A computational approach to explore the functional missense mutations in the spindle check point protein Mad1

Abstract

In this work, the most detrimental missense mutations of Mad1 protein that cause various types of cancer were identified computationally and the substrate binding efficiencies of those missense mutations were analyzed. Out of 13 missense mutations, I Mutant 2.0, SIFT and PolyPhen programs identified 3 variants that were less stable, deleterious and damaging respectively. Subsequently, modeling of these 3 variants was performed to understand the change in their conformations with respect to the native Mad1 by computing their root mean squared deviation (RMSD). Furthermore, the native protein and the 3 mutants were docked with the binding partner Mad2 to explain the substrate binding efficiencies of those detrimental missense mutations. The docking studies identified that all the 3 mutants caused lower binding affinity for Mad2 than the native protein. Finally, normal mode analysis determined that the loss of binding affinity of these 3 mutants was caused by altered flexibility in the amino acids that bind to Mad2 compared with the native protein. Thus, the present study showed that majority of the substrate binding amino acids in those 3 mutants displayed loss of flexibility, which could be the theoretical explanation of decreased binding affinity between the mutant Mad1 and Mad2.

Keywords

missense mutation / Mad1 / Mad2 / spindle check point / cancer

References

[1] Bava K A, Gromiha M M, Uedaira H, Kitajima K, Sarai A (2004). ProTherm, version 4.0: thermodynamic database for proteins and mutants. Nucleic Acids Res, 32(90001 Database issue): D120–D121

[2] Berman H M, Battistuz T, Bhat T N, Bluhm W F, Bourne P E, Burkhardt K, Feng Z, Gilliland G L, Iype L, Jain S, Fagan P, Marvin J, Padilla D, Ravichandran V, Schneider B, Thanki N, Weissig H, Westbrook J D, Zardecki C (2002). The Protein Data Bank. Acta Crystallogr D Biol Crystallogr, 58(Pt 6 No 1): 899–907

[3] Bharadwaj R, Yu H (2004). The spindle checkpoint, aneuploidy, and cancer. Oncogene, 23(11): 2016–2027

[4] Boeckmann B, Bairoch A, Apweiler R, Blatter M C, Estreicher A, Gasteiger E, Martin M J, Michoud K, O’Donovan C, Phan I, Pilbout S, Schneider M (2003). The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Res, 31(1): 365–370

[5] Brady D M, Hardwick K G (2000). Complex formation between Mad1p, Bub1p and Bub3p is crucial for spindle checkpoint function. Curr Biol, 10(11): 675–678

[6] Cahill D P, Lengauer C, Yu J, Riggins G J, Willson J K, Markowitz S D, Kinzler K W, Vogelstein B (1998). Mutations of mitotic checkpoint genes in human cancers. Nature, 392(6673): 300–303

[7] Capriotti E, Fariselli P, Casadio R (2005). I-Mutant2.0: predicting stability changes upon mutation from the protein sequence or structure. Nucleic Acids Res, 33(Web Server Web Server issue): W306-10

[8] Carlson H A, McCammon J A (2000). Accommodating protein flexibility in computational drug design. Mol Pharmacol, 57(2): 213–218

[9] Chao W C, Kulkarni K, Zhang Z, Kong E H, Barford D (2012). Structure of the mitotic checkpoint complex. Nature, 484(7393): 208–213

[10] Chen R H, Shevchenko A, Mann M, Murray A W (1998). Spindle checkpoint protein Xmad1 recruits Xmad2 to unattached kinetochores. J Cell Biol, 143(2): 283–295

[11] Chen R H, Waters J C, Salmon E D, Murray A W (1996). Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores. Science, 274(5285): 242–246

[12] Chung E, Chen R H (2002). Spindle checkpoint requires Mad1-bound and Mad1-free Mad2. Mol Biol Cell, 13(5): 1501–1511

[13] Connolly M L (1983). Solvent-accessible surfaces of proteins and nucleic acids. Science, 221(4612): 709–713

[14] Delarue M, Dumas P (2004). On the use of low-frequency normal modes to enforce collective movements in refining macromolecular structural models. Proc Natl Acad Sci USA, 101(18): 6957–6962

[15] Duhovny D, Nussinov R, Wolfson H J (2002). Efficient unbound docking of rigid molecules. In: Proceedings of the 2nd Workshop on Algorithms in Bioinformatics (WABI) Lecture Notes in Computer Science, Rome, Italy, 2452: 185–200

[16] Fava L L, Kaulich M, Nigg E A, Santamaria A (2011). Probing the in vivo function of Mad1:C-Mad2 in the spindle assembly checkpoint. EMBO J, 30(16): 3322–3336

[17] Gemma A, Seike M, Seike Y, Uematsu K, Hibino S, Kurimoto F, Yoshimura A, Shibuya M, Harris C C, Kudoh S (2000). Somatic mutation of the hBUB1 mitotic checkpoint gene in primary lung cancer. Genes Chromosomes Cancer, 29(3): 213–218

[18] Han J H, Kerrison N, Chothia C, Teichmann S A (2006). Divergence of interdomain geometry in two-domain proteins. Structure, 14(5): 935–945

[19] Han S, Park K, Kim H Y, Lee M S, Kim H J, Kim Y D, Yuh Y J, Kim S R, Suh H S (2000). Clinical implication of altered expression of Mad1 protein in human breast carcinoma. Cancer, 88(7): 1623–1632

[20] Hardwick K G, Weiss E, Luca F C, Winey M, Murray A W (1996). Activation of the budding yeast spindle assembly checkpoint without mitotic spindle disruption. Science, 273(5277): 953–956

[21] Hinkle A, Tobacman L S (2003). Folding and function of the troponin tail domain. Effects of cardiomyopathic troponin T mutations. J Biol Chem, 278(1): 506–513

[22] Hoyt M A, Totis L, Roberts B T S (1991). S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell, 66(3): 507–517

[23] Hwang L H, Lau L F, Smith D L, Mistrot C A, Hardwick K G, Hwang E S, Amon A, Murray A W (1998). Budding yeast Cdc20: a target of the spindle checkpoint. Science, 279(5353): 1041–1044

[24] Jallepalli P V, Lengauer C (2001). Chromosome segregation and cancer: cutting through the mystery. Nat Rev Cancer, 1(2): 109–117

[25] Kim S H, Lin D P, Matsumoto S, Kitazono A, Matsumoto T (1998). Fission yeast Slp1: an effector of the Mad2-dependent spindle checkpoint. Science, 279(5353): 1045–1047

[26] Li R, Murray A W (1991). Feedback control of mitosis in budding yeast. Cell, 66(3): 519–531

[27] Lindahl E, Azuara C, Koehl P, Delarue M (2006). NOMAD-Ref: visualization, deformation and refinement of macromolecular structures based on all-atom normal mode analysis. Nucleic Acids Res, 34(Web Server Web Server issue): W52-6

[28] Lopes C S, Sunkel C E (2003). The spindle checkpoint: from normal cell division to tumorigenesis. Arch Med Res, 34(3): 155–165

[29] Michael S C, Gary J G (1995).Microinjection of mitotic cells with the 3F3/2 Anti-phosphoepitope antibody delays the onset of anaphase. J Cell Biol, 129 (5): 1195–1204

[30] Mimori K, Inoue H, Alder H, Ueo H, Tanaka Y, Mori M (2001). Mutation analysis of hBUB1, human mitotic checkpoint gene in multiple carcinomas. Oncol Rep, 8(1): 39–42

[31] Ng P C, Henikoff S (2001). Predicting deleterious amino acid substitutions. Genome Res, 11(5): 863–874

[32] Ng P C, Henikoff S (2003). SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res, 31(13): 3812–3814

[33] Nomoto S, Haruki N, Takahashi T, Masuda A, Koshikawa T, Takahashi T, Fujii Y, Osada H, Takahashi T (1999). Search for in vivo somatic mutations in the mitotic checkpoint gene, hMAD1, in human lung cancers. Oncogene, 18(50): 7180–7183

[34] Ohshima K, Haraoka S, Yoshioka S, Hamasaki M, Fujiki T, Suzumiya J, Kawasaki C, Kanda M, Kikuchi M (2000). Mutation analysis of mitotic checkpoint genes (hBUB1 and hBUBR1) and microsatellite instability in adult T-cell leukemia/lymphoma. Cancer Lett, 158(2): 141–150

[35] Peters J M (2006). The anaphase promoting complex/cyclosome: a machine designed to destroy. Nat Rev Mol Cell Biol, 7(9): 644–656

[36] Peters J M (2008). Checkpoint control: the journey continues. Curr Biol, 18(4): R170–R172

[37] Rajasekaran R, Priya Doss C G, Sudandiradoss C, Ramanathan K, Sethumadhavan R (2008). In silico analysis of structural and functional consequences in p16INK4A by deleterious nsSNPs associated CDKN2A gene in malignant melanoma. Biochimie, 90(10): 1523–1529

[38] Ramensky V, Bork P, Sunyaev S (2002). Human non-synonymous SNPs: server and survey. Nucleic Acids Res, 30(17): 3894–3900

[39] Reis R M, Nakamura M, Masuoka J, Watanabe T, Colella S, Yonekawa Y, Kleihues P, Ohgaki H (2001). Mutation analysis of hBUB1, hBUBR1 and hBUB3 genes in glioblastomas. Acta Neuropathol, 101(4): 297–304

[40] Sato M, Sekido Y, Horio Y, Takahashi M, Saito H, Minna J D, Shimokata K, Hasegawa Y (2000). Infrequent mutation of the hBUB1 and hBUBR1 genes in human lung cancer. Jpn J Cancer Res, 91(5): 504–509

[41] Schneidman-Duhovny D, Inbar Y, Nussinov R, Wolfson H J (2005). PatchDock and SymmDock: servers for rigid and symmetric docking. Nucleic Acids Res, 33(Web Server issue): W363-7

[42] Suhre K, Sanejouand Y H (2004). ElNemo: a normal mode web server for protein movement analysis and the generation of templates for molecular replacement. Nucleic Acids Res, 32( Web Server issue): W610-4

[43] Tina K G, Bhadra R, Srinivasan N (2007). PIC: Protein Interactions Calculator. Nucleic Acids Res, 35(Web Server issue): W473-6

[44] Tsukasaki K, Miller C W, Greenspun E, Eshaghian S, Kawabata H, Fujimoto T, Tomonaga M, Sawyers C, Said J W, Koeffler H P (2001). Mutations in the mitotic check point gene, MAD1L1, in human cancers. Oncogene, 20(25): 3301–3305

[45] Varfolomeev S D, Uporov I V, Fedorov E V (2002). Bioinformatics and molecular modeling in chemical enzymology. Active sites of hydrolases. Biochemistry (Mosc), 67(10): 1099–1108

[46] Wang X, Jin D Y, Ng R W, Feng H, Wong Y C, Cheung A L, Tsao S W (2002). Significance of MAD2 expression to mitotic checkpoint control in ovarian cancer cells. Cancer Res, 62(6): 1662–1668

[47] Yip Y L, Famiglietti M, Gos A, Duek P D, David F P, Gateau A, Bairoch A (2008). Annotating single amino acid polymorphisms in the UniProt/Swiss-Prot knowledgebase. Hum Mutat, 29(3): 361–366

[48] Yip Y L, Scheib H, Diemand A V, Gattiker A, Famiglietti L M, Gasteiger E, Bairoch A (2004). The Swiss-Prot variant page and the ModSNP database: a resource for sequence and structure information on human protein variants. Hum Mutat, 23(5): 464–470

[49] Yu H (2002). Regulation of APC-Cdc20 by the spindle checkpoint. Curr Opin Cell Biol, 14(6): 706–714

[50] Zhang C, Vasmatzis G, Cornette J L, DeLisi C (1997). Determination of atomic desolvation energies from the structures of crystallized proteins. J Mol Biol, 267(3): 707–726

Acknowledgement

The authors thank the management of Vellore Institute of Technology University for providing the facilities to carry out this work.

Compliance with ethics guidelines

Merlin Lopus, Chandrasekaran P, Sreevishnupriya K, R. Rajasekaran, Ramanathan K, ShanthiV declare that they have no conflict of interest.

This article does not contain any studies with human or animal subjects performed by the any of the authors.

RIGHTS & PERMISSIONS

2014 Higher Education Press and Springer-Verlag Berlin Heidelberg