The characterization and crystallization of a virally encoded Ustilago maydis KP4 toxin

Gu, Fei; Sullivan, Tiffany S.; Che, Zhiwei; Ganesa, Chandrashekar; Flurkey, William H.; Bozarth, R. F.; Smith, Thomas J.

doi:10.1016/0022-2836(94)90048-5

Cited by 14 publications

(11 citation statements)

References 35 publications

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“…KP4 was purified as reported previously (Gu et al ., 1994; 1995). Briefly, the toxin was isolated from the supernatant of the KP4 toxin expressing strains of U. maydis (or S. cerevisiae ) grown in complete U. maydis media (2.5% bacto peptone, 1% dextrose, 0.15% ammonium nitrate, 0.1% yeast extract) for 7–10 days.…”

Section: Methodsmentioning

confidence: 99%

KP4 fungal toxin inhibits growth in Ustilago maydis by blocking calcium uptake

Gage

Bruenn

Fischer

et al. 2001

Molecular Microbiology

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SummaryKP4 is a virally encoded fungal toxin secreted by the P4 killer strain of Ustilago maydis. From our previous structural studies, it seemed unlikely that KP4 acts by forming channels in the target cell membrane. Instead, KP4 was proposed to act by blocking fungal calcium channels, as KP4 was shown to inhibit voltage-gated calcium channels in rat neuronal cells, and its effects on fungal cells were abrogated by exogenously added calcium. Here, we extend these studies and demonstrate that KP4 acts in a reversible manner on the cell membrane and does not kill the cells, but rather inhibits cell division. This action is mimicked by EGTA and is abrogated specifically by low concentrations of calcium or non-specifically by high ionic strength buffers. We also demonstrate that KP4 affects 45 Ca uptake in U. maydis. Finally, we show that cAMP and a cAMP analogue, N 6,2 0 -O-dibutyryladenosine 3 0 :5 0 -cyclic monophosphate, both abrogate KP4 effects. These results suggest that KP4 may inhibit cell growth and division by blocking calciumregulated signal transduction pathways.

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Section: Methodsmentioning

confidence: 99%

KP4 fungal toxin inhibits growth in Ustilago maydis by blocking calcium uptake

Gage

Bruenn

Fischer

et al. 2001

Molecular Microbiology

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“…KP4 was purified as reported previously (Gu et al, 1994(Gu et al, , 1995. In brief, the toxin was isolated from the supernatant of the KP4 toxin expressing strains of U. maydis (or S. cerevisiae) grown in complete U. maydis media (2.5% bacto-peptone, 1% dextrose, 0.15% ammonium nitrate, 0.1% yeast extract) for 7 to 10 days.…”

Section: Kp4 Purificationmentioning

confidence: 99%

The Virally Encoded Fungal Toxin KP4 Specifically Blocks L-Type Voltage-Gated Calcium Channels

et al. 2002

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KP4 is a virally encoded fungal toxin secreted by the P4 killer strain of Ustilago maydis. Previous studies demonstrated that this toxin inhibits growth of the target fungal cells by blocking calcium uptake rather than forming channels, as had been suggested previously. Unexpectedly, this toxin was also shown to inhibit voltage-gated calcium channel activity in mammalian cells. We used whole-cell patch-clamp techniques to further characterize this activity against mammalian cells. KP4 is shown to specifically block L-type calcium channels with weak voltage dependence to the block. Because KP4 activity is abrogated by calcium, KP4 probably binds competitively with calcium to the channel exterior. Finally, it is shown that chemical reagents that modify lysine residues reduce KP4 activity in both patch-clamp experiments on mammalian cells and in fungal killing assays. Because the only lysine residue is K42, this residue seems to be crucial for both mammalian and fungal channel activity. Our results defining the type of mammalian channel affected by this fungal toxin further support our contention that KP4 inhibits fungal growth by blocking transmembrane calcium flux through fungal calcium channels, and imply a high degree of structural homology between these fungal and mammalian calcium channels.

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“….100 25-50 (Fontecilla-Camps, 1989;Gu et al, 1994;Gage et al, 2001). Second, Arg-38 lies in a homologous region shown to influence the antifungal activity of Rs-AFP2 (De Samblanx et al, 1997).…”

Section: Arg-38 Is Important For Antifungal Activity Of Msdef1mentioning

confidence: 99%

“…KP4 was purified as reported previously (Gu et al, 1994(Gu et al, , 1995. Briefly, the toxin was isolated from the supernatant of the KP4 toxin expressing strains of Ustilago maydis strain P4 using ion-exchange chromatography similar to that described above.…”

Section: Purification Of Kp4mentioning

confidence: 99%

Differential Antifungal and Calcium Channel-Blocking Activity among Structurally Related Plant Defensins

et al. 2004

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Plant defensins are a family of small Cys-rich antifungal proteins that play important roles in plant defense against invading fungi. Structures of several plant defensins share a Cys-stabilized a/b-motif. Structural determinants in plant defensins that govern their antifungal activity and the mechanisms by which they inhibit fungal growth remain unclear. Alfalfa (Medicago sativa) seed defensin, MsDef1, strongly inhibits the growth of Fusarium graminearum in vitro, and its antifungal activity is markedly reduced in the presence of Ca 21. By contrast, MtDef2 from Medicago truncatula, which shares 65% amino acid sequence identity with MsDef1, lacks antifungal activity against F. graminearum. Characterization of the in vitro antifungal activity of the chimeras containing portions of the MsDef1 and MtDef2 proteins shows that the major determinants of antifungal activity reside in the carboxy-terminal region (amino acids 31-45) of MsDef1. We further define the active site by demonstrating that the Arg at position 38 of MsDef1 is critical for its antifungal activity. Furthermore, we have found for the first time, to our knowledge, that MsDef1 blocks the mammalian L-type Ca 21 channel in a manner akin to a virally encoded and structurally unrelated antifungal toxin KP4 from Ustilago maydis, whereas structurally similar MtDef2 and the radish (Raphanus sativus) seed defensin Rs-AFP2 fail to block the L-type Ca 21 channel. From these results, we speculate that the two unrelated antifungal proteins, KP4 and MsDef1, have evolutionarily converged upon the same molecular target, whereas the two structurally related antifungal plant defensins, MtDef2 and Rs-AFP2, have diverged to attack different targets in fungi.

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The characterization and crystallization of a virally encoded Ustilago maydis KP4 toxin

Cited by 14 publications

References 35 publications

KP4 fungal toxin inhibits growth in Ustilago maydis by blocking calcium uptake

KP4 fungal toxin inhibits growth in Ustilago maydis by blocking calcium uptake

The Virally Encoded Fungal Toxin KP4 Specifically Blocks L-Type Voltage-Gated Calcium Channels

Differential Antifungal and Calcium Channel-Blocking Activity among Structurally Related Plant Defensins

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