Structural analysis of the multienzyme aminoacyl‐tRNA synthetase complex: A three‐domain model based on reversible chemical crosslinking

Abstract

A subset of eukaryotic aminoacyl-tRNA synthetases (a-RS) are contained in a multienzyme complex for which little structural detail is known. Three reversible chemical crosslinking reagents have been used to investigate the arrangement of polypeptides within this particle as isolated from rabbit reticulocytes. Identification of the crosslinked protein pairs was accomplished by two-dimensional SDS diagonal gel electrophoresis. Seventeen neighboring protein pairs have been identified. Eight are seen with at least two reagents: K-RS:p38, D-RS:K-RS, R-RS dimer, K-RS dimer, K-RS:Q-RS, E/P-RS:K-RS, E/P-RS:I-RS, and Q-RS with one of the nonsynthetase proteins. Nine more are observed with one reagent: D-RS dimer, R-RS:p43, D-RS:Q-RS, D-RS:M-RS, K-RS:L-RS, I-RS:R-RS, D-RS:E/P-RS, I-RS:Q-RS, I-RS:L-RS. One trimeric association is seen: E/P-RS:I-RS:L-RS.The observed neighboring protein pairs suggest that the polypeptides within the aminoacyl-tRNA synthetase complex are distributed in three structural domains of similar mass. These can be arranged in a U-shaped particle in which each ''arm'' is considered a domain and the third forms the "base" of the structure. The arms have been termed domain I (D-RS, M-RS, Q-RS) and domain I1 (K-RS, R-RS), with domain III (E/P-RS, I-RS, L-RS) assigned to the base. The smaller proteins (p38, p43) may bridge the domains. This proposed spatial relationship of these domains, as well as their compositions, are consistent with earlier studies. Thus, this study provides an initial three-dimensional working model of the arrangement of polypeptides within the multienzyme aminoacyl-tRNA synthetase complex.Keywords: aminoacyl-tRNA synthetase complex; multienzyme complex; protein-protein interaction; protein structure; reversible chemical crosslinking Aminoacyl-tRNA synthetases are a family of enzymes characterized by their role in coupling each amino acid with the correct tRNA for subsequent use in protein biosynthesis. In addition to this fundamental role in translation, these enzymes are widely studied as models for the evolution of modular proteins and for insight into the mechanisms of RNA-protein recognition. These enzymes also have additional cellular functions, such as synthesis of signaling dinucleotides. There are several recent reviews of these topics (Mirande, 1991;Schulman, 1991;Cusack, 1995).One of the most intriguing and least understood properties of aminoacyl-tRNA synthetases from higher eukaryotes is the existence of a high molecular mass multienzyme complex of a subset Reprint requests to: Mona T. Norcum, Biochemistry Department, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216-4505; e-mail: mnorcum@fiona.umsmed.edu.Abbreviations: a-RS, aminoacyl-tRNA synthetase, a is the one-letter ambo acid abbreviation; cis-DDP, cis-dichlorodiammine platinum; EGS, erhylene glycobis(succinimydy1succinate); SMPT, 4-succinimydyl oxycarbonyl-2-pyridyldithiotoluene. of these proteins (reviewed in Kisselev & Wolfson, 1994;Yang, 1996). As isolated fr...

A Three-dimensional Working Model of the Multienzyme Complex of Aminoacyl-tRNA Synthetases Based on Electron Microscopic Placements of tRNA and Proteins

“…However, by combining the information in TABLE ONE, previous results, and the new data, it is possible to make some reasonable predictions. For example, interactions between LeuRS and GluProRS have been demonstrated by chemical cross-linking (14) and genetic analyses (15,16). Together with IleRS, these same two enzymes form a distinct sub-complex of the multisynthetase particle (13).…”

“…Isolated protein was then fractionated by gel filtration HPLC. SDS-PAGE analysis of fractions that eluted in the position corresponding to a mass of ϳ1 ϫ 10 6 Da exhibited the band pattern characteristic of the multiprotein particle (14). Both control and tagged complex contained additional non-stoichiometric proteins as compared with those from the standard procedure.…”

“…Ultrastructure-Because the studies reported below each began with much less cellular material than our usual preparations of multisynthetase complex (14), it was of importance to develop an appropriately scaled isolation method that was rapid and required only a few steps. Briefly, lysate from ϳ1 g of wet weight of control or transfected 293F cells was applied to a 1.5-ml column of lysine-agarose, washed with 25 mM lysine, and then step-eluted with a solution of total yeast tRNA.…”

“…Placement of the protein components within particular areas of the structure was begun some time ago through use of chaotropic salts to prepare and characterize sub-complexes (13). Then, a number of nearest-neighbor protein interactions within the isolated complex were detected via chemical cross-linking studies (14). Using an in vivo approach, a number of potential component interactions have been identified by yeast two-hybrid genetic screens (15,16).…”

“…The auxiliary proteins are at the heart of the complex with p38 interactions responsible for connecting the two domains. The second model divides the multisynthetase complex into three domains (14). The supporting data include characterization of sub-complexes, reversible chemical cross-linking experiments, and electron microscopy.…”