Abstract
EngA proteins form a unique family of bacterial GTPases with two GTP-binding domains in tandem, namely GD1 and GD2, followed by a KH (K-homology) domain. They have been shown to interact with the bacterial ribosome and to be involved in its biogenesis. Most prokaryotic EngA possess a high GTPase activity in contrast to eukaryotic GTPases that act mainly as molecular switches. Here, we have purified and characterized the GTPase activity of the Bacillus subtilis EngA and two shortened EngA variants that only contain GD1 or GD2-KH. Interestingly, the GTPase activity of GD1 alone is similar to that of the whole EngA, whereas GD2-KH has a 150-fold lower GTPase activity. At physiological concentration, potassium strongly stimulates the GTPase activity of each protein construct. Interestingly, it affects neither the affinities for nucleotides nor the monomeric status of EngA or the GD1 domain. Thus, potassium likely acts as a chemical GTPase-activating element as proposed for another bacterial GTPase like MnmE. However, unlike MnmE, potassium does not promote dimerization of EngA. In addition, we solved two crystal structures of full-length EngA. One of them contained for the first time a GTP-like analogue bound to GD2 while GD1 was free. Surprisingly, its overall fold was similar to a previously solved structure with GDP bound to both sites. Our data indicate that a significant structural change must occur upon K+ binding to GD2, and a comparison with T. maritima EngA and MnmE structures allowed us to propose a model explaining the chemical basis for the different GTPase activities of GD1 and GD2.
Highlights
GTPases form one of the most widely spread protein families in all kingdoms of life and they play vital functions in many cellular processes, including translation, cellular trafficking and cell division [1,2]
Variants, GD1 and GD2-KH were overexpressed in E. coli and purified to near homogeneity (Fig. 1)
We checked the effect of K+ on the GTPase activity of B. subtilis EngA, and test whether K+ was able to affect the GTPase activity of either variant
Summary
GTPases form one of the most widely spread protein families in all kingdoms of life and they play vital functions in many cellular processes, including translation, cellular trafficking and cell division [1,2] In eukaryotes, these enzymes have usually a very low intrinsic GTPase activity which can be strongly stimulated, up to several orders of magnitude, by additional protein partners known as GAP (‘GTPase Activating Protein’) [3]. These enzymes have usually a very low intrinsic GTPase activity which can be strongly stimulated, up to several orders of magnitude, by additional protein partners known as GAP (‘GTPase Activating Protein’) [3] Another class of GTPases has been described in which the catalytic activity is promoted upon a nucleotide-dependent dimerization [4]. The catalytic residue(s) can be stabilized by a protein partner (or a second subunit in the case of a dimer) when they are already present in an otherwise flexible part of the active site [3,4]
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