributes to the understanding of the molecular function of Mob1 proteins.

Crystal structure of the N-terminal dimerisation domain of VicH, the H-NS-like protein of Vibrio cholerae.

The histone-like nucleoid structuring (H-NS) protein is a global modulator of gene expression in Gram-negative bacteria. VicH, the H-NS protein of Vibrio cholerae, regulates the expression of certain major virulence determinants implicated in the pathogenesis of cholera. We present here the 2.5A crystal structure of the N-terminal oligomerisation domain of VicH (VicH_Nt). VicH_Nt adopts the same fold and dimeric assembly as the NMR structure of Escherichia coli H-NS_Nt, thus validating this fold against conflicting data. The structural similarity of V.cholerae VicH_Nt and E.coli H-NS_Nt, despite differences in origin, system of expression, experimental conditions and techniques used, indicates that the fold determined in our studies is robust to experimental conditions. Structural analysis and homology modelling were carried out to further elucidate the molecular basis of the functional polyvalence of the N-terminal domain. Our analysis of members of the H-NS superfamily supports the suggestion that the oligomerisation function of H-NS_Nt is conserved even in more distantly related proteins.