MS – University of Connecticut, Storrs, CT
PhD – Yale University, New Haven, CT
My laboratory seeks to understand the molecular basis of regulation of cell division and apoptosis, with special emphasis on the role of tumor suppressor proteins in these vital biological processes. We apply structural biology and biophysical techniques (NMR spectroscopy, x-ray crystallography, calorimetry, AUC, etc.), as well as biochemical and cell biological methods, to study the details of biomolecular mechanisms from the test tube to cells. Of particular interest is the role of intrinsic protein flexibility in regulatory mechanisms.
p21 and p27 are small proteins that regulate cyclin-dependent kinases (Cdks), the master timekeepers of cell division. p21 and p27 are intrinsically unfolded, or disordered, in isolation and fold upon binding their cyclin/Cdk targets. Past studies have shown that the folding-upon-binding process is sequential and that this mediates specificity for particular Cdk/cyclin complexes. Recently, we discovered that tyrosine kinases (e.g. the leukemogenic kinase, BCR-ABL) directly regulate the inhibitory activity and stability of p27, leading to the concept that p27, and other intrinsically disordered proteins, serve as molecular conduits for signal transduction. In addition to BCR-ABL, a number of other oncogenic tyrosine kinases (e.g. Src and Lyn) have recently been shown to phosphorylate p27 in association with human cancer. Our continuing studies are focused on fully understanding the mechanistic role of the p27 signaling conduit in tumorigenesis.
A new project focuses on regulation of the intrinsic apoptotic pathway by the proteins, p53, BCL-xL and PUMA. Past studies by the Green laboratory at St. Jude have shown that PUMA is unique amongst BH3-only, pro-apoptotic proteins in causing the release of p53 from inactive complexes with BCL-xL. However, the molecular details of this process have remained a mystery. Using NMR spectroscopy, x-ray crystallography, and a plethora of biochemical and cellular assays, we have discovered a novel molecular mechanism by which PUMA releases p53 from BCL-xL. This mechanism involves drastic, PUMA-induced structural remodeling of BCL-xL. Continuing studies seek to understand the generality of this mechanism in apoptotic signaling. Further, we seek to understand at the atomic level how binding of PUMA to BCL-xL brings about the gross structural rearrangement that releases p53. Mutagenesis, calorimetry, biochemical and cellular assays, x-ray crystallography and NMR spectroscopy are being applied to understand the structural, dynamic and thermodynamic features of the PUMA-induced p53 release mechanism.
Mitrea DM, Yoon MK, Ou L, Kriwacki RW. Disorder-function relationships for the cell cycle regulatory proteins p21 and p27. Biol Chem 393(4):259-74, 2012.
Follis AV, Galea CA, Kriwacki RW. Intrinsic protein flexibility in regulation of cell proliferation: advantages for signaling and opportunities for novel therapeutics. Adv Exp Med Biol 725:27-49, 2012.
Zhang J, Ding L, Holmfeldt L, Wu G, Heatley SL, Payne-Turner D, Easton J, Chen X, Wang J, Rusch M, Lu C, Chen SC, Wei L, Collins-Underwood JR, Ma J, Roberts KG, Pounds SB, Ulyanov A, Becksfort J, Gupta P, Huether R, Kriwacki RW, Parker M, McGoldrick DJ, Zhao D, Alford D, Espy S, Bobba KC, Song G, Pei D, Cheng C, Roberts S, Barbato MI, Campana D, Coustan-Smith E, Shurtleff SA, Raimondi SC, Kleppe M, Cools J, Shimano KA, Hermiston ML, Doulatov S, Eppert K, Laurenti E, Notta F, Dick JE, Basso G, Hunger SP, Loh ML, Devidas M, Wood B, Winter S, Dunsmore KP, Fulton RS, Fulton LL, Hong X, Harris CC, Dooling DJ, Ochoa K, Johnson KJ, Obenauer JC, Evans WE, Pui CH, Naeve CW, Ley TJ, Mardis ER, Wilson RK, Downing JR, Mullighan CG. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature 481(7380):157-63, 2012.
Kriwacki RW, Yoon MK. Cell biology. Fishing in the nuclear pore. Science Jul 1;333(6038):44-5, 2011. PMID: 21719663.
Dunker AK, Kriwacki RW. The orderly chaos of proteins. Sci Am Apr;304(4):68-73, 2011. PMID: 21495485.
Wang Y, Fisher JC, Mathew R, Ou L, Otieno S, Sublet J, Xiao L, Chen J, Roussel MF, Kriwacki RW. Intrinsic disorder mediates the diverse regulatory functions of the Cdk inhibitor p21. Nat Chem Biol Apr;7(4):214-21, 2011. PMID: 21358637. PMCID: PMC3124363.
Galea CA, High AA, Obenauer JC, Mishra A, Park CG, Punta M, Schlessinger A, Ma J, Rost B, Slaughter CA, Kriwacki RW. Large-scale analysis of thermostable, mammalian proteins provides insights into the intrinsically disordered proteome. J Proteome Res Jan;8(1):211-26, 2009. PMID: 19067583. PMCID: PMC2760310.
Chipuk JE, Fisher JC, Dillon CP, Kriwacki RW, Kuwana T, Green DR. Mechanism of apoptosis induction by inhibition of the anti-apoptotic BCL-2 proteins. Proc Natl Acad Sci USA Dec 23;105(51):20327-32, 2008. PMID: 19074266. PMCID: PMC2629294.
Sivakolundu SG, Nourse A, Moshiach S, Bothner B, Ashley C, Satumba J, Lahti J, Kriwacki RW. Intrinsically unstructured domains of Arf and Hdm2 form bimolecular oligomeric structures in vitro and in vivo. J Mol Biol Dec 5;384(1):240-54, 2008.PMID: 18809412. PMCID: PMC2612038.
Galea CA, Wang Y, Sivakolundu SG, Kriwacki RW. Regulation of cell division by intrinsically unstructured proteins: intrinsic flexibility, modularity, and signaling conduits. Biochemistry 22;47(29):7598-609, 2008. .PMID: 18627125. PMCID: PMC2580775.
Galea CA, Nourse A, Wang Y, Sivakolundu SG, Heller WT, Kriwacki RW. Role of intrinsic flexibility in signal transduction mediated by the cell cycle regulator, p27 Kip1. J Mol Biol Feb 22;376(3):827-38, 2008. PMID: 18177895. PMCID: PMC2350195.
Grimmler M, Wang Y, Mund T, Cilensek Z, Keidel EM, Waddell MB, Jäkel H, Kullmann M, Kriwacki RW, Hengst L. Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases. Cell Jan 26;128(2):269-802007. PMID: 17254966
Galea CA, Pagala VR, Obenauer JC, Park CG, Slaughter CA, Kriwacki RW. Proteomic studies of the intrinsically unstructured mammalian proteome. J Proteome Res Oct;5(10):2839-48, 2006. PMID: 17022655.
Wang Y, Filippov I, Richter C, Luo R, Kriwacki RW. Solution NMR studies of an intrinsically unstructured protein within a dilute, 75 kDa eukaryotic protein assembly; probing the practical limits for efficiently assigning polypeptide backbone resonances. Chembiochem Dec;6(12):2242-6, 2005. PMID: 16270364
Galea C, Bowman P, Kriwacki RW. Disruption of an intermonomer salt bridge in the p53 tetramerization domain results in an increased propensity to form amyloid fibrils. Protein Sci Dec;14(12):2993-3003, 2005. PMID: 16260757. PMCID: PMC2253254.
Sivakolundu SG, Bashford D, Kriwacki RW. Disordered p27Kip1 exhibits intrinsic structure resembling the Cdk2/cyclin A-bound conformation. J Mol Biol Nov 11;353(5):1118-28, 2005. PMID: 16214166.
Lacy ER, Wang Y, Post J, Nourse A, Webb W, Mapelli M, Musacchio A, Siuzdak G, Kriwacki RW. Molecular basis for the specificity of p27 toward cyclin-dependent kinases that regulate cell division. J Mol Biol Jun 17;349(4):764-73, 2005. PMID: 15890360.
Luo R, Mann B, Lewis WS, Rowe A, Heath R, Stewart ML, Hamburger AE, Sivakolundu S, Lacy ER, Bjorkman PJ, Tuomanen E, Kriwacki RW. Solution structure of choline binding protein A, the major adhesin of Streptococcus pneumoniae. EMBO J Jan 12;24(1):34-43, 2005. Epub 2004 Dec 16. PMID: 15616594. PMCID: PMC544903.
Lacy ER, Filippov I, Lewis WS, Otieno S, Xiao L, Weiss S, Hengst L, Kriwacki RW. p27 binds cyclin-CDK complexes through a sequential mechanism involving binding-induced protein folding. Nat Struct Mol Biol Apr;11(4):358-64, 2004. PMID: 15024385.
Bothner B, Aubin Y, Kriwacki RW. Peptides derived from two dynamically disordered proteins self-assemble into amyloid-like fibrils. J Am Chem Soc Mar 19;125(11):3200-1, 2003. PMID: 12630860.
Lee AS, Galea C, DiGiammarino EL, Jun B, Murti G, Ribeiro RC, Zambetti G, Schultz CP, Kriwacki RW. Reversible amyloid formation by the p53 tetramerization domain and a cancer-associated mutant. J Mol Biol Mar 28;327(3):699-709, 2003. PMID: 12634062.
Somerville L, Krynetski EY, Krynetskaia NF, Beger RD, Zhang W, Marhefka CA, Evans WE, Kriwacki RW. Structure and dynamics of thioguanine-modified duplex DNA. J Biol Chem Jan 10;278(2):1005-11, 2003. PubMed PMID: 12401802.
DiGiammarino EL, Lee AS, Cadwell C, Zhang W, Bothner B, Ribeiro RC, Zambetti G, Kriwacki RW. A novel mechanism of tumorigenesis involving pH-dependent destabilization of a mutant p53 tetramer. Nat Struct Biol Jan;9(1):12-6, 2002. PMID: 11753428.
Kriwacki RW, Hengst L, Tennant L, Reed SI, Wright PE. Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity. Proc Natl Acad Sci USA Oct 15;93(21):11504-9, 1996. PMID: 8876165. PMCID: PMC38087.
Last update: May 2012