Substrate Preference of Transglutaminase 2 Revealed by Logistic Regression Analysis and Intrinsic Disorder Examination Eva Csosz 1 ⁎, Peter Bagossi 1 , Zoltan Nagy 2 , Zsuzsanna Dosztanyi 3 , Istvan Simon 3 and Laszlo Fesus 1,4 ⁎ 1 Department of Biochemistry and Molecular Biology, University of Debrecen, Egyetem ter 1, Life Science Building, 4010 Debrecen, Hungary 2 Department of Computer Science, University of Debrecen, Debrecen, Hungary 3 Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary 4 Apoptosis and Genomics Research Group of the Hungarian Academy of Sciences, Debrecen, Hungary Received 16 July 2008; received in revised form 12 August 2008; accepted 12 August 2008 Available online 22 August 2008 Tissue transglutaminase (TG2) catalyzes the Ca 2+ -dependent posttransla- tional modification of proteins via formation of isopeptide bonds between their glutamine and lysine residues. Although substrate specificity of TG2 has been studied repeatedly at the sequence level, no clear consensus sequences have been determined so far. With the use of the extensive structural information on TG2 substrate proteins listed in TRANSDAB Wiki database†, a slight preference of TG2 for glutamine and lysine residues situated in turns could be observed. When the spatial environment of the favored glutamine and lysine residues was analyzed with logistic regres- sion, the presence of specific amino acid patterns was identified. By using the occurrence of the predictor amino acids as selection criteria, several poly- peptides were predicted and later identified as novel in vitro substrates for TG2. By studying the sequence of TG2 substrate proteins lacking available crystal structure, the strong favorable influence on substrate selection of the presence of substrate glutamine and lysine residues in intrinsically disordered regions could also be revealed. The collected structural data have provided novel understanding of how this versatile enzyme selects its substrates in various cell compartments and tissues. © 2008 Elsevier Ltd. All rights reserved. Edited by B. Honig Keywords: glutamine and lysine site; TG2; IUP; substrate preference; 3D structure Introduction Transglutaminases (EC 2.3.2.13) catalyze the Ca 2+ - dependent posttranslational modification of proteins via an acyl-transfer reaction between the γ-carbox- amide group of glutamine (Q-donor) and the ɛ-amino group of lysine (K-donor) residues or primary amines, which leads to the formation of a protease-resistant ɛ (γ-glutamyl)lysine isopeptide bond. 1–3 Tissue trans- glutaminase (TG2) is ubiquitously expressed in the cells, acting at various locations as a multifunctional protein. 4,5 It can modify cytoskeletal proteins (actin, myosin, ROCK2) with a role in cell motility and adhesion. 6,7 TG2 influences inflammatory cytokine production by cross-linking free inhibitory kappa B protein (IκB)α, leading to NFκB translocation to the nucleus, 8 and by cross-linking and, thus, enhancing the activity of annexin I. 9 Depending on the cell type, TG2 can exert pro-apoptotic or anti-apoptotic effects, 10 and as soon as apoptosis starts and the *Corresponding authors. E-mail addresses: cseva@dote.hu; fesus@dote.hu. † http://genomics.dote.hu/wiki Abbreviations used: AUC, area under the curve; CD, delta carbon; IDR, intrinsically disordered region; IGFBP, insulin-like growth factor binding protein; IκB, inhibitory kappa B protein; NZ, zeta nitrogen; ROC, receiver operating characteristic; TG2, tissue transglutaminase; VIP, vasoactive intestinal peptide; 3D, three-dimensional; 5-BPA, 5-biotinamido-penthylamine. doi:10.1016/j.jmb.2008.08.026 J. Mol. Biol. (2008) 383, 390–402 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.