Research
Our group uses biochemical and biophysical methods to study the structure, dynamics, and function of protein complexes. We are particularly interested in how intrinsically disordered regions in these proteins affect their activities.
Yeast cadmium factor 1 (Ycf1p) is an ATP binding cassette (ABC) transporter found in the yeast cells that is responsible for sequestering Cd2+ and other metal ions in the vacuole, thereby detoxifying the cytoplasm. Like other ABC proteins, Ycf1p contains a core structure comprised of two transmembrane domains (TMD1, TMD2) and two nucleotide binding domains (NBD1, NBD2). As a member of the C subfamily of ABC proteins, which also include the homologous human multidrug resistant proteins (MRPs), Ycf1p contains an additional TMD (TMD0) that is connected to the ABC core structure by the L0 linker. We solved the first high-resolution structure of Ycf1p, showing how TMD0 associates with the ABC core in Ycf1p (and in MRP1) (2021). We also show that mature Ycf1p, which is proteolytically cleaved on a vacuolar loop, forms a stable dimer that associates through protein-protein and protein-lipid interactions, mainly involving TMD0 (2024). Our structure of dimeric Ycf1p accurately predicted the structure of dimeric MRP1. Current work examines how phosphorylation of the intrinsically disordered regulatory (R) region alters its interactions with other regions of the protein.
ATP-sensitive potassium (KATP) channels are large macromolecular complexes comprised of four inward-rectifying Kir6 subunits (Kir6.1, Kir6.2) and four sulphonylurea receptors (SUR1, SUR2 isoforms). The SUR proteins are members of the C subfamily of ABC proteins, but unlike most members of the family, SUR proteins do not possess any transporter functions. Instead, SUR protein activity regulates opening and closing of the Kir6 pore. Like other ABC proteins, and other membrane proteins, SUR proteins contain a number of regulatory intrinsically disordered regions, such as the L1 linker that connects TMD1 to NBD1. Our past work showed that, although intrinsically disordered, the L1 linker possesses residual structure, makes transient interactions with NBD1, and contains a regulatory hot-spot (2015, 2018). Phosphorylation of L1 disrupts in structure and NBD1 interactions, leading to increased ATP binding at NBD1. We showed that mutations in SUR1 NBD1 cause gross structural changes in the NBD and compromise its ATP binding, even if the mutation is not near the ATP binding site (2017). These studies are possible because we can generate stable, well-folded samples of the NBDs (2011, 2014), a first for SUR NBDs. Current work is focussed on studying how the L1 linker interacts with an intrinsically disordered regulatory (R) loop located within NBD1, and the effects of disease-causing mutations in the L1 linker, NBD1, and NBD2 affect their structures and interactions. We are also studying the intrinsically disordered C-terminal tail of the Kir6 pore and its interactions with the SUR protein.
Ni(II) uptake and delivery in E. coli cells requires the concerted action of many proteins, including dedicated uptake systems in the periplasm and inner membrane, as well as metallochaperones and storage proteins in the cytoplasm. We showed how NikA, a periplasmic protein that is part of the NikABCDE ABC importer, preferentially interacts with the Ni(II)-(L-His)2 complex over other metal-amino acid ligands (2022). We also showed that the intrinsically disordered N-terminal extension of the cytoplasmic metallochaperone HypB possesses residual structure, and interacts with the HypB G-domain and also with the metallochaperone SlyD (2025). Notably, binding of SlyD to the HypB NTE affects residues in the HypB G-domain that also respond to Ni(II) and GDP binding. The data give rise to a model explaining the mechanism by which the HypB NTE connects the activities of the HypB G-domain and SlyD.
Projects
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Resources
Fluorimeter
Chromatography System (one of three in the lab)
Example of researcher lab benches
ABE Site Operations
NMR center
Collaborators
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Publications
A complete list of publications is found on Pubmed.
35. Structure of a dimeric full-length ABC transporter.
Bickers SC, Benlekbir S, Rubinstein JL, Kanelis V. Nat Commun. 2024 Nov 16;15(1):9946. doi: 10.1038/s41467-024-54147-8
34. Law WWH*, Kanelis V*, Zamble DB. Biochemical studies highlight determinants for metal selectivity in the Escherichia coli periplasmic solute binding protein NikA. Metallomics 2022, 14 (11), mfac084, https://doi.org/10.1093/mtomcs/mfac084
33. Editorial for the special issue "A Celebration of Women in Biophysical Chemistry".
Musselman C, Kanelis V, Lecomte S, Strodel B. Biophys Chem. 2023 Jan;292:106929. doi: 10.1016/j.bpc.2022.106929.
32. Woloschuk, R. M., Reed, P. M. M., Jaikaran, A. S. I., Demmans, K. Z., Youn, J., Kanelis, V., Uppalapati, M., and Woolley, G. A. (2021) Structure-based design of a photoswitchable affibody scaffold, Protein Sci 30, 2359-2372, 10.1002/pro.4196.
31. Bickers, S. C., Benlekbir, S., Rubinstein, J. L.*, and Kanelis, V*. (2021) Structure of Ycf1p reveals the transmembrane domain TMD0 and the regulatory region of ABCC transporters, Proc. Natl. Acad. Sci. U. S. A. 118, 10.1073/pnas.2025853118.
30. Weiditch, S. A., Bickers, S. C., Bona, D., Maxwell, K. L., and Kanelis, V. (2020) Hk97 gp74 possesses an α-helical insertion in the ββα-fold that affects its metal binding, cos site digestion, and in vivo activities, J. Bacteriol. 202, 10.1128/JB.00644-19.
29. Bickers, S. C., Sayewich, J. S., and Kanelis, V. (2020) Intrinsically disordered regions regulate the activities of atp binding cassette transporters, Biochim. Biophys. Acta Biomembr. 1862, 183202, 10.1016/j.bbamem.2020.183202.
Invited Review that was also peer-reviewed.
28. Weiditch, S. A., Seraphim, T. V., Houry, W. A., and Kanelis, V. (2019) Strategies for purification of the bacteriophage HK97 small and large terminase subunits that yield pure and homogeneous samples that are functional, Protein Expr. Purif. 160, 45-55, 10.1016/j.pep.2019.03.017.
27. Sooklal, C. R., Lopez-Alonso, J. P., Papp, N., and Kanelis, V. (2018) Phosphorylation alters the residual structure and interactions of the regulatory L1 linker connecting NBD1 to the membrane-bound domain in SUR2B, Biochemistry 57, 6278-6292, 10.1021/acs.biochem.8b00503.
26. Zhao, J., Beyrakhova, K., Liu, Y., Alvarez, C. P., Bueler, S. A., Xu, L., Xu, C., Boniecki, M. T., Kanelis, V., Luo, Z. Q., Cygler, M., and Rubinstein, J. L. (2017) Molecular basis for the binding and modulation of v-atpase by a bacterial effector protein, PLoS Pathog .13, e1006394, 10.1371/journal.ppat.1006394.
25. Kanelis, V. (2017) From ions to insulin, Elife 6, 10.7554/eLife.25159.
Invited commentary
24. Alvarez, C. P., Stagljar, M., Muhandiram, D. R., and Kanelis, V. (2017) Hyperinsulinism-causing mutations cause multiple molecular defects in SUR1 NBD1, Biochemistry 56, 2400-2416, 10.1021/acs.biochem.6b00681.
23. de Araujo, E. D., Alvarez, C. P., Lopez-Alonso, J. P., Sooklal, C. R., Stagljar, M., and Kanelis, V. (2015) Phosphorylation-dependent changes in nucleotide binding, conformation, and dynamics of the first nucleotide binding domain (NBD1) of the sulfonylurea receptor 2B (SUR2B), J. Biol. Chem. 290, 22699-22714, 10.1074/jbc.M114.636233.
22. de Araujo, E. D. and Kanelis, V. (2014) Successful development and use of a thermodynamic stability screen for optimizing the yield of nucleotide binding domains, Protein Expr. Purif. 103, 38-47, 10.1016/j.pep.2014.08.006.
21. Kala, S., Cumby, N., Sadowski, P. D., Hyder, B. Z., Kanelis, V., Davidson, A. R., and Maxwell, K. L. (2014) Hnh proteins are a widespread component of phage DNA packaging machines, Proc. Natl. Acad. Sci. U. S. A.111, 6022-6027, 10.1073/pnas.1320952111.
20. Lopez-Alonso, J. P., de Araujo, E. D., and Kanelis, V. (2012) NMR and fluorescence studies of drug binding to the first nucleotide binding domain of SUR2A, Biochemistry 51, 9211-9222, 10.1021/bi301019e
19. Moodley, S., Maxwell, K. L., and Kanelis, V. (2012) The protein gp74 from the bacteriophage HK97 functions as a HNH endonuclease, Protein Sci 21, 809-818, 10.1002/pro.2064.
18. de Araujo, E. D., Ikeda, L. K., Tzvetkova, S., and Kanelis, V. (2011) The first nucleotide binding domain of the sulfonylurea receptor 2A contains regulatory elements and is folded and functions as an independent module, Biochemistry 50, 6655-6666, 10.1021/bi200434d.
17. Kanelis, V., Chong, P.A., and Forman-Kay, J.D. (2011) NMR Spectroscopy to study the dynamics and interactions of CFTR. Methods Mol Biol 741, 377-403, 10.1007/978-1-61779-117-8_25
16. Pell, L. G., Gasmi-Seabrook, G. M., Morais, M., Neudecker, P., Kanelis, V., Bona, D., Donaldson, L. W., Edwards, A. M., Howell, P. L., Davidson, A. R., and Maxwell, K. L. (2010) The solution structure of the C-terminal Ig-like domain of the bacteriophage lambda tail tube protein, J. Mol. Biol. 403, 468-479, 10.1016/j.jmb.2010.08.044.
15. Kanelis, V., Hudson, R. P., Thibodeau, P. H., Thomas, P. J., and Forman-Kay, J. D. (2010) NMR evidence for differential phosphorylation-dependent interactions in WT and ΔF508 CFTR, EMBO J. 29, 263-277, 10.1038/emboj.2009.329.
14. Pell, L. G., Kanelis, V., Donaldson, L. W., Howell, P. L., and Davidson, A. R. (2009) The phage lambda major tail protein structure reveals a common evolution for long-tailed phages and the type VI bacterial secretion system, Proc Natl Acad Sci U S A 106, 4160-4165, 10.1073/pnas.0900044106
(* This paper was selected for a Commentary in PNAS.)
13. Bruce, M. C., Kanelis, V., Fouladkou, F., Debonneville, A., Staub, O., and Rotin, D. (2008) Regulation of NEDD4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain, Biochem. J. 415, 155-163, 10.1042/BJ20071708.
12. Baker, J. M., Hudson, R. P., Kanelis, V., Choy, W. Y., Thibodeau, P. H., Thomas, P. J., and Forman-Kay, J. D. (2007) CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices, Nat. Struct. Mol. Biol. 14, 738-745, 10.1038/nsmb1278.
11. Tugarinov, V., Kanelis, V., and Kay, L. E. (2006) Isotope labeling strategies for the study of high-molecular-weight proteins by solution nmr spectroscopy, Nat. Protoco1., 749-754, 10.1038/nprot.2006.101.
10. Chitayat, S., Kanelis, V., Koschinsky, M. L., and Smith, S. P. (2007) Nuclear magnetic resonance (nmr) solution structure, dynamics, and binding properties of the kringle IV type 8 module of apolipoprotein(a), Biochemistry 46, 1732-1742, 10.1021/bi061814g.
9. Kanelis, V., Bruce, M. C., Skrynnikov, N. R., Rotin, D., and Forman-Kay, J. D. (2006) Structural determinants for high-affinity binding in a NEDD4 WW3* domain-Comm PY motif complex, Structure 14, 543-553, 10.1016/j.str.2005.11.018.
8. Korzhnev, D. M., Kloiber, K., Kanelis, V., Tugarinov, V., and Kay, L. E. (2004) Probing slow dynamics in high molecular weight proteins by methyl-trosy NMR spectroscopy: Application to a 723-residue enzyme, J. Am. Chem. Soc. 126, 3964-3973, 10.1021/ja039587i.
7. Henry, P. C., Kanelis, V., O'Brien, M. C., Kim, B., Gautschi, I., Forman-Kay, J., Schild, L., and Rotin, D. (2003) Affinity and specificity of interactions between NEDD4 isoforms and the epithelial Na+ channel, J. Biol. Chem. 278, 20019-20028, 10.1074/jbc.M211153200.
6. Kanelis, V., Forman-Kay, J. D., and Kay, L. E. (2001) Multidimensional NMR methods for protein structure determination, IUBMB Life 52, 291-302, 10.1080/152165401317291147.
5. Rotin, D., Kanelis, V., and Schild, L. (2001) Trafficking and cell surface stability of ENaC, Am. J. Physiol. Renal. Physiol. 281, F391-399, 10.1152/ajprenal.2001.281.3.F391.
4. Kanelis, V., Rotin, D., and Forman-Kay, J. D. (2001) Solution structure of a NEDD4 WW domain-ENaC peptide complex, Nat. Struct. Biol. 8, 407-412, 10.1038/87562.
3. Kanelis, V., Donaldson, L., Muhandiram, D. R., Rotin, D., Forman-Kay, J. D., and Kay, L. E. (2000) Sequential assignment of proline-rich regions in proteins: Application to modular binding domain complexes, J. Biomol. NMR 16, 253-259, 10.1023/a:1008355012528.
2. Staub, O., Abriel, H., Plant, P., Ishikawa, T., Kanelis, V., Saleki, R., Horisberger, J. D., Schild, L., and Rotin, D. (2000) Regulation of the epithelial Na+ channel by NEDD4 and ubiquitination, Kidney Int. 57, 809-815, 10.1046/j.1523-1755.2000.00919.x.
1. Kanelis, V., Farrow, N. A., Kay, L. E., Rotin, D., and Forman-Kay, J. D. (1998) NMR studies of tandem ww domains of NEDD4 in complex with a PY motif-containing region of the epithelial sodium channel, Biochem. Cell Biol. 76, 341-350, 10.1139/bcb-76-2-3-341
To be added