<i>Staphylococcus</i> <i>aureus</i> Sortase A Exists as a Dimeric Protein In Vitro

Lu, Changsheng; Zhu, Jie; Wang, Yun; Umeda, Aiko; Cowmeadow, Roshani B.; Lai, Eric; Moreno, Gabrielle N; Person, Maria D.; Zhang, Zhiwen

doi:10.1021/bi700519w

Cited by 36 publications

(43 citation statements)

References 42 publications

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“…Calcium concentrations required for stimulation of sortase A activity are found under physiological conditions in host tissues, and these ions are involved in structural rearrangements of a disordered loop covering the active site that enable substrate binding (Ilangovan et al, 2001b;Zong et al, 2004a;Naik et al, 2006). Interestingly, sortase also forms dimers in vitro, a function that may stimulate catalysis (Lu et al, 2007).…”

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confidence: 99%

Sortase as a Target of Anti-Infective Therapy

2008

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confidence: 99%

Sortase as a Target of Anti-Infective Therapy

2008

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“…Thus, the full length SrtA forms stronger dimer on the cell membrane than its truncated form SrtA Á59 in solution with the dissociation constant measured at 55 mM. 18 Several factors may contribute to the enhanced dimerization of full-length SrtA expressed on S. aureus cell membrane. The transmembrane domain of SrtA would provide additional hydrophobic interaction.…”

Section: Discussionmentioning

confidence: 91%

“…In vitro expression and purification of SrtA ÁN59 and SrtA ÁN59, NKY was the same as previously described. 18 Chemical cross-linking in S. aureus S. aureus cells were cultured as described above. EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride, Sigma, St. Louis, MO) was added to the media to a final concentration of 0.7 mg/mL.…”

Section: Expression and Purification Of Srta Proteinsmentioning

confidence: 99%

Equilibrium of sortase A dimerization on Staphylococcus aureus cell surface mediates its cell wall sorting activity

Zhu

Xiang

Jiang

et al. 2015

Exp Biol Med (Maywood)

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Staphylococcus aureus sortase A (SrtA) transpeptidase is a therapeutically important membrane-bound enzyme in Gram-positive bacteria, which organizes the covalently attached cell surface proteins on the peptidoglycan cell wall of the organism. Here, we report the direct observation of the highly selective homo-dimerization of SrtA on the cell membrane. To address the biological significance of the dimerization towards enzyme function, site-directed mutagenesis was performed to generate a SrtA mutant, which exists as monomer on the cell membrane. We observed that the cell surface display of adhesive proteins in S. aureus cells expressing monomeric SrtA mutant is more prominent than the cells expressing the wild-type enzyme. A cell-based invasion assay was also performed to evaluate the activities of wild-type SrtA and its monomeric mutant as well. Our data demonstrated that S. aureus cells expressing SrtA in monomeric form invade host mammalian cells more efficiently than those expressing wild-type SrtA in dimer-monomer equilibrium. The results suggested that the monomeric form of SrtA is more active than the dimeric form of the enzyme in terms of cell surface display of virulence factors for infection. This is the first study to present the oligomerization of SrtA and its related biological function on the cell membrane. Study of SrtA dimerization has implications for understanding its catalytic mechanism at the cellular level as well as the development of novel anti-infective agents.

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“…SrtA-ΔN59 have been expressed and used in previous studies since it retains the same transpeptidation activity as the full-length SrtA enzyme. 15,17 Expression vector pET28b-SrtA was constructed for expression of soluble SrtA with a His6 tag at its C -terminal. The proteins were expressed in E. coli cells and in one step, purified in large amounts.…”

Section: Resultsmentioning

confidence: 99%

End-Point Immobilization of Recombinant Thrombomodulin via Sortase-Mediated Ligation

et al. 2012

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We report an enzymatic end-point modification and immobilization of recombinant human thrombomodulin (TM), a cofactor for activation of anticoagulant protein C pathway via thrombin. First, a truncated TM mutant consisting of epidermal growth factor-like domains 4–6 (TM456) with a conserved pentapeptide LPETG motif at its C-terminal was expressed and purified in E. coli. Next, the truncated TM456 derivative was site-specifically modified with N-terminal diglycine containing molecules such as biotin and the fluorescent probe dansyl via sortase A (SrtA) mediated ligation (SML). The successful ligations were confirmed by SDS-PAGE and fluorescence imaging. Finally, the truncated TM456 was immobilized onto N-terminal diglycine-functionalized glass slide surface via SML directly. Alternatively, the truncated TM456 was biotinylated via SML and then immobilized onto streptavidin-functionalized glass slide surface indirectly. The successful immobilizations were confirmed by fluorescence imaging. The bioactivity of the immobilized truncated TM456 was further confirmed by protein C activation assay, in which enhanced activation of protein C by immobilized recombinant TM was observed. The sortase A-catalyzed surface ligation took place under mild conditions and is rapid occurring in a single step without prior chemical modification of the target protein. This site-specific covalent modification leads to molecules being arranged in a definitively ordered fashion and facilitating the preservation of the protein’s biological activity.

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Staphylococcus aureus Sortase A Exists as a Dimeric Protein In Vitro

Cited by 36 publications

References 42 publications

Sortase as a Target of Anti-Infective Therapy

Sortase as a Target of Anti-Infective Therapy

Equilibrium of sortase A dimerization on Staphylococcus aureus cell surface mediates its cell wall sorting activity

End-Point Immobilization of Recombinant Thrombomodulin via Sortase-Mediated Ligation

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