"Snapshots" of Ispinesib-induced Conformational Changes in the Mitotic Kinesin Eg5

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2013 May 10

PMID 23658017

Citations 13

Authors

Hung Yi Kristal Kaan

Jennifer Major

Katarzyna Tkocz

Frank Kozielski

Steven S Rosenfeld

Affiliations

Soon will be listed here.

Abstract

Kinesins comprise a superfamily of molecular motors that drive a wide variety of cellular physiologies, from cytoplasmic transport to formation of the bipolar spindle in mitosis. These differing roles are reflected in corresponding polymorphisms in key kinesin structural elements. One of these is a unique loop and stem motif found in all kinesins and referred to as loop 5 (L5). This loop is longest in the mitotic kinesin Eg5 and is the target for a number of small molecule inhibitors, including ispinesib, which is being used in clinical trials in patients with cancer. In this study, we have used x-ray crystallography to identify a new structure of an Eg5-ispinesib complex and have combined this with transient state kinetics to identify a plausible sequence of conformational changes that occur in response to ispinesib binding. Our results demonstrate that ispinesib-induced structural changes in L5 from Eg5 lead to subsequent changes in the conformation of the switch II loop and helix and in the neck linker. We conclude that L5 in Eg5 simultaneously regulates the structure of both the ATP binding site and the motor's mechanical elements that generate force.

Citing Articles

Design, synthesis, molecular docking, and studies of 2-mercaptoquinazolin-4(3)-ones as potential anti-breast cancer agents.

Alossaimi M, Riadi Y, Alnuwaybit G, Md S, Alkreathy H, Elekhnawy E Saudi Pharm J. 2024; 32(3):101971.

PMID: 38357701 PMC: 10864842. DOI: 10.1016/j.jsps.2024.101971.

Drug resistance dependent on allostery: A P-loop rigor Eg5 mutant exhibits resistance to allosteric inhibition by STLC.

Indorato R, DeBonis S, Garcia-Saez I, Skoufias D Front Oncol. 2022; 12:965455.

PMID: 36313676 PMC: 9597087. DOI: 10.3389/fonc.2022.965455.

Optical Control of Mitosis with a Photoswitchable Eg5 Inhibitor.

Impastato A, Shemet A, Veprek N, Saper G, Hess H, Rao L Angew Chem Int Ed Engl. 2021; 61(9):e202115846.

PMID: 34958711 PMC: 9533678. DOI: 10.1002/anie.202115846.

Inhibitor library screening identifies ispinesib as a new potential chemotherapeutic agent for pancreatic cancers.

Murase Y, Ono H, Ogawa K, Yoshioka R, Ishikawa Y, Ueda H Cancer Sci. 2021; 112(11):4641-4654.

PMID: 34510663 PMC: 8586681. DOI: 10.1111/cas.15134.

In Vitro FRET- and Fluorescence-Based Assays to Study Protein Conformation and Protein-Protein Interactions in Mitosis.

Ems-McClung S, Walczak C Methods Mol Biol. 2019; 2101:93-122.

PMID: 31879900 PMC: 7189611. DOI: 10.1007/978-1-0716-0219-5_7.

References

Dale R, Eisinger J, Blumberg W . The orientational freedom of molecular probes. The orientation factor in intramolecular energy transfer. Biophys J. 1979; 26(2):161-93. PMC: 1328514. DOI: 10.1016/S0006-3495(79)85243-1. View

Maliga Z, Mitchison T . Small-molecule and mutational analysis of allosteric Eg5 inhibition by monastrol. BMC Chem Biol. 2006; 6:2. PMC: 1448180. DOI: 10.1186/1472-6769-6-2. View

Tcherniuk S, van Lis R, Kozielski F, Skoufias D . Mutations in the human kinesin Eg5 that confer resistance to monastrol and S-trityl-L-cysteine in tumor derived cell lines. Biochem Pharmacol. 2009; 79(6):864-72. DOI: 10.1016/j.bcp.2009.11.001. View

ROSENFELD S, Rener B, Correia J, Mayo M, Cheung H . Equilibrium studies of kinesin-nucleotide intermediates. J Biol Chem. 1996; 271(16):9473-82. DOI: 10.1074/jbc.271.16.9473. View

Cruickshank D . Remarks about protein structure precision. Acta Crystallogr D Biol Crystallogr. 1999; 55(Pt 3):583-601. DOI: 10.1107/s0907444998012645. View

Turner J, Anderson R, Guo J, Beraud C, Fletterick R, Sakowicz R . Crystal structure of the mitotic spindle kinesin Eg5 reveals a novel conformation of the neck-linker. J Biol Chem. 2001; 276(27):25496-502. DOI: 10.1074/jbc.M100395200. View

Larson A, Naber N, Cooke R, Pate E, Rice S . The conserved L5 loop establishes the pre-powerstroke conformation of the Kinesin-5 motor, eg5. Biophys J. 2010; 98(11):2619-27. PMC: 2877332. DOI: 10.1016/j.bpj.2010.03.014. View

Battye T, Kontogiannis L, Johnson O, Powell H, Leslie A . iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr. 2011; 67(Pt 4):271-81. PMC: 3069742. DOI: 10.1107/S0907444910048675. View

Goulet A, Behnke-Parks W, Sindelar C, Major J, Rosenfeld S, Moores C . The structural basis of force generation by the mitotic motor kinesin-5. J Biol Chem. 2012; 287(53):44654-66. PMC: 3531780. DOI: 10.1074/jbc.M112.404228. View

10.

Behnke-Parks W, Vendome J, Honig B, Maliga Z, Moores C, Rosenfeld S . Loop L5 acts as a conformational latch in the mitotic kinesin Eg5. J Biol Chem. 2010; 286(7):5242-53. PMC: 3037637. DOI: 10.1074/jbc.M110.192930. View

11.

Burris 3rd H, Jones S, Williams D, Kathman S, Hodge J, Pandite L . A phase I study of ispinesib, a kinesin spindle protein inhibitor, administered weekly for three consecutive weeks of a 28-day cycle in patients with solid tumors. Invest New Drugs. 2010; 29(3):467-72. DOI: 10.1007/s10637-009-9374-x. View

12.

Cochran J, Gilbert S . ATPase mechanism of Eg5 in the absence of microtubules: insight into microtubule activation and allosteric inhibition by monastrol. Biochemistry. 2005; 44(50):16633-48. PMC: 2270472. DOI: 10.1021/bi051724w. View

13.

Nesmelov Y, Agafonov R, Negrashov I, Blakely S, Titus M, Thomas D . Structural kinetics of myosin by transient time-resolved FRET. Proc Natl Acad Sci U S A. 2011; 108(5):1891-6. PMC: 3033248. DOI: 10.1073/pnas.1012320108. View

14.

Garcia-Saez I, Yen T, Wade R, Kozielski F . Crystal structure of the motor domain of the human kinetochore protein CENP-E. J Mol Biol. 2004; 340(5):1107-16. DOI: 10.1016/j.jmb.2004.05.053. View

15.

Mayer T, Kapoor T, Haggarty S, King R, Schreiber S, Mitchison T . Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science. 1999; 286(5441):971-4. DOI: 10.1126/science.286.5441.971. View

16.

Winn M, Ballard C, Cowtan K, Dodson E, Emsley P, Evans P . Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 2011; 67(Pt 4):235-42. PMC: 3069738. DOI: 10.1107/S0907444910045749. View

17.

Trybus K, Taylor E . Transient kinetics of adenosine 5'-diphosphate and adenosine 5'-(beta, gamma-imidotriphosphate) binding to subfragment 1 and actosubfragment 1. Biochemistry. 1982; 21(6):1284-94. DOI: 10.1021/bi00535a028. View

18.

Lad L, Luo L, Carson J, Wood K, Hartman J, Copeland R . Mechanism of inhibition of human KSP by ispinesib. Biochemistry. 2008; 47(11):3576-85. DOI: 10.1021/bi702061g. View

19.

Schuttelkopf A, van Aalten D . PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr D Biol Crystallogr. 2004; 60(Pt 8):1355-63. DOI: 10.1107/S0907444904011679. View

20.

Beer T, Goldman B, Synold T, Ryan C, Vasist L, Van Veldhuizen Jr P . Southwest Oncology Group phase II study of ispinesib in androgen-independent prostate cancer previously treated with taxanes. Clin Genitourin Cancer. 2008; 6(2):103-9. DOI: 10.3816/CGC.2008.n.016. View