Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Structural Diversity and Functional Versatility Among Phenylalanyl-tRNA Synthetases in Primary Kingdoms
The aminoacyl-tRNA synthetases (aaRSs) ensure the fidelity of the genetic code translation, covalently attaching appropriate amino acids to the corresponding nucleic acid adaptor molecules - tRNA. Phenylalanyl-tRNA synthetase (PheRS) is the enzyme responsible for specific incorporation of amino acid phenylalanine into protein sequence and structure. PheRS ? the largest and complex enzyme among the 19 other members of aaRS family, has (αβ)2 subunit organization and its structure was first solved for the T. thermophilus enzyme . Phylogenetic and structural analysis suggest that there are three major forms of PheRS: a) heterodimeric (αβ)2 bacterial; b) heterodimeric (αβ)2 archaeal/eukaryotic-cytosolic; and c) monomeric mitochondrial.
Two crystal structures of PheRS from different compartments of eukaryotic cell have been determined very recently. While the total length of the (αβ)2 human cytosolic enzyme is made up of 2194 residues, the mature mitochondrial PheRS is the smallest known monomeric aminoacylation system consisting of 415 amino acids only and, in fact it is a chimera of the catalytic α-subunit and the anticodon-binding domain from β-subunit of the bacterial enzyme. All three enzymes catalyzing the same enzymatic reaction demonstrate, however, remarkable diversity in their structural organization (see Figure 1) [2, 3]. Although basic architecture of the core domains (A1 and A2 from α-subunit and B6 and B7 from β-subunit) that have been implicated in formation of four-helix bundle interface of the heterodimer is well conserved in cytosolic enzymes, the unique peptide extensions and shortenings have been found at the N- and C-terminal ends of the human enzyme. These features suggest a subsidiary and essential changes in the structure of human enzyme as compared to the Th. thermophilus one and lead us to conclusion that modes of binding and recognition of cognate tRNAPhe are different in prokaryotes and eukaryotes. This, in turn, testifies that PheRS holds a unique position among the other 20 aaRSs. Moreover, as regards to proofreading activity associated with a distinct active site, where misactivated tyrosyl-adenylate or misaminoacylated Tyr-tRNAPhe have to be hydrolyzed, PheRSs from different compartments also differ widely. Thus, eukaryotic and prokaryotic cytosolic enzymes are capable to deacylate Tyr-tRNAPhe while mitochondrial PheRS is unable to do this due to the absence of the editing module. Transition from heterodimeric subunit organization of PheRSs in cytoplasm to monomeric in mitochondria most likely is accompanied by changes in dynamic characteristics of PheRS-tRNAPhe complex formation. We hypothesize that during the transfer to the tRNA-free state, mitochondrial enzyme exhibits both ??open?? and ??closed?? conformations. Contrary to cytoplasmic enzymes that retain their 3D-structure upon tRNA binding, complex formation in mitochondria must be accompanied by considerable rearrangement (hinge-type rotation through 160°) of the anticodon-binding domain. We also show that PheRSs demonstrate a marked degree of natural plasticity within the active site: the amino acid binding pocket is capable of binding both the noncognate tyrosine and its unnatural derivatives i.e., substrates of larger size than cognate phenylalanine.
References and Footnotes
1Department of Structural Biology