Abstract
We present the crystal structure and biochemical characterization of Escherichia coli YbiB, a member of the hitherto uncharacterized TrpD2 protein family. Our results demonstrate that the functional diversity of proteins with a common fold can be far greater than predictable by computational annotation. The TrpD2 proteins show high structural homology to anthranilate phosphoribosyltransferase (TrpD) and nucleoside phosphorylase class II enzymes but bind with high affinity (KD = 10-100 nM) to nucleic acids without detectable sequence specificity. The difference in affinity between single- and double-stranded DNA is minor. Results suggest that multiple YbiB molecules bind to one longer DNA molecule in a cooperative manner. The YbiB protein is a homodimer that, therefore, has two electropositive DNA binding grooves. But due to negative cooperativity within the dimer, only one groove binds DNA in in vitro experiments. A monomerized variant remains able to bind DNA with similar affinity, but the negative cooperative effect is eliminated. The ybiB gene forms an operon with the DNA helicase gene dinG and is under LexA control, being induced by DNA-damaging agents. Thus, speculatively, the TrpD2 proteins may be part of the LexA-controlled SOS response in bacteria.
Highlights
YbiB belongs to the uncharacterized family of TrpD2 proteins
To investigate whether TrpD2 shares a catalytic activity with the latter enzymes, recombinant YbiB was tested for TrpD activity, nucleotide salvage activity, and nucleoside phosphorylase activity
This fold is organized into non-sequential N- and C-terminal domains, where the smaller N-terminal ␣-helical domain is formed by six helices and provides the contact interface for homodimerization, whereas the larger C-terminal ␣/domain comprises a central -sheet that is surrounded by seven additional ␣-helices
Summary
YbiB belongs to the uncharacterized family of TrpD2 proteins. Results: YbiB binds to DNA with high affinity. The ybiB gene is under LexA control and induced by DNA-damaging agents. TrpD2 was initially found as a duplicate of the TrpD enzyme, termed TrpD (on average 17% sequence identity) This enzyme, from the metabolic pathway of tryptophan biosynthesis, catalyzes the condensation of the nitrogenated base anthranilate and the phosphoribosyl donor 5Ј-phosphoribosyl␣1-pyrophosphate (PRPP).. Despite sharing a low sequence similarity (on average 12% sequence identity) and displaying some significant differences, TrpD and NP-II proteins are both members of the phosphoribosyl transferase class III superfamily and have a similar fold, their active sites share common features, and both enzymes have a similar quaternary structure, where homodimers are formed by head-to-head interactions of equivalent secondary structure elements [2, 3]. Despite having a common fold, TrpD2 enzymes do not share catalytic functions with TrpD and NP-II enzymes but are a new class of DNA-binding proteins. A participation of the TrpD2 proteins in the LexA-controlled mechanism of SOS response in bacteria might be hypothesized, but this remains to be confirmed
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