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Conformational and oligomeric effects on the cysteine pK(a) of tryparedoxin peroxidase.

ABSTRACT: Typical 2-Cys peroxiredoxins (Prxs) are peroxidases which regulate cell signaling pathways, apoptosis, and differentiation. These enzymes are obligate homodimers, and can form decamers in solution. During catalysis, Prxs exhibit cysteine-dependent reactivity which requires the deprotonation of the peroxidatic cysteine (C(p)) supported by a lowered pK(a) in the initial step. We present the results of molecular dynamics simulations combined with pKa calculations on the monomeric, dimeric and decameric forms of one typical 2-Cys Prx, the tryparedoxin peroxidase from Trypanosoma cruzi (PDB id, 1uul). The calculations indicate that C(p) (C52) pK(a) values are highly affected by oligomeric state; an unshifted C(p) pK(a) (approximately 8.3, comparable to the pK(a) of isolated cysteine) is calculated for the monomer. In the dimers, starting with essentially identical structures, the C(p)s evolve dynamically asymmetric pK(a)s during the simulations; one subunit's C(p) pK(a) is shifted downward at a time, while the other is unshifted. However, when averaged over time, or multiple simulations, the two subunits within a dimer exhibit the same C(p), showing no preference for a lowered pK(a) in either subunit. Two conserved pathways that communicate the asymmetric pK(a)s between C(p)s of different subunits can be identified. In the decamer, all the C(p) pK(a)s are shifted downward, with slight asymmetry in the dimers which form the decamers. Structural analyses implicate oligomerization effects as responsible for these oligomeric state-dependent C(p) pK(a) shifts. The intra-dimer and the inter-dimer subunit contacts in the decamer restrict the conformations of the side chains of several residues (T49, T54 and E55) calculated to be key in shifting the C(p) pK(a). In addition, the backbone fluctuations of a few residues (M46, D47 and F48) result in a different electrostatic environment for the C(p) in dimers relative to the monomers. These side chain and backbone interactions which contribute to pK(a) modulation indicate the importance of oligomerization to the function of the typical 2-Cys Prxs.

SUBMITTER: Yuan Y

PROVIDER: S-EPMC2874197 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Conformational and oligomeric effects on the cysteine pK(a) of tryparedoxin peroxidase.

Yuan Ye Y Knaggs Michael H M Poole Leslie B L Fetrow Jacquelyn S J Salsbury Freddie R F

Journal of biomolecular structure & dynamics 20100801 1

Typical 2-Cys peroxiredoxins (Prxs) are peroxidases which regulate cell signaling pathways, apoptosis, and differentiation. These enzymes are obligate homodimers, and can form decamers in solution. During catalysis, Prxs exhibit cysteine-dependent reactivity which requires the deprotonation of the peroxidatic cysteine (C(p)) supported by a lowered pK(a) in the initial step. We present the results of molecular dynamics simulations combined with pKa calculations on the monomeric, dimeric and decam ...[more]

PMID: 20476795

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Project description:Leishmaniasis is a neglected disease caused by Leishmania, an intracellular protozoan parasite which possesses a unique thiol metabolism based on trypanothione. Trypanothione is used as a source of electrons by the tryparedoxin/tryparedoxin peroxidase system (TXN/TXNPx) to reduce the hydroperoxides produced by macrophages during infection. This detoxification pathway is not only unique to the parasite but is also essential for its survival; therefore, it constitutes a most attractive drug target. Several forms of TXNPx, with very high sequence identity to one another, have been found in Leishmania strains, one of which has been used as a component of a potential anti-leishmanial polyprotein vaccine. The structures of cytosolic TXN and TXNPx from L. major (LmTXN and LmTXNPx) offer a unique opportunity to study peroxide reduction in Leishmania parasites at a molecular level, and may provide new tools for multienzyme inhibition-based drug discovery. Structural analyses bring out key structural features to elucidate LmTXN and LmTXNPx function. LmTXN displays an unusual N-terminal ?-helix which allows the formation of a stable domain-swapped dimer. In LmTXNPx, crystallized in reducing condition, both the locally unfolded (LU) and fully folded (FF) conformations, typical of the oxidized and reduced protein respectively, are populated. The structural analysis presented here points to a high flexibility of the loop that includes the peroxidatic cysteine which facilitates Cys52 to form an inter-chain disulfide bond with the resolving cysteine (Cys173), thereby preventing over-oxidation which would inactivate the enzyme. Analysis of the electrostatic surface potentials of both LmTXN and LmTXNPx unveils the structural elements at the basis of functionally relevant interaction between the two proteins. Finally, the structural analysis of TXNPx allows us to identify the position of the epitopes that make the protein antigenic and therefore potentially suitable to be used in an anti-leishmanial polyprotein vaccine.

Project description:Cutaneous leishmaniasis (CL) remains a globally spreading public health problem. Among Latin America countries, Brazil has the greatest number of recorded CL cases with several Leishmania species being associated with human cases. Laboratory diagnosis is one of the major challenges to disease control due to the low accuracy of parasitological techniques, the restricted use of molecular techniques, and the importance of differential diagnosis with regard to several dermatological and systemic diseases. In response, we have developed and validated an immunohistochemistry (IHC) technique for CL diagnosis using anti-mTXNPx monoclonal antibody (mAb). Recombinant Leishmania-mTXNPx was produced and used as an immunogen for mAb production through the somatic hybridization technique. The viability of mAb labeling of Leishmania amastigotes was tested by IHC performed with skin biopsies from hamsters experimentally infected with Leishmania amazonensis, Leishmania braziliensis, and Leishmania guyanensis. The enzymes horseradish peroxidase (IHC-HRP) and alkaline phosphatase (IHC-AP), both biotin-free polymer detection systems, were used in the standardization step. The IHC was further validated with skin biopsies from 49 CL patients diagnosed by clinical examination and quantitative real-time polymerase chain reaction and from 37 patients presenting other dermatological infectious diseases. Other parasitological techniques, such as direct examination and culture, were also performed for confirmed CL patients. Histopathology and IHC were performed for all included patients. Overall, the highest sensitivity was observed for IHC-AP (85.7%), followed by IHC-HRP (79.6%), direct examination (77.6%), histopathological examination (HE; 65.3%), and in vitro culture (49%). Only IHC and HE presented specificity over 90% and were able to detect CL patients regardless of parasite burden (odds ratio > 1.94; 95%CI: 0.34-11.23). A significant increase in positivity rates was observed when IHC-AP was combined with direct examination (95.9%) and HE (93.9%). The IHC techniques evaluated in here detected the main Leishmania species causing CL in Brazil and can support diagnostic strategies for controlling this neglected disease, especially if used in combination with other approaches for an integrative laboratorial diagnosis.

Dataset Information

Conformational and oligomeric effects on the cysteine pK(a) of tryparedoxin peroxidase.

Publications

Conformational and oligomeric effects on the cysteine pK(a) of tryparedoxin peroxidase.

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