Poster Presentation 50th Lorne Proteins Conference 2025

Exploring the Stability of the Plakin-Repeat Domain of Plectin. (#352)

Laura J Walsham 1 , Christian G Hartinger 1 , Matthew P Sullivan 1 , David C Goldstone 1
  1. University of Auckland, Auckland, New Zealand

Cancer is a leading cause of death worldwide. Cancer treatment relies heavily on chemotherapy, with non-specific platinum-based compounds being utilised in 50% of cancer treatments1. Platinum-based compounds cause a broad range of side- and off-target effects that limit their use. Consequently, there is a need for targeted chemotherapeutic treatments. The novel antiproliferative agent plecstatin‑1 unexpectedly targets a single protein, plectin2. However, the exact plecstatin-1 binding site remains unknown. Plectin is a large multidomain cytoskeletal protein that facilitates interactions between actin, microtubules, and intermediate filaments3. Plectin is involved in the invasion and metastasis of cancer cells, is mis‑expressed in some cancers, and has been explored as a diagnostic biomarker4,5.

Plectin comprises a globular N-terminal head and a C-terminal tail linked via a coiled-coil. The N‑terminal region has been well-studied, and structures of its plakin and actin-binding domains have been determined. However, less is known about the structure of the C‑terminal tail. The C-terminal tail consists of six plakin-repeat domains (PRD)6, for which we have a construct library. The resultant plakin‑repeat domains have variable solubility, and we have not yet obtained a crystal structure. AlphaFold modelling of these domains revealed a conserved globular core with highly variable and potentially flexible N- and C‑termini. Five truncation constructs were designed, expressed, and purified for each of the M (1st PRD) and P (4th PRD) domains to investigate whether truncations of these domains may improve the solubility and ease of crystallisation. Differential scanning fluorimetry was used to investigate the stability of soluble constructs, revealing that the original M domain was the most stable. In contrast, truncation of the P domain appeared to improve protein stability. These results will inform the design of further truncation constructs of other insoluble plakin-repeat domains to elucidate the plecstatin‑1 binding site on plectin.

  1. Galanski, M. S.; Jakupec, M. A.; Keppler, B. K. Update of the Preclinical Situation of Anticancer Platinum Complexes: Novel Design Strategies and Innovative Analytical Approaches. Curr. Med. Chem. 2005, 12 (18), 2075–2094.
  2. Meier, S. M.; Kreutz, D.; Winter, L.; Klose, M. H. M.; Cseh, K.; Weiss, T.; Bileck, A.; Alte, B.; Mader, J. C.; Jana, S.; Chatterjee, A.; Bhattacharyya, A.; Hejl, M.; Jakupec, M. A.; Heffeter, P.; Berger, W.; Hartinger, C. G.; Keppler, B. K.; Wiche, G.; Gerner, C. An Organoruthenium Anticancer Agent Shows Unexpected Target Selectivity For Plectin. Angew. Chem. Int. Ed. 2017, 56 (28), 8267–8271.
  3. Wiche, G. Role of Plectin in Cytoskeleton Organization and Dynamics. J. Cell Sci. 1998, 111 (17), 2477–2486.
  4. Buckup, M.; Rice, M. A.; Hsu, E.-C.; Garcia-Marques, F.; Liu, S.; Aslan, M.; Bermudez, A.; Huang, J.; Pitteri, S. J.; Stoyanova, T. Plectin Is a Regulator of Prostate Cancer Growth and Metastasis. Oncogene 2021, 40 (3), 663–676.
  5. Bausch, D.; Thomas, S.; Mino-Kenudson, M.; Fernández-del, C. C.; Bauer, T. W.; Williams, M.; Warshaw, A. L.; Thayer, S. P.; Kelly, K. A. Plectin-1 as a Novel Biomarker for Pancreatic Cancer. Clin. Cancer Res. 2011, 17 (2), 302–309.
  6. Janda, L.; Damborský, J.; Rezniczek, G. A.; Wiche, G. Plectin Repeats and Modules: Strategic Cysteines and Their Presumed Impact on Cytolinker Functions. BioEssays 2001, 23 (11), 1064–1069.