TY - JOUR
T1 - Mycobacterium tuberculosis UvrB forms dimers in solution and interacts with UvrA in the absence of ligands
AU - Lahiri, Samarpita
AU - Rizzi, Menico
AU - Rossi, Franca
AU - Miggiano, Riccardo
N1 - Publisher Copyright:
© 2017 Wiley Periodicals, Inc.
PY - 2018/1
Y1 - 2018/1
N2 - During its life cycle Mycobacterium tuberculosis (MTB) must face a variety of environmental and endogenous physical and chemical stresses that could produce genotoxic damage. However, MTB possesses efficient systems to counteract the harmful effects of DNA-damaging assaults. The nucleotide excision repair (NER) is a highly conserved multi-enzymatic cascade that is initiated by the concerted action of three core proteins, that is UvrA, UvrB, and UvrC. Although the functional roles of these enzymes are well characterized, the intra-pathway coordination of the NER components and the dynamics of their association is still a matter of debate. In the presented study, we analyzed the hydrodynamic properties and the oligomeric state of the MTB UvrB protein (MtUvrB) that we expressed and purified to homogeneity in a tag-free form. Our results show that, differently to what has been previously observed for the His-tagged version of the protein, MtUvrB forms dimers in solution, which are characterized by an elongated shape, as determined by small-angle X-ray scattering analysis. Moreover, to gain insights into the mycobacterial UvrA/UvrB lesion sensing/tracking complex we adopted a size-exclusion chromatography-based approach, revealing that the two proteins interact in the absence of ligands, leading to the assembling of A2B2 hetero-tetramers in solution. Surface plasmon resonance analysis showed that the dissociation constant of the MtUvrA/MtUvrB complex falls in the low micromolar range that could represent the basis for a fine modulation of the complex architecture accompanying the multi-step DNA repair activity of mycobacterial NER.
AB - During its life cycle Mycobacterium tuberculosis (MTB) must face a variety of environmental and endogenous physical and chemical stresses that could produce genotoxic damage. However, MTB possesses efficient systems to counteract the harmful effects of DNA-damaging assaults. The nucleotide excision repair (NER) is a highly conserved multi-enzymatic cascade that is initiated by the concerted action of three core proteins, that is UvrA, UvrB, and UvrC. Although the functional roles of these enzymes are well characterized, the intra-pathway coordination of the NER components and the dynamics of their association is still a matter of debate. In the presented study, we analyzed the hydrodynamic properties and the oligomeric state of the MTB UvrB protein (MtUvrB) that we expressed and purified to homogeneity in a tag-free form. Our results show that, differently to what has been previously observed for the His-tagged version of the protein, MtUvrB forms dimers in solution, which are characterized by an elongated shape, as determined by small-angle X-ray scattering analysis. Moreover, to gain insights into the mycobacterial UvrA/UvrB lesion sensing/tracking complex we adopted a size-exclusion chromatography-based approach, revealing that the two proteins interact in the absence of ligands, leading to the assembling of A2B2 hetero-tetramers in solution. Surface plasmon resonance analysis showed that the dissociation constant of the MtUvrA/MtUvrB complex falls in the low micromolar range that could represent the basis for a fine modulation of the complex architecture accompanying the multi-step DNA repair activity of mycobacterial NER.
KW - DNA repair
KW - nucleotide excision repair
KW - size-exclusion chromatography
KW - small-angle X-ray scattering
KW - surface plasmon resonance
KW - tuberculosis
UR - http://www.scopus.com/inward/record.url?scp=85033553262&partnerID=8YFLogxK
U2 - 10.1002/prot.25412
DO - 10.1002/prot.25412
M3 - Article
SN - 0887-3585
VL - 86
SP - 98
EP - 109
JO - Proteins: Structure, Function and Bioinformatics
JF - Proteins: Structure, Function and Bioinformatics
IS - 1
ER -