Christian Dumas

The crystal structure of HIV-1 Nef protein bound to the Fyn kinase SH3 domain suggests a role for this complex in altered T cell receptor signaling

BACKGROUNDnHuman immunodeficiency virus (HIV) Nef protein accelerates virulent progression of acquired immunodeficiency syndrome (AIDS) by its interaction with specific cellular proteins involved in signal transduction and host cell activation. Nef has been shown to bind specifically to a subset of the Src family of kinases. The structures of free Nef and Nef bound to Src homology region 3 (SH3) domain are important for the elucidation of how the affinity and specificity for the Src kinase family SH3 domains are achieved, and also for the development of potential drugs and vaccines against AIDS.nnnRESULTSnWe have determined the crystal structures of the conserved core of HIV-1 Nef protein alone and in complex with the wild-type SH3 domain of the p59fyn protein tyrosine kinase (Fyn), at 3.0 A resolution. Comparison of the bound and unbound Nef structures revealed that a proline-rich motif (Pro-x-x-Pro), which is implicated in SH3 binding, is partially disordered in the absence of the binding partner; this motif only fully adopts a left-handed polyproline type II helix conformation upon complex formation with the Fyn SH3 domain. In addition, the structures show how an arginine residue (Arg77) of Nef interacts with Asp 100 of the so-called RT loop within the Fyn SH3 domain, and triggers a hydrogen-bond rearrangement which allows the loop to adapt to complement the Nef surface. The Arg96 residue of the Fyn SH3 domain is specifically accommodated in the same hydrophobic pocket of Nef as the isoleucine residue of a previously described Fyn SH3 (Arg96-->lle) mutant that binds to Nef with higher affinity than the wild type.nnnCONCLUSIONSnThe three-dimensional structures support evidence that the Nef-Fyn complex forms in vivo and may have a crucial role in the T cell perturbating action of Nef by altering T cell receptor signaling. The structures of bound and unbound Nef reveal that the multivalency of SH3 binding may be achieved by a ligand induced flexibility in the RT loop. The structures suggest possible targets for the design of inhibitors which specifically block Nef-SH3 interactions.

X-ray structure of nucleoside diphosphate kinase.

The X‐ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution. The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold. Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta‐sheet. The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position. Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase. Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro‐‐‐‐Ser substitution in Killer of prune (K‐pn) mutants of the highly homologous Drosophila enzyme. Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three‐dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins.

Three-dimensional structure of nucleoside diphosphate kinase.

Three-dimensional structures are known from X-ray studies of the nucleoside diphosphate(NDP) kinase of many organisms from bacteria to human. All NDP kinases have subunits ofabout 150 residues with a very similar fold based on the αβ sandwich orferredoxin fold.This fold is found in many nucleotide or polynucleotide-binding proteins with no sequencerelationship to NDP kinase. This common fold is augmented here with specificfeatures: asurface α-helix hairpin, the Kpn loop, and the C-terminal extension. The α-helix hairpin andKpn loop make up the nucleotide binding site, which is unique to NDP kinase and differentfrom that of other kinases or ATPases. The Kpn loop and the C-terminal extension are alsoinvolved in the quaternary structure. Whereas all known eukaryotic NDP kinases, includingmitochondral enzymes, are hexamers, some bacterial enzymes are tetramers. However,hexameric and tetrameric NDP kinases are built from the same dimer. The structural environmentof the active histidine is identical in all. The nucleotide binding site is also fully conserved,except for a feature implicating C-terminal residues in the hexamer, but not in the tetramer.Structural data on the native and phosphorylated enzyme, complexes with substrates, inhibitor,and a transition state analog, give a solid basis to a mechanism of phosphate transfer in whichthe largest contributors to catalysis are the 3′-OH of the sugar and the bound Mg2+ in thenucleotide substrate. In contrast, we still lack structural data relating to DNA binding andother functions of NDP kinases.

Interaction of Calmodulin, a Sorting Nexin and Kinase-Associated Protein Phosphatase with the Brassica oleracea S Locus Receptor Kinase

Recognition of self-pollen during the self-incompatibility response in Brassica oleracea is mediated by the binding of a secreted peptide (the S locus cysteine-rich protein) to the S locus receptor kinase (SRK), a member of the plant receptor kinase (PRK) superfamily. Here, we describe the characterization of three proteins that interact with the cytosolic kinase domain of SRK. A B. oleracea homolog of Arabidopsis kinase-associated protein phosphatase was shown to interact with and dephosphorylate SRK and was itself phosphorylated by SRK. Yeast (Saccharomyces cerevisiae) two-hybrid screens identified two additional interactors, calmodulin and a sorting nexin, both of which have been implicated in receptor kinase down-regulation in animals. A calmodulin-binding site was identified in sub-domain VIa of the SRK kinase domain. The binding site is conserved and functional in several other members of the PRK family. The sorting nexin also interacted with diverse members of the PRK family, suggesting that all three of the interacting proteins described here may play a general role in signal transduction by this family of proteins.

Mutation of a Conserved Residue (D123) Required for Oligomerization of Human Immunodeficiency Virus Type 1 Nef Protein Abolishes Interaction with Human Thioesterase and Results in Impairment of Nef Biological Functions

ABSTRACT Nef is a myristoylated protein of 27 to 35 kDa that is conserved in primate lentiviruses. In vivo, Nef is required for high viral load and full pathological effects. In vitro, Nef has at least four activities: induction of CD4 and major histocompatibility complex (MHC) class I downregulation, enhancement of viral infectivity, and alteration of T-cell activation pathways. We previously reported that the Nef protein from human immunodeficiency virus type 1 interacts with a novel human thioesterase (hTE). In the present study, by mutational analysis, we identified a region of the Nef core, extending from the residues D108 to W124, that is involved both in Nef-hTE interaction and in Nef-induced CD4 downregulation. This region of Nef is located on the oligomer interface and is in close proximity to the putative CD4 binding site. One of the mutants carrying a mutation in this region, targeted to the conserved residue D123, was also found to be defective in two other functions of Nef, MHC class I downmodulation and enhancement of viral infectivity. Furthermore, mutation of this residue affected the ability of Nef to form dimers, suggesting that the oligomerization of Nef may be critical for its multiple functions.

Adenosine 5'-diphosphate binding and the active site of nucleoside diphosphate kinase.

The X-ray structure of nucleoside diphosphate kinase (NDP kinase) from the slime mold Dictyostelium discoideum has been determined to 2.2-A resolution and refined to an R-factor of 0.19 with and without bound ADP-Mg2+. The nucleotide binds near His 122, a residue which becomes phosphorylated during the catalytic cycle. The mode of binding is different from that observed in other phosphokinases, and it involves no glycine-rich sequence. The adenine base makes only nonpolar contacts with the protein. It points outside, explaining the lack of specificity of NDP kinase toward the base. The ribose 2- and 3-hydroxyls and the pyrophosphate moiety are H-bonded to polar side chains. A Mg2+ ion bridges the alpha- to the beta-phosphate which approaches the imidazole group of His 122 from the N delta side. The geometry at the active site in the ADP-Mg2+ complex suggests a mechanism for catalysis whereby the gamma-phosphate of a nucleoside triphosphate can be transferred onto His 122 with a minimum of atomic motion.

Crystal structure of the Awd nucleotide diphosphate kinase from Drosophila

BACKGROUNDnNucleotide diphosphate kinase (NDP kinase) is a phosphate transfer enzyme involved in cell regulation and in animal development. Drosophila NDP kinase is the product of the abnormal wing disc (awd) developmental gene, a point mutation in which can produce the killer of prune (K-pn) conditional lethal phenotype. The highly homologous mammalian genes control metastasis and a human NDP kinase acts as a transcription factor.nnnRESULTSnThe X-ray structure of the Awd protein prepared from Drosophila was solved at 2.4 A resolution by molecular replacement from the homologous Dictyostelium protein. Both are hexamers, and both have the same fold and the same active site. Subunit contacts differ as a result of sequence changes in the carboxy-terminal segment and in the loop that is the site of the K-pn mutation.nnnCONCLUSIONSnRegulatory properties of animal NDP kinases depend on interactions with other macromolecules, such as DNA and the product of the Drosophila prune gene. The Awd structure suggests an allosteric mechanism of action of NDP kinase where DNA is the effector and the protein undergoes a major conformational change, possibly dissociating to dimers.

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.

Crystal structure of a new RNA-binding domain from the antiterminator protein SacY of Bacillus subtilis

SacY belongs to a family of, at present, seven bacterial transcriptional antiterminators. The RNA‐binding and antitermination capacity of SacY resides in the 55 amino acids at the N‐terminal [SacY(1‐55)]. The crystal structure at 2 Å resolution shows that SacY(1‐55) forms a dimer in the crystal, in accordance with the NMR solution structure. The structure of the monomer is a four‐stranded β‐sheet with a simple β1β2β3β4 topology. One side of the sheet is covered by a long surface loop and the other side forms the dimer interface. The dimer is stabilized by the orthogonal stacking of the two β‐sheets. The crystal structure is in excellent agreement with the NMR solution structure (r.m.s. distance for Cα coordinates is 1.3 Å). The structure of SacY(1‐55) reveals a new RNA‐binding motif.

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.