Structural model of human dUTPase in complex with a novel proteinaceous inhibitor

Nyíri, Kinga; Mertens, Haydyn D. T.; Tihanyi, Borbála; Nagy, Gergely; Kőhegyi, Bianka; Matejka, Judit; Harris, Mary; Szabó, J.; Papp-Kádár, Veronika; Németh-Pongrácz, V.; Ozohanics, Olivér; Vékey, Károly; Svergun, Dmitri I.; Borysik, Antoni J.; Vértessy, Beáta G.

doi:10.1038/s41598-018-22145-8

Cited by 17 publications

(51 citation statements)

References 77 publications

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“…Conserved dUTPase sequence motifs were identified based on earlier studies on dUTPases [2,26]. Recent hydrogen deuterium exchange-mass spectrometry (HDX-MS) measurements on the interaction surface of human dUTPase and protein Stl were also taken into consideration in the sequence alignment [23].…”

Section: Methodsmentioning

confidence: 99%

“…In our previous studies, we reported insights into Stl-inhibition of dUTPases from staphylococcal, human, mycobacterial and Drosophila sources [20,22,23,24]. We observed a generally valid inhibition pattern characterized by a strong dUTPase:Stl complex with dissociation constant in the nanomolar range, and provided a detailed elucidation of the molecular mechanism of interaction and inhibition for the Φ11 dUTPase:Stl system [20].…”

Section: Introductionmentioning

confidence: 96%

“…Previous publications of our research group showed that protein Stl has a potent cross-species inhibitory effect on some other trimeric dUTPase homologues from Mycobacteria , Drosophila and human sources as well [22,23,24]. Efficiency of Stl-induced inhibition on dUTPase activity varies among the complexes with different trimeric dUTPase homologues, and the exact structural background of these species-specific differences in Stl binding are yet to be investigated in details.…”

Section: Introductionmentioning

confidence: 99%

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The Role of a Key Amino Acid Position in Species-Specific Proteinaceous dUTPase Inhibition

et al. 2019

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Protein inhibitors of key DNA repair enzymes play an important role in deciphering physiological pathways responsible for genome integrity, and may also be exploited in biomedical research. The staphylococcal repressor StlSaPIbov1 protein was described to be an efficient inhibitor of dUTPase homologues showing a certain degree of species-specificity. In order to provide insight into the inhibition mechanism, in the present study we investigated the interaction of StlSaPIbov1 and Escherichia coli dUTPase. Although we observed a strong interaction of these proteins, unexpectedly the E. coli dUTPase was not inhibited. Seeking a structural explanation for this phenomenon, we identified a key amino acid position where specific mutations sensitized E. coli dUTPase to StlSaPIbov1 inhibition. We solved the three-dimensional (3D) crystal structure of such a mutant in complex with the substrate analogue dUPNPP and surprisingly found that the C-terminal arm of the enzyme, containing the P-loop-like motif was ordered in the structure. This segment was never localized before in any other E. coli dUTPase crystal structures. The 3D structure in agreement with solution phase experiments suggested that ordering of the flexible C-terminal segment upon substrate binding is a major factor in defining the sensitivity of E. coli dUTPase for StlSaPIbov1 inhibition.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

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The Role of a Key Amino Acid Position in Species-Specific Proteinaceous dUTPase Inhibition

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“…The subunits of this protein interact through twisted β -sheets thus resulting in burying the hydrophobic surfaces about the axis [ 123 ]. It converts deoxyuridine triphosphate to deoxyuridine monophosphate [ 124 ] and prevents DNA uracilation, and maintains genome integrity [ 125 ]. It consists of three active sites and requires divalent Mg 2+ for its activity.…”

Section: Treatment Strategies For Organophosphorus Poisoningmentioning

confidence: 99%

Organophosphorus Nerve Agents: Types, Toxicity, and Treatments

Mukherjee

Gupta

2020

Journal of Toxicology

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Organophosphorus compounds are extensively used worldwide as pesticides which cause great hazards to human health. Nerve agents, a subcategory of the organophosphorus compounds, have been produced and used during wars, and they have also been used in terrorist activities. These compounds possess physiological threats by interacting and inhibiting acetylcholinesterase enzyme which leads to the cholinergic crisis. After a general introduction, this review elucidates the mechanisms underlying cholinergic and noncholinergic effects of organophosphorus compounds. The conceivable treatment strategies for organophosphate poisoning are different types of bioscavengers which include stoichiometric, catalytic, and pseudocatalytic. The current research on the promising treatments specifically the catalytic bioscavengers including several wild-type organophosphate hydrolases such as paraoxonase and phosphotriesterase, phosphotriesterase-like lactonase, methyl parathion hydrolase, organophosphate acid anhydrolase, diisopropyl fluorophosphatase, human triphosphate nucleotidohydrolase, and senescence marker protein has been widely discussed. Organophosphorus compounds are reported to be the nonphysiological substrate for many mammalian organophosphate hydrolysing enzymes; therefore, the efficiency of these enzymes toward these compounds is inadequate. Hence, studies have been conducted to create mutants with an enhanced rate of hydrolysis and high specificity. Several mutants have been created by applying directed molecular evolution and/or targeted mutagenesis, and catalytic efficiency has been characterized. Generally, organophosphorus compounds are chiral in nature. The development of mutant enzymes for providing superior stereoselective degradation of toxic organophosphorus compounds has also been widely accounted for in this review. Existing enzymes have shown limited efficiency; hence, more effective treatment strategies have also been critically analyzed.

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“…Different strains of S. aureus encode numerous mutated versions of SAUGI [18]. While the exact biological role of SAUGI is still unclear, it is highly interesting to note that S. aureus also encodes an inhibitory protein for dUT-Pase, namely Stl [23][24][25][26][27].…”

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Mass spectrometry‐based analysis of macromolecular complexes ofStaphylococcus aureusuracil‐DNA glycosylase and its inhibitor reveals specific variations due to naturally occurring mutations

et al. 2019

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The base excision repair pathway plays an important role in correcting damage induced by either physiological or external effects. This repair pathway removes incorrect bases from the DNA . The uracil base is among the most frequently occurring erroneous bases in DNA , and is cut out from the phosphodiester backbone via the catalytic action of uracil‐ DNA glycosylase. Uracil excision repair is an evolutionarily highly conserved pathway and can be specifically inhibited by a protein inhibitor of uracil‐ DNA glycosylase. Interestingly, both uracil‐ DNA glycosylase ( Staphylococcus aureus uracil‐ DNA glycosylase; SAUDG ) and its inhibitor ( S. aureus uracil‐ DNA glycosylase inhibitor; SAUGI ) are present in the staphylococcal cell. The interaction of these two proteins effectively decreases the efficiency of uracil‐ DNA excision repair. The physiological relevance of this complexation has not yet been addressed in detailed; however, numerous mutations have been identified within SAUGI . Here, we investigated whether these mutations drastically perturb the interaction with SAUDG . To perform quantitative analysis of the macromolecular interactions, we applied native mass spectrometry and demonstrated that this is a highly efficient and specific method for determination of dissociation constants. Our results indicate that several naturally occurring mutations of SAUGI do indeed lead to appreciable changes in the dissociation constants for complex formation. However, all of these K d values remain in the nanomolar range and therefore the association of these two proteins is preserved. We conclude that complexation is most likely preserved even with the naturally occurring mutant uracil‐ DNA glycosylase inhibitor proteins.

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Structural model of human dUTPase in complex with a novel proteinaceous inhibitor

Cited by 17 publications

References 77 publications

The Role of a Key Amino Acid Position in Species-Specific Proteinaceous dUTPase Inhibition

The Role of a Key Amino Acid Position in Species-Specific Proteinaceous dUTPase Inhibition

Organophosphorus Nerve Agents: Types, Toxicity, and Treatments

Mass spectrometry‐based analysis of macromolecular complexes ofStaphylococcus aureusuracil‐DNA glycosylase and its inhibitor reveals specific variations due to naturally occurring mutations

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