Evolution of a molecular switch: universal bacterial GTPases regulate ribosome function

111

Bacillus subtilis YlqF belongs to the Era/Obg subfamily of small GTP-binding proteins and is essential for bacterial growth. Here we report that YlqF participates in the late step of 50 S ribosomal subunit assembly. YlqF was co-fractionated with the 50 S subunit, depending on the presence of noncleavable GTP analog. Moreover, the GTPase activity of YlqF was stimulated specifically by the 50 S subunit in vitro. Dimethyl sulfate footprinting analysis disclosed that YlqF binds to a unique position in 23 S rRNA. Yeast two-hybrid data revealed interactions between YlqF and the B. subtilis L25 protein (Ctc). The interaction was confirmed by the pull-down assay of the purified proteins. Specifically, YlqF is positioned around the A-site and P-site on the 50 S subunit. Proteome analysis of the abnormal 50 S subunits that accumulated in YlqF-depleted cells showed that L16 and L27 proteins, located near the YlqF-binding domain, are missing. Our results collectively indicate that YlqF will organize the late step of 50 S ribosomal subunit assembly.

mentioning

confidence: 89%

The GTP-binding Protein YlqF Participates in the Late Step of 50 S Ribosomal Subunit Assembly in Bacillus subtilis

Matsuo

Morimoto²,

Kuwano

et al. 2006

111

“…Strikingly, most of these GTPases are universally conserved and can be assigned to four main ancestral groups: elongation factors (EF-G, EF-Tu, and IF2), protein secretion factors (FtsY and Ffh), Era-related GTPases (Era, EngA, and ThdF/TrmE), and Obg-re-lated proteins (Obg and YchF) (5,6). Mutational analyses in many organisms have shown that most of these GTPases are essential (7,10).…”

mentioning

confidence: 99%

Human OLA1 Defines an ATPase Subfamily in the Obg Family of GTP-binding Proteins

Koller-Eichhorn

Marquardt

Gail

et al. 2007

100

140

Purine nucleotide-binding proteins build the large family of P-loop GTPases and related ATPases, which perform essential functions in all kingdoms of life. The Obg family comprises a group of ancient GTPases belonging to the TRAFAC (for translation factors) class and can be subdivided into several distinct protein subfamilies. The founding member of one of these subfamilies is the bacterial P-loop NTPase YchF, which had so far been assumed to act as GTPase. We have biochemically characterized the human homologue of YchF and found that it binds and hydrolyzes ATP more efficiently than GTP. For this reason, we have termed the protein hOLA1, for human Obg-like ATPase 1. Further biochemical characterization of YchF proteins from different species revealed that ATPase activity is a general but previously missed feature of the YchF subfamily of Obg-like GTPases. To explain ATP specificity of hOLA1, we have solved the x-ray structure of hOLA1 bound to the nonhydrolyzable ATP analogue AMPPCP. Our structural data help to explain the altered nucleotide specificity of YchF homologues and identify the Ola1/YchF subfamily of the Obg-related NTPases as an exceptional example of a single protein subfamily, which has evolved altered nucleotide specificity within a distinct protein family of GTPases.

“…The to as the phosphate binding or P-loop, or alternatively, the G 1 motif of bacterial GTPases (13) …”

mentioning

confidence: 99%

Insight into the Role of Escherichia coli MobB in Molybdenum Cofactor Biosynthesis Based on the High Resolution Crystal Structure

McLuskey

Harrison

Schüttelkopf

et al. 2003

Two proteins, which are co-transcribed in Escherichia coli (MobA and MobB), are involved in the attachment of a nucleotide moiety to the molybdenum cofactor to form active molybdopterin guanine dinucleotide. Although not essential for this process, the dimeric MobB increases the activation of molybdoenzymes, incorporating this cofactor by a mechanism that is not understood. The structure of MobB has been elucidated in two crystal forms, one of which has provided a model at 1.9-Å resolution with R work and R free values of 21.5 and 28.7%, respectively. The MobB subunit displays an ␣/␤-fold arranged into a major and minor domain, the latter of which is inserted between the major and minor domains of the partner subunit, creating an elongated dimer constructed around a 16-stranded ␤-sheet. Structural homologues have been identified, and they include a number of nucleotide-binding proteins. Comparisons indicate that although the phosphate-binding regions are highly conserved, MobB lacks the elements of structure required to interact with and efficiently bind a nucleotide base. In the present structure, a sulfate is bound to the Walker A phosphate-binding motif of MobB. The possibility that MobB forms a complex with the nucleotidebinding MobA, the protein with which it is co-transcribed, is explored, and modeling suggests that such a MobA:MobB complex is feasible. This hypothesis is supported by recent biochemical evidence indicating that MobB interacts with several proteins involved in various stages of molybdenum cofactor biosynthesis including MobA. We propose therefore that MobB is an adapter protein that acts in concert with MobA to achieve the efficient biosynthesis and utilization of molybdopterin guanine dinucleotide.Molybdenum is an essential trace element associated with a diverse range of redox-active enzymes (1). Such molybdoenzymes catalyze basic metabolic reactions in the carbon, nitrogen, and sulfur cycles exploiting the redox chemistry of this second row transition metal. With the exception of the nitrogenases that contain an iron-molybdenum-sulfur cluster, molybdenum is integrated into the enzymes as the molybdenum cofactor (Moco), 1 which contains mononuclear molybdenum coordinated to an organic cofactor termed molybdopterin (MPT).