The interaction between fMet-tRNA f Met and Bacillus stearothermophilus translation initiation factor IF2 has been characterized. We demonstrate that essentially all thermodynamic determinants governing the stability and the speci®city of this interaction are localized within the acceptor hexanucleotide fMet-3¢ACCAAC of the initiator tRNA and a fairly small area at the surface of the b-barrel structure of the 90-amino acid C-terminal domain of IF2 (IF2 C-2). A weak but speci®c interaction between IF2 C-2 and formyl-methionyl was also demonstrated. The surface of IF2 C-2 interacting with fMet-tRNA f Met has been mapped using two independent approaches, sitedirected mutagenesis and NMR spectroscopy, which yielded consistent results. The binding site comprises C668 and G715 located in a groove accommodating the methionyl side-chain, R700, in the vicinity of the formyl group, Y701 and K702 close to the acyl bond between fMet and tRNA f Met , and the surface lined with residues K702-S660, along which the acceptor arm of the initiator tRNA spans in the direction 3¢ to 5¢. Keywords: NMR spectroscopy/protein±RNA interaction/ site-directed mutagenesis/translation initiation IntroductionThe speci®c recognition of fMet-tRNA f Met by initiation factor IF2 represents one of the most important interactions occurring during translation initiation in bacteria (for reviews see Spurio et al., 1993; Schmitt et al., 1996;Gualerzi et al., 2000). This interaction determines the accuracy in the selection of the correct initiation site of both leadered (containing a 5¢-UTR) and unleadered mRNAs (Grill et al., 2000 and references therein), in the speed and ef®ciency of both 30S and 70S initiation complex formation (Gualerzi et al., 1986) and in the formation of the ®rst peptide bond (initiation dipeptide), which marks the transition from the initiation to the elongation phase of translation (La Teana et al., 1996;Toms Ïic et al., 2000).Important progress in the elucidation of the IF2 structure was made recently. The molecular dissection of Bacillus stearothermophilus IF2 (82 kDa) allowed the identi®cation of three domains in the molecule (the N-terminal domain, the central G-domain and the carboxyl-terminal C-domain), the site responsible for the recognition and binding of fMet-tRNA f Met being located in the 24.5 kDa C-terminal part of the protein (IF2 C) (Gualerzi et al., 1991). Further studies have shown that IF2 C is constituted by two domains of approximately equal size (IF2 C-1 and IF2 C-2) (Misselwitz et al., 1997). IF2 C-2, of 110 residues, was found to contain all the structural determinants involved in the recognition of fMet-tRNA f Met , and its complex with fMet-tRNA f Met displayed the same stability and properties as those formed by intact IF2 and IF2 C Spurio et al., 2000). The three dimensional (3D) solution structure of B.stearothermophilus IF2 C-2 determined by multinuclear NMR spectroscopy consists of a compact b-barrel, structurally homologous to domains II of elongation factors EF-Tu and EF-G, despite the lack of an...

The three-dimensional structure of the fMet-tRNA(fMet) -binding domain of translation initiation factor IF2 from Bacillus stearothermophilus has been determined by heteronuclear NMR spectroscopy. Its structure consists of six antiparallel beta-strands, connected via loops, and forms a closed beta-barrel similar to domain II of elongation factors EF-Tu and EF-G, despite low sequence homology. Two structures of the ternary complexes of the EF-Tu small middle dotaminoacyl-tRNA small middle dot GDP analogue have been reported and were used to propose and discuss the possible fMet-tRNA(fMet)-binding site of IF2.

kappa-Conotoxin PVIIA from the venom of Conus purpurascens is the first cone snail toxin that was described to block potassium channels. We synthesized chemically this toxin and showed that its disulfide bridge pattern is similar to those of omega- and delta-conotoxins. kappa-conotoxin competes with radioactive alpha-dendrotoxin for binding to rat brain synaptosomes, confirming its capacity to bind to potassium channels; however, it behaves as a weak competitor. The three-dimensional structure of kappa-conotoxin PVIIA, as elucidated by NMR spectroscopy and molecular modeling, comprises two large parallel loops stabilized by a triple-stranded antiparallel beta-sheet and three disulfide bridges. The overall fold of kappa-conotoxin is similar to that of calcium channel-blocking omega-conotoxins but differs from those of potassium channel-blocking toxins from sea anemones, scorpions, and snakes. Local topographies of kappa-conotoxin PVIIA that might account for its capacity to recognize Kv1-type potassium channels are discussed.

The advantages of using off-resonance rf fields in heteronuclear self-relaxation experiments are explored on a fully (15)N-enriched protein. It is firstly shown that in the absence of slow motions the longitudinal and transverse (15)N self-relaxation rate values derived with this method are in agreement with the ones measured by the classical inversion-recovery and Carr-Purcell-Meiboom-Gill (CPMG) sequences, respectively. Secondly, by comparing the (15)N transverse self-relaxation rates obtained by the proposed off-resonance sequence and by the CPMG sequence, 11 residues out of the 61 of toxin α are shown to exhibit a chemical exchange phenomenon in water on a time scale ranging from 1 µs to 100 ms. By varying the effective field amplitude, chemical exchange processes involving these residues are measured and the corresponding correlation times are evaluated without having assumed any motion model. Similar, though less precise, results are given by the analysis of the (15)N off-resonance self-relaxation rates on the basis of the Lipari-Szabo model to describe the fast internal dynamics of toxin α.