The Role of the S-S Bridge in Retroviral Protease Function and Virion Maturation

Zábranská, Helena; Tůma, Roman; Kluh, I.; Svatoš, Aleš; Ruml, Tomáš; Hrabal, Richard; Pichová, Iva

doi:10.1016/j.jmb.2006.11.005

Cited by 10 publications

(10 citation statements)

References 37 publications

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“…The D26N or C7A/D26N/C106A mutations were introduced into the previously described plasmid pBPS13ATG using the QuikChange Site-Directed Mutagenesis Kit (Zá branská et al, 2007) and were verified by DNA sequencing. The expression of M-PMV PRs was performed in Escherichia coli BL21 (DE3) cells under the conditions described previously (Zá branská et al, 2007).…”

Section: Cloning Expression and Purificationmentioning

confidence: 99%

“…In the virions, M-PMV PR is excised autocatalytically from the Gag-Pro polyprotein in a 17 kDa form (per monomer) that undergoes further C-terminal processing, yielding a 13 kDa PR form with comparable catalytic efficiency (Zá branský et al, 1998). In vitro, the 13 kDa (114-residue) form can autodigest even further, producing a 12 kDa (107-residue) polypeptide, which is, however, more than ten times less active (Zá branská et al, 2007). Precise structural characterization of a retroviral protease monomer is also attractive from the point of view of the design of dimerization inhibitors as new-generation drugs against retroviral infection-related diseases, including AIDS.…”

Section: Introductionmentioning

confidence: 99%

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Comparison of a retroviral protease in monomeric and dimeric states

Wosicki

Gilski

Zábranská

et al. 2019

Acta Cryst Sect D Struct Biol

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Retroviral proteases (RPs) are of high interest owing to their crucial role in the maturation process of retroviral particles. RPs are obligatory homodimers, with a pepsin-like active site built around two aspartates (in DTG triads) that activate a water molecule, as the nucleophile, under two flap loops. Mason-Pfizer monkey virus (M-PMV) is unique among retroviruses as its protease is also stable in the monomeric form, as confirmed by an existing crystal structure of a 13 kDa variant of the protein (M-PMV PR) and its previous biochemical characterization. In the present work, two mutants of M-PMV PR, D26N and C7A/D26N/C106A, were crystallized in complex with a peptidomimetic inhibitor and one mutant (D26N) was crystallized without the inhibitor. The crystal structures were solved at resolutions of 1.6, 1.9 and 2.0 Å , respectively. At variance with the previous study, all of the new structures have the canonical dimeric form of retroviral proteases. The protomers within a dimer differ mainly in the flap-loop region, with the most extreme case observed in the apo structure, in which one flap loop is well defined while the other flap loop is not defined by electron density. The presence of the inhibitor molecules in the complex structures was assessed using polder maps, but some details of their conformations remain ambiguous. In all of the presented structures the active site contains a water molecule buried deeply between the Asn26-Thr27-Gly28 triads of the protomers. Such a water molecule is completely unique not only in retropepsins but also in aspartic proteases in general. The C7A and C106A mutations do not influence the conformation of the protein. The Cys106 residue is properly placed at the homodimer interface area for a disulfide cross-link, but the reducing conditions of the crystallization experiment prevented S-S bond formation. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Acta_Cryst_D:S2059798319011355.

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Section: Cloning Expression and Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Comparison of a retroviral protease in monomeric and dimeric states

Wosicki

Gilski

Zábranská

et al. 2019

Acta Cryst Sect D Struct Biol

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“…During maturation of the Gag–Pro polyprotein, the GPD remains attached transiently to the C-terminal part of the PR (PR17), from which it is later detached by PR itself, yielding ΔGPDPR13. Both PR17 and ΔGPDPR13 are present in released virions, and display similar proteolytic activity and substrate specificity (Zábranská et al , 2007; Zábranský et al , 1998). Although the role of the GPD in the MPMV life cycle has been studied (Křízová et al , 2012; Svec et al , 2004; Zábranská et al , 2007), its precise function remains unclear.…”

Section: Full Textmentioning

confidence: 99%

Breast cancer-associated protein – a novel binding partner of Mason-Pfizer monkey virus protease

Rumlová

Křížová

Hadravová

et al. 2014

Journal of General Virology

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We identified breast cancer-associated protein (BCA3) as a novel binding partner of MasonPfizer monkey virus (MPMV) protease (PR). The interaction was confirmed by coimmunoprecipitation and immunocolocalization of MPMV PR and BCA3. Full-length but not Cterminally truncated BCA3 was incorporated into MPMV virions. We ruled out the potential role of the G-patch domain, a glycine-rich domain located at the C terminus of MPMV PR, in BCA3 interaction and virion incorporation. Expression of BCA3 did not affect MPMV particle release and proteolytic processing; however, it slightly increased MPMV infectivity.

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“…In the intramolecular context, the Cys7-Cys106 disulfide stabilizes the monomeric fold, while in the intermolecular context it would be expected to reinforce the dimer. Indeed, it has been shown that in the C7A/ C106A mutant the enzymatic activity is reduced in vitro by 60% (Zá branská et al, 2007). However, in vivo these mutations do not influence Gag processing and virus infectivity.…”

Section: Structural Consequences Of the Monomeric Foldmentioning

confidence: 99%

High-resolution structure of a retroviral protease folded as a monomer

Gilski

Kazmierczyk

Krzywda

et al. 2011

Acta Crystallogr D Biol Cryst

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Mason-Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsinlike aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N-and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C deviations are large and the active-site 'DTG' loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å ) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections, including AIDS.

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The Role of the S-S Bridge in Retroviral Protease Function and Virion Maturation

Cited by 10 publications

References 37 publications

Comparison of a retroviral protease in monomeric and dimeric states

Comparison of a retroviral protease in monomeric and dimeric states

Breast cancer-associated protein – a novel binding partner of Mason-Pfizer monkey virus protease

High-resolution structure of a retroviral protease folded as a monomer

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