A retroviral-like metal binding motif in an aminoacyl-tRNA synthetase is important for tRNA recognition.

Abstract

The gag genes of retrovnruses encode nucleocapsid proteins that package genomic RNA and are essential for viral infectivity. These RNA binding proteins have a Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys zinc binding motif that is distinct from the typical zinc-finger motif Cys-Xaa2-Cys-Xaa12.14-His-Xaa2-His that is found in some transcriptional activators. Escherichia coli alanyl-tRNA synthetase contains a zinc-binding Cys-Xaa2-Cys-Xaa6-His-Xaa2-His motif that resembles that of retroviral nucleic acid binding proteins. We show here that, for alanyltRNA synthetase, the metal bound at the retroviral-like metal binding motif is important specifically for tRNA recognition and not for amino acid activation. Moreover, the enzymetRNA interaction is strongly dependent on the geometry of metal coordination to the protein. These and additional experiments collectively suggest a role for the retroviral-like metal binding motif in RNA recognition and, further, raise the possibility that the protein-bound metal itself participates in an RNA interaction.The gag genes of retroviruses encode nucleocapsid (NC) proteins that package genomic RNA and are essential for viral infectivity (1, 2). The gag polypeptides contain one or two copies of the sequence motif Cys-Xaa2-Cys-Xaa4-HisXaa4-Cys (CCHC boxes) (3, 4). This motif is distinct from the structures of DNA-binding zinc fingers exemplified by ADR1 (5), Xfin (6), and Zif268 (7) or the binuclear cluster of six cysteines such as found in GAL4 (8). The high-resolution NMR structure of the first zinc binding domain from the NC protein of human immunodeficiency virus has a tetracoordinate bound zinc surrounded by a compact structure that is devoid of any regular secondary structure (9). Mutational analysis of NCs indicates that the cysteines and histidines in the CCHC box are essential for viral function (10-12). However, because there is no biochemical activity that can be conveniently assayed, the involvement of the CCHC box in RNA recognition has not been clarified.Escherichia coli alanyl-tRNA synthetase contains the retroviral-like Cys-Xaa2-Cys-Xaa6-His-Xaa2-His motif that was recognized by Berg (13) as a potential metal binding site.Atomic absorption analysis established that the enzyme has 1 mol of tightly bound zinc per mol of polypeptide (14). A synthetic peptide model ofthe Cys-Xaa2-Cys-Xaa6-His-Xaa2-His motif binds Zn2+ or Co2+ stoichiometrically and the visible spectrum of the Co2'-bound peptide has the d-orbital splitting that is characteristic of tetrahedral geometry and also has a metal-to-sulfur charge transfer band (14). The peptide-Co2+ dissociation constant of 1 ,uM is similar to that for Co2+ complexes with the Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys-containing peptides derived from NC sequences (15). Mutational analysis established that the Cys-Xaa2-Cys-Xaa6-His-Xaa2-His motif that starts at position 178 in alanyl-tRNA synthetase is essential for enzyme structure and activity (14). Collectively the results suggest that this motif is an essential portion of the metal binding do...

The Cellular and Molecular Mechanisms of Immuno-Suppression by Human Type 1 Regulatory T Cells

“…It is well documented that a deficiency in T reg frequency or number, or a defect in their function can lead to inflammation and/or autoimmune diseases (Roncarolo and Levings, 2000; Roncarolo and Battaglia, 2007; Sakaguchi et al, 2008). Over the years, several types of T reg populations have been identified: TGF-β secreting, Type 3 helper cells (Th3; Miller et al, 1992), CD8 + CD28 − T cells (Liu et al, 1998), HLA-E-specific CD8 + T cells (Jiang et al, 2010), etc. but, to date, the best characterized are the FOXP3 + T reg (Hori et al, 2003; Khattri et al, 2003) and the CD4 + IL-10-producing Tr1 cells (Groux et al, 1997; Barrat et al, 2002; Akdis et al, 2004).…”

Effect of rearrangements and duplications of the Cys-His motifs of Rous sarcoma virus nucleocapsid protein

“…In vitro studies suggested that retroviral NC may be involved in positioning the tRNA primer of reverse transcription at the primer binding site in the leader (26): furthermore, the NC mutant PrC-1 was shown by strong stop analysis to be deficient in this function. A number of aminoacyl-tRNA synthetases have a Cys-His motif, and this sequence appears important for tRNA binding but not for aminoacylation (24).…”

Dispensability of zinc and the putative zinc-binding domain in bacterial glutamyl-tRNA synthetase

“…For example, zinc is intimately related with cognate ligand recognition in Escherichia coli threonyl-tRNA synthetase [ 1 ] and cysteinyl-tRNA synthetase [ 2 , 3 ]. Similarly, a retrovirus like zinc-coordinating motif is important in tRNA recognition in E. coli alanyl-tRNA synthetase [ 4 ]. Chemical or mutational modification of the zinc-coordinating ligands in isoleucyl-tRNA synthetase (of E. coli and Thermus thermophlilus ) affects the enzyme efficiency [ 5 , 6 ].…”