Etudes structurales d'un moteur moléculaire : la myosine

Les moteurs moléculaires sont des protéines capables de produire une force : elles peuvent hydrolyser un nucléotide, l'ATP, et convertir l'énergie chimique libérée en énergie mécanique. Cette caractéristique intéressante est partagée par trois grandes familles de moteurs moléculaires, les myosines, les kinésines et les dynéines. Les myosines, notre famille préférée, interviennent dans une kyrielle de fonctions cellulaires comme la contraction musculaire, l'ouïe, la vue, la pigmentation de la peau, la digestion, le développement cérébral, le trafic intracellulaire, la division cellulaire ou bien la phagocytose. Pour comprendre les bases de leur génération de force, et pour à plus long terme utiliser les moteurs moléculaires comme cible thérapeutique, les myosines de classe II et V ont été étudiées pour leurs caractéristiques particulières. Ces myosines partagent le même mécanisme de production de force, même si elles possèdent des fonctions très différentes dans la cellule. Les résultats cinétiques et structuraux de ces deux classes de myosines ont permis de mieux comprendre le cycle catalytique de la myosine avec son partenaire, l'actine. De nouveaux états conformationnels de myosine V, isolés par cristallographie, ont permis de décrire les éléments structuraux responsables de l'interaction forte de la myosine et de l'actine, ainsi que l'effet du nucléotide sur le complexe actomyosine. Les études menées sur différents mutants de myosine II ont de plus apporté quelques éléments de réponse sur une étape clé de la production de force des myosines ; la libération de l'un des produits d'hydrolyse.Molecular motors are proteins that are able to produce a force : they convert the chemical energy released from ATP hydrolysis into mechanical force. This interesting feature is shared among three molecular motors families : myosins, kinesins and dyneins. Myosins, our favorite family, are involved in a litany of cellular functions as muscular contraction, hearing, vision, skin pigmentation, digestion, brain development, intracellular traffic, cellular division or phagocytosis. To understand the basis of force generation, and one day to use molecular motors as therapeutic targets, class II and V myosins were studied for their interesting features. These myosins share the same production force mechanism, but they have totally different functions in the cell (one forms filaments and contracts muscular fibers whereas the other is a dimer and carries vesicules).Kinetic and structural results on those two myosins classes helped to better understand the catalytic cycle of myosin with its partner, actin. Some new conformational states of myosin V, isolated by crystallography, allowed us to describe the structural elements responsible of the strong interaction between myosin and actin, and the effect of the nucleotide on the actomyosin complex. The studies lead on different myosin II mutants gave some parts of answer on a key step on myosin production force ; one of the hydrolysis products release. These mutants participated to a better understanding of the aim of some residues in the kinetic differences within the myosin superfamily.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Start Codon Recognition in Eukaryotic and Archaeal Translation Initiation: A Common Structural Core

International audienceUnderstanding molecular mechanisms of ribosomal translation sheds light on the emergence and evolution of protein synthesis in the three domains of life. Universally, ribosomal translation is described in three steps: initiation, elongation and termination. During initiation, a macromolecular complex assembled around the small ribosomal subunit selects the start codon on the mRNA and defines the open reading frame. In this review, we focus on the comparison of start codon selection mechanisms in eukaryotes and archaea. Eukaryotic translation initiation is a very complicated process, involving many initiation factors. The most widespread mechanism for the discovery of the start codon is the scanning of the mRNA by a pre-initiation complex until the first AUG codon in a correct context is found. In archaea, long-range scanning does not occur because of the presence of Shine-Dalgarno (SD) sequences or of short 5' untranslated regions. However, archaeal and eukaryotic translation initiations have three initiation factors in common: e/aIF1, e/aIF1A and e/aIF2 are directly involved in the selection of the start codon. Therefore, the idea that these archaeal and eukaryotic factors fulfill similar functions within a common structural ribosomal core complex has emerged. A divergence between eukaryotic and archaeal factors allowed for the adaptation to the long-range scanning process versus the SD mediated prepositioning of the ribosome

tRNA accommodation during archaeal translation initiation

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Cryo-EM study of an archaeal 30S initiation complex gives insights into evolution of translation initiation

International audienceArchaeal translation initiation occurs within a macromolecular complex containing the small ribosomal subunit (30S) bound to mRNA, initiation factors aIF1, aIF1A and the ternary complex aIF2:GDPNP:Met-tRNAiMet. Here, we determine the cryo-EM structure of a 30S:mRNA:aIF1A:aIF2:GTP:Met-tRNAiMet complex from Pyrococcus abyssi at 3.2 Å resolution. It highlights archaeal features in ribosomal proteins and rRNA modifications. We find an aS21 protein, at the location of eS21 in eukaryotic ribosomes. Moreover, we identify an N-terminal extension of archaeal eL41 contacting the P site. We characterize 34 N4-acetylcytidines distributed throughout 16S rRNA, likely contributing to hyperthermostability. Without aIF1, the 30S head is stabilized and initiator tRNA is tightly bound to the P site. A network of interactions involving tRNA, mRNA, rRNA modified nucleotides and C-terminal tails of uS9, uS13 and uS19 is observed. Universal features and domain-specific idiosyncrasies of translation initiation are discussed in light of ribosomal structures from representatives of each domain of life

Role of aIF1 in Pyrococcus abyssi translation initiation

International audienceIn archaeal translation initiation, a preinitiation complex (PIC) made up of aIF1, aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNA i Met) and mRNA bound to the small ribosomal subunit is responsible for start codon selection. Many archaeal mRNAs contain a Shine-Dalgarno (SD) sequence allowing the PIC to be prepositioned in the vicinity of the start codon. Nevertheless, cryo-EM studies have suggested local scanning to definitely establish base pairing of the start codon with the tRNA anticodon. Here, using flu-orescence anisotropy, we show that aIF1 and mRNA have synergistic binding to the Pyrococcus abyssi 30S. Stability of 30S:mRNA:aIF1 strongly depends on the SD sequence. Further, toeprinting experiments show that aIF1-containing PICs display a dynamic conformation with the tRNA not firmly accommodated in the P site. AIF1-induced destabilization of the PIC is favorable for proofreading erroneous initiation complexes. After aIF1 departure, the stability of the PIC increases reflecting initiator tRNA fully base-paired to the start codon. Altogether, our data support the idea that some of the main events governing start codon selection in eukaryotes and archaea occur within a common structural and functional core. However, idiosyncratic features in loop 1 sequence involved in 30S:mRNA binding suggest adjustments of e/aIF1 functioning in the two domains

The structure of an E. coli tRNA f Met A 1-U 72 variant shows an unusual conformation of the A 1-U 72 base pair

International audienceTranslation initiation in eukaryotes and archaea involves a methionylated initiator tRNA delivered to the ribosome in a ternary complex with e/aIF2 and GTP. Eukaryotic and archaeal initiator tRNAs contain a highly conserved A 1-U 72 base pair at the top of the acceptor stem. The importance of this base pair to discriminate initiator tRNAs from elongator tRNAs has been established previously using genetics and biochemistry. However, no structural data illustrating how the A 1-U 72 base pair participates in the accurate selection of the initiator tRNAs by the translation initiation systems are available. Here, we describe the crystal structure of a mutant E. coli initiator tRNA f Met A 1-U 72 , aminoacylated with methionine, in which the C 1 : A 72 mismatch at the end of the tRNA acceptor stem has been changed to an A 1-U 72 base pair. Sequence alignments show that the mutant E. coli tRNA is a good mimic of archaeal initiator tRNAs. The crystal structure, determined at 2.8 Å resolution, shows that the A 1-U 72 pair adopts an unusual arrangement. A 1 is in a syn conformation and forms a single H-bond interaction with U 72. This interaction requires protonation of the N1 atom of A 1. Moreover, the 5 ′ phosphoryl group folds back into the major groove of the acceptor stem and interacts with the N7 atom of G 2. A possible role of this unusual geometry of the A 1-U 72 pair in the recognition of the initiator tRNA by its partners during eukaryotic and archaeal translation initiation is discussed

Three myosin V structures delineate essential features of chemo-mechanical transduction

The molecular motor, myosin, undergoes conformational changes in order to convert chemical energy into force production. Based on kinetic and structural considerations, we assert that three crystal forms of the myosin V motor delineate the conformational changes that myosin motors undergo upon detachment from actin. First, a motor domain structure demonstrates that nucleotide-free myosin V adopts a specific state (rigor-like) that is not influenced by crystal packing. A second structure reveals an actomyosin state that favors rapid release of ADP, and differs from the rigor-like state by a P-loop rearrangement. Comparison of these structures with a third structure, a 2.0 Å resolution structure of the motor bound to an ATP analog, illuminates the structural features that provide communication between the actin interface and nucleotide-binding site. Paramount among these is a region we name the transducer, which is composed of the seven-stranded β-sheet and associated loops and linkers. Reminiscent of the β-sheet distortion of the F1-ATPase, sequential distortion of this transducer region likely controls sequential release of products from the nucleotide pocket during force generation

Recent Advances in Archaeal Translation Initiation

International audienceTranslation initiation (TI) allows accurate selection of the initiation codon on a messenger RNA (mRNA) and defines the reading frame. In all domains of life, translation initiation generally occurs within a macromolecular complex made up of the small ribosomal subunit, the mRNA, a specialized methionylated initiator tRNA, and translation initiation factors (IFs). Once the start codon is selected at the P site of the ribosome and the large subunit is associated, the IFs are released and a ribosome competent for elongation is formed. However, even if the general principles are the same in the three domains of life, the molecular mechanisms are different in bacteria, eukaryotes, and archaea and may also vary depending on the mRNA. Because TI mechanisms have evolved lately, their studies bring important information about the evolutionary relationships between extant organisms. In this context, recent structural data on ribosomal complexes and genome-wide studies are particularly valuable. This review focuses on archaeal translation initiation highlighting its relationships with either the eukaryotic or the bacterial world. Eukaryotic features of the archaeal small ribosomal subunit are presented. Ribosome evolution and TI mechanisms diversity in archaeal branches are discussed. Next, the use of leaderless mRNAs and that of leadered mRNAs having Shine-Dalgarno sequences is analyzed. Finally, the current knowledge on TI mechanisms of SD-leadered and leaderless mRNAs is detailed