Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Aug 24;6(3):145.
doi: 10.3390/jof6030145.

Histidine-Rich Defensins from the Solanaceae and Brasicaceae Are Antifungal and Metal Binding Proteins

Mark R Bleackley  1 , Shaily Vasa  1 , Peta J Harvey  2 , Thomas M A Shafee  1 , Bomai K Kerenga  1 , Tatiana P Soares da Costa  1 , David J Craik  2 , Rohan G T Lowe  1 , Marilyn A Anderson  1
Affiliations

Histidine-Rich Defensins from the Solanaceae and Brasicaceae Are Antifungal and Metal Binding Proteins

Mark R Bleackley et al. J Fungi (Basel). .

Abstract

Plant defensins are best known for their antifungal activity and contribution to the plant immune system. The defining feature of plant defensins is their three-dimensional structure known as the cysteine stabilized alpha-beta motif. This protein fold is remarkably tolerant to sequence variation with only the eight cysteines that contribute to the stabilizing disulfide bonds absolutely conserved across the family. Mature defensins are typically 46-50 amino acids in length and are enriched in lysine and/or arginine residues. Examination of a database of approximately 1200 defensin sequences revealed a subset of defensin sequences that were extended in length and were enriched in histidine residues leading to their classification as histidine-rich defensins (HRDs). Using these initial HRD sequences as a query, a search of the available sequence databases identified over 750 HRDs in solanaceous plants and 20 in brassicas. Histidine residues are known to contribute to metal binding functions in proteins leading to the hypothesis that HRDs would have metal binding properties. A selection of the HRD sequences were recombinantly expressed and purified and their antifungal and metal binding activity was characterized. Of the four HRDs that were successfully expressed all displayed some level of metal binding and two of four had antifungal activity. Structural characterization of the other HRDs identified a novel pattern of disulfide linkages in one of the HRDs that is predicted to also occur in HRDs with similar cysteine spacing. Metal binding by HRDs represents a specialization of the plant defensin fold outside of antifungal activity.

Keywords: antifungal; histidine; metal binding; plant defensin.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Histidine-rich defensins (HRDs) are a rare subset of the plant defensins. (A) Histidine-frequency in plant defensins (log scale). Grey shading represents the bulk of defensin sequences. Blue represents sequences identified as histidine rich. (B) Sequence space of 1901 defensins with HRDs (≥6 His) highlighted in blue. (C) Multiple sequence alignment of five example HRDs and a typical antimicrobial defensin (NaD1) for comparison. Cysteines are drawn in yellow, histidines in blue, all other residues in grey. Inter-cysteine loops are numbered below as per NaD1. (D) Average histidine number per loop for all defensins, HRDs in the Solanaceae cluster, HRDs in the Brassicaceae cluster, and HRDs that sit outside of those clusters. For alignment overviews see Figure S2. (E) Tissues where gene expression has been reported in the literature [20,30,31,32,33], including HRDs and NaD1. Check marks indicate tissues where expression has been detected. No data were available for CrD26. For more detail on AtD90, AtD212, and SlD26, see Figure S3.
Figure 2
Figure 2
NiCl2 induced precipitation of AtD212. (A) AtD212 was mixed with a range of concentrations of NiCl2 and assessed for precipitation by the eye. Precipitate formed at NiCl2 concentrations of 0.31 mM and above. The samples were centrifuged and the precipitate (B) and supernatant (C) were analyzed by SDS-PAGE. The majority of HRD protein was in the precipitate. At concentrations where there was no precipitation the majority of HRD was in the supernatant. Lane 9 on both gels is 40 µM AtD90 run as a control.
Figure 3
Figure 3
Structural analysis of histidine-rich defensins. Overlay of the 20 lowest-energy states for (A) SlD26 and (B) AtD90 with disulfides in yellow, beta strands in blue, and alpha helices in red. The differences in disulfide connectivity when comparing SlD26 and AtD90 are shown in panel (C). Numbers indicate the order of cysteine residues in the defensin amino acid sequence.
Figure 4
Figure 4
Histidine side-chain orientation. Defensins are shown with disulfides in yellow and histidines in blue (with their nitrogen atoms in dark blue). (A) The NaD1 dimer (from PDB:4CQK). (B) SlD26, (C) AtD90, (D) a homology model of NbD2. For flexibility of histidine sidechains see Figure S5).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Parisi K., Shafee T.M.A., Quimbar P., van der Weerden N.L., Bleackley M.R., Anderson M.A. The Evolution, Function and Mechanisms of Action for Plant Defensins. Semin. Cell Dev. Biol. 2019;88:107–118. doi: 10.1016/j.semcdb.2018.02.004. - DOI - PubMed
    1. Shafee T.M., Lay F.T., Phan T.K., Anderson M.A., Hulett M.D. Convergent Evolution of Defensin Sequence, Structure and Function. Cell. Mol. Life Sci. 2017;4:663–682. doi: 10.1007/s00018-016-2344-5. - DOI - PMC - PubMed
    1. Ishaq N., Bilal M., Iqbal H. Medicinal Potentialities of Plant Defensins: A Review with Applied Perspectives. Medicines. 2019;6:29. doi: 10.3390/medicines6010029. - DOI - PMC - PubMed
    1. Van der Weerden N.L., Anderson M.A. Plant Defensins: Common Fold, Multiple Functions. Fungal Biol. Rev. 2013;26:121–131. doi: 10.1016/j.fbr.2012.08.004. - DOI
    1. Shafee T.M., Robinson A.J., van der Weerden N., Anderson M.A. Structural Homology Guided Alignment of Cysteine Rich Proteins. SpringerPlus. 2016;5:1–7. - PMC - PubMed

LinkOut - more resources