Current Science

Open Access Open Access  Restricted Access Subscription Access

A crystal form of PSMD10Gankyrin with channels accessible to small molecules


Affiliations
1 Protein Interactome Laboratory for Structural and Functional Biology, Advanced Centre for Treatment, Research and Education in Cancer, Kharghar, Navi Mumbai 412 100, India; and Homi Bhabha National Institute, 2nd Floor, BARC Training School Complex, Anushaktinagar, Mumbai 400 094, India
 

New crystal forms and conditions that aid in rapid formation of crystals would ease the efforts in drug discovery. In addition, if such new crystal forms also yielded high-resolution protein structures, then they can become better templates for screening of drugs using computational tools with better outcome. Such structures are also essential for unambiguous determination of side-chain positions such that subtle conformational changes attributed to mutations, protein dynamics and interactions are true to the proposed mechanism. In this study, we have identified a buffer cocktail which enables crystallization of PSMD10Gankyrin in a novel crystal form. PSMD10Gankyrin is important in the biology of the proteasome assembly and functions of the ubiquitin proteasome pathway. It is also a sought-after therapeutic oncoprotein in multiple cancers. This crystal form yielded a high-resolution structure of PSMD10Gankyrin solved at 1.71 Å. The protein in the crystal is relatively less densely packed with its symmetry-related neighbours. Channels seen all around the protein would guide soaked small molecules to the exposed binding sites. We show that the Alphafold predicted model can be used as an molecular replacement ensemble to solve structures. We also highlight the differences bet­ween the current structure and the Alphafold structure. Thus, the crystal form of PSMD10Gankyrin provides novel insights and opportunities for drug discovery.

Keywords

Crystal forms, drug discovery, protein structures, small molecules.
User
Notifications
Font Size

  • Bedford, L., Paine, S., Sheppard, P. W., Mayer, R. J. and Roelofs, J., Assembly, structure, and function of the 26S proteasome. Trends Cell Biol., 2010, 20, 391–401.

  • Higashitsuji, H. et al., Reduced stability of retinoblastoma protein by gankyrin, an oncogenic ankyrin-repeat protein overexpressed in hepatomas. Nature Med., 2000, 6, 96–99.

  • Fu, X. Y., Wang, H. Y., Tan, L., Liu, S. Q., Cao, H. F. and Wu, M. C., Overexpression of p28/gankyrin in human hepatocellular carcinoma and its clinical significance. World J. Gastroenterol., 2002, 8, 638–643.

  • Park, T. J., Kim, H. S., Byun, K. H., Jang, J. J., Lee, Y. S. and Lim, I. K., Sequential changes in hepatocarcinogenesis induced by diethylnitrosamine plus thioacetamide tamide in fischer 344 rats: induction of gankyrin expression in liver fibrosis, pRB degradation in cirrhosis, and methylation of p16INK4A exon 1 in hepatocellular carcino. Mol. Carcinog., 2001, 30, 138–150.

  • Meng, Y. et al., Gankyrin promotes the proliferation of human pancreatic cancer. Cancer Lett., 2010, 297, 9–17.

  • Man, J. et al., Gankyrin plays an essential role in Ras-induced tumorigenesis through regulation of the RhoA/ROCK pathway in mammalian cells. J. Clin. Invest., 2010, 120, 2829–2841.

  • Higashitsuji, H. et al., The oncoprotein gankyrin binds to MDM2/ HDM2, enhancing ubiquitylation and degradation of p53. Cancer Cell, 2005, 8, 75–87.

  • Higashitsuji, H., Liu, Y., Mayer, R. J. and Fujita, J., The oncoprotein gankyrin negatively regulates both p53 and RB by enhancing proteasomal degradation. Cell Cycle, 2005, 4, 1335–1337.

  • Nanaware, P. P., Ramteke, M. P., Somavarapu, A. K. and Venkatraman, P., Discovery of multiple interacting partners of gankyrin, a proteasomal chaperone and an oncoprotein – evidence for a common hot spot site at the interface and its functional relevance. Proteins Struct. Funct. Bioinformat., 2014, 82, 1283–1300.

  • Sahu, I., Nanaware, P., Mane, M., Mulla, S. W., Roy, S. and Venkatraman, P., Role of a 19S proteasome subunit-PSMD10Gankyrin in neurogenesis of human neural progenitor cells. Int. J. Stem Cells, 2019, 12, 463–473.

  • Krzywda, S. et al., The crystal structure of Gankyrin, an oncoprotein found in complexes with cyclin-dependent kinase 4, a 19 S proteasomal ATPase regulator, and the tumor suppressors Rb and p53. J. Biol. Chem., 2004, 279, 1541–1545.

  • Nakamura, Y. et al., Structure of the oncoprotein gankyrin in complex with S6 ATPase of the 26S proteasome. Structure, 2007, 15, 179–189.

  • Modi, K., Dalvi, S. and Venkatraman, P., Two negatively charged invariant residues influence ligand binding and conformational dynamics of 14-3-3ζ. FEBS Lett., 2020, 594, 878–886.

  • Winter, G., xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr., 2010, 43, 186–190.

  • Winn, M. D. et al., Overview of the CCP4 suite and current developments. Acta Crystallogr. Sect. D, 2011, 67, 235–242.

  • Liebschner, D. et al., Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. Sect. D, 2019, 75, 861–877.

  • Murshudov, G. N., Vagin, A. A. and Dodson, E. J., Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. Sect. D, 1997, 53, 240–255.

  • Emsley, P., Lohkamp, B., Scott, W. G. and Cowtan, K., Features and development of Coot. Acta Crystallogr. Sect. D, 2010, 66, 486–501.

  • Pettersen, E. F. et al., UCSF Chimera – a visualization system for exploratory research and analysis. J. Comput. Chem., 2004, 25, 1605–1612.

  • Schrödinger, LLC, The {PyMOL} Molecular Graphics System, Version 1.8, 2015.

  • Jumper, J. et al., Highly accurate protein structure prediction diction with AlphaFold. Nature, 2021, 596, 583–589.

  • Flower, T. G. and Hurley, J. H., Crystallographic molecular replacement using an in silico-generated search model of SARSCoV-2 ORF8. Protein Sci., 2021, 30, 728–734.

  • Matthews, B. W., Solvent content of protein crystals. J. Mol. Biol., 1968, 33, 491–497.

  • Juers, D. H. and Ruffin, J., MAP-CHANNELS: a computation tool to aid in the visualization and characterization of solvent channels in macromolecular crystals. J. Appl. Crystallogr. Sect., 2014, 47, 2105–2108.

  • Li, J. et al., Gankyrin, a biomarker for epithelial carcinogenesis, is overexpressed in human oral cancer. Anticancer Res., 2011, 31, 2683–2692.

  • Rickman, D. S. et al., Prediction of future metastasis and molecular characterization of head and neck squamous-cell carcinoma based on transcriptome and genome analysis by microarrays. Oncogene, 2008, 27, 6607–6622.

  • Zhen, C. et al., Gankyrin promotes breast cancer cell metastasis by regulating Rac1 activity. Oncogene, 2012, 32(29), 3452–3460.

  • Chattopadhyay, A. et al., Discovery of a small-molecule binder of the oncoprotein gankyrin that modulates gankyrin activity in the cell. Sci. Rep., 2016, 6, 1–11.

  • Kanabar, D. et al., Structural modification of the aryl sulfonate ester of cjoc42 for enhanced gankyrin binding and anti-cancer activity. Bioorg. Med. Chem. Lett., 2020, 30, 126889.

  • Mukund Sudharsan, M. G., Chikhale, R., Nanaware, P. P., Dalvi, S. and Venkatraman, P., A druggable pocket on PSMD10 Gankyrin that can accommodate an interface peptide and doxorubicin. Eur. J. Pharmacol., 2022, 915, 174718.

  • Müller, I., Guidelines for the successful generation of protein–ligand complex crystals. Acta Crystallogr. Sect. D, 2017, 73, 79–92.

  • Serquera, D., Lee, W., Settanni, G., Marszalek, P. E., Paci, E. and Ltzhaki, L. S., Mechanical unfolding of an ankyrin repeat protein. Biophys. J., 2010, 1294–1301.


Abstract Views: 194

PDF Views: 97




  • A crystal form of PSMD10Gankyrin with channels accessible to small molecules

Abstract Views: 194  |  PDF Views: 97

Authors

M. G. Mukund Sudharsan
Protein Interactome Laboratory for Structural and Functional Biology, Advanced Centre for Treatment, Research and Education in Cancer, Kharghar, Navi Mumbai 412 100, India; and Homi Bhabha National Institute, 2nd Floor, BARC Training School Complex, Anushaktinagar, Mumbai 400 094, India
Prasanna Venkatraman
Protein Interactome Laboratory for Structural and Functional Biology, Advanced Centre for Treatment, Research and Education in Cancer, Kharghar, Navi Mumbai 412 100, India; and Homi Bhabha National Institute, 2nd Floor, BARC Training School Complex, Anushaktinagar, Mumbai 400 094, India

Abstract


New crystal forms and conditions that aid in rapid formation of crystals would ease the efforts in drug discovery. In addition, if such new crystal forms also yielded high-resolution protein structures, then they can become better templates for screening of drugs using computational tools with better outcome. Such structures are also essential for unambiguous determination of side-chain positions such that subtle conformational changes attributed to mutations, protein dynamics and interactions are true to the proposed mechanism. In this study, we have identified a buffer cocktail which enables crystallization of PSMD10Gankyrin in a novel crystal form. PSMD10Gankyrin is important in the biology of the proteasome assembly and functions of the ubiquitin proteasome pathway. It is also a sought-after therapeutic oncoprotein in multiple cancers. This crystal form yielded a high-resolution structure of PSMD10Gankyrin solved at 1.71 Å. The protein in the crystal is relatively less densely packed with its symmetry-related neighbours. Channels seen all around the protein would guide soaked small molecules to the exposed binding sites. We show that the Alphafold predicted model can be used as an molecular replacement ensemble to solve structures. We also highlight the differences bet­ween the current structure and the Alphafold structure. Thus, the crystal form of PSMD10Gankyrin provides novel insights and opportunities for drug discovery.

Keywords


Crystal forms, drug discovery, protein structures, small molecules.

References





DOI: https://doi.org/10.18520/cs%2Fv122%2Fi6%2F674-681