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• W2078230526 abstract "Chromosomal translocations leading to overexpression of p14TCL1 and its homologue p13MTCP1 are hallmarks of several human T-cell malignancies (1Pekarsky Y. Hallas C. Croce C.M. J. Am. Med. Assoc. 2001; 286: 2308-2314Crossref PubMed Scopus (65) Google Scholar). p14TCL1/p13MTCP1 co-activate protein kinase B (PKB, also named Akt) by binding to its pleckstrin homology (PH) domain, suggesting that p14TCL1/p13MTCP1 induce T-cell leukemia by promoting anti-apoptotic signals via PKB (2Laine J. Kunstle G. Obata T. Noguchi M. J. Biol. Chem. 2002; 277: 3743-3751Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 3Pekarsky Y. Hallas C. Croce C.M. Oncogene. 2001; 20: 5638-5643Crossref PubMed Scopus (80) Google Scholar). Here we combined fluorescence anisotropy, NMR, and small angle x-ray-scattering measurements to determine the affinities, molecular interfaces, and low resolution structure of the complex formed between PKBβ-PH and p14TCL1/p13MTCP1. We show that p14TCL1/p13MTCP1 target PKB-PH at a site that has not yet been observed in PH-protein interactions. Located opposite the phospholipid binding pocket and distal from known protein-protein interaction sites on PH domains, the binding of dimeric TCL1 proteins to this site would allow the crosslinking of two PKB molecules at the cellular membrane in a preactivated conformation without disrupting certain PH-ligand interactions. Thus this interaction could serve to strengthen membrane association, promote trans-phosphorylation, hinder deactivation of PKB, and involve PKB in a multi-protein complex, explaining the array of known effects of TCL1. The binding sites on both proteins present attractive drug targets against leukemia caused by TCL1 proteins. Chromosomal translocations leading to overexpression of p14TCL1 and its homologue p13MTCP1 are hallmarks of several human T-cell malignancies (1Pekarsky Y. Hallas C. Croce C.M. J. Am. Med. Assoc. 2001; 286: 2308-2314Crossref PubMed Scopus (65) Google Scholar). p14TCL1/p13MTCP1 co-activate protein kinase B (PKB, also named Akt) by binding to its pleckstrin homology (PH) domain, suggesting that p14TCL1/p13MTCP1 induce T-cell leukemia by promoting anti-apoptotic signals via PKB (2Laine J. Kunstle G. Obata T. Noguchi M. J. Biol. Chem. 2002; 277: 3743-3751Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 3Pekarsky Y. Hallas C. Croce C.M. Oncogene. 2001; 20: 5638-5643Crossref PubMed Scopus (80) Google Scholar). Here we combined fluorescence anisotropy, NMR, and small angle x-ray-scattering measurements to determine the affinities, molecular interfaces, and low resolution structure of the complex formed between PKBβ-PH and p14TCL1/p13MTCP1. We show that p14TCL1/p13MTCP1 target PKB-PH at a site that has not yet been observed in PH-protein interactions. Located opposite the phospholipid binding pocket and distal from known protein-protein interaction sites on PH domains, the binding of dimeric TCL1 proteins to this site would allow the crosslinking of two PKB molecules at the cellular membrane in a preactivated conformation without disrupting certain PH-ligand interactions. Thus this interaction could serve to strengthen membrane association, promote trans-phosphorylation, hinder deactivation of PKB, and involve PKB in a multi-protein complex, explaining the array of known effects of TCL1. The binding sites on both proteins present attractive drug targets against leukemia caused by TCL1 proteins. Protein kinase B (PKB) 1The abbreviations used are: PKB, protein kinase B; PH, pleckstrin homology; PtdIns-P, inositol phospholipid; TCL1, T-cell leukemia-1; p14TCL1, the 14-kDa protein product of the TCL1 gene; p13MTCP1, the 13-kDa product of the MTCP1 gene; PDB, Protein Data Bank; R2, relaxation rate; SAXS, small angle x-ray scattering. is a 60-kDa member of the AGC superfamily of serine/threonine kinases composed of a amino-terminal PH domain and linked to a kinase domain by a 30 amino acid linker. PKB is frequently called Akt, because it is a mammalian homologue of v-Akt, a viral oncogene isolated from the Akt8 virus that causes T-cell leukemia in mice (4Staal S.P. Hartley J.W. Rowe W.P. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3065-3067Crossref PubMed Scopus (213) Google Scholar, 5Bellacosa A. Franke T.F. Gonzalez-Portal M.E. Datta K. Taguchi T. Gardner J. Cheng J.Q. Testa J.R. Tsichlis P.N. Oncogene. 1993; 8: 745-754PubMed Google Scholar, 6Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Crossref PubMed Scopus (793) Google Scholar, 7Staal S.P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5034-5037Crossref PubMed Scopus (646) Google Scholar). Actually, the PKB family comprises three members, PKBα, PKBβ, and PKBγ (Akt1, Akt2, and Akt3), all of which display, despite some idiosyncrasies, a high level of functional redundancy (Fig. 1) (8Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1040) Google Scholar). Protein kinase B is a central component of phosphoinositide 3′-kinase signaling pathways and has emerged as a pivotal regulator of many cellular processes including apoptosis, proliferation, differentiation, and metabolism (3Pekarsky Y. Hallas C. Croce C.M. Oncogene. 2001; 20: 5638-5643Crossref PubMed Scopus (80) Google Scholar, 9Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar, 10Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3721) Google Scholar). Deregulation of members of the PKB family has been associated with human pathologies such as cancer and diabetes (8Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1040) Google Scholar). PKB activation in response to growth factors and other extracellular stimuli involves membrane recruitment of PKB triggered by inositol phospholipid (PtdIns-P) binding of its amino-terminal pleckstrin homology (PH) domain. At the membrane, PKB is activated by a partially defined process involving lipid-mediated dimerization and phosphorylation of two critical residues, Thr308/Thr309/Thr305 in the kinase activation segment and Ser473/Ser474/Ser472 in the COOH-terminal hydrophobic motif, on PKBα, PKBβ, and PKBγ, respectively (6Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Crossref PubMed Scopus (793) Google Scholar, 11Andjelkovic M. Jakubowicz T. Cron P. Ming X.F. Han J.W. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (430) Google Scholar, 12Coffer P.J. Woodgett J.R. Eur. J. Biochem. 1991; 201: 475-481Crossref PubMed Scopus (389) Google Scholar, 13Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1880) Google Scholar, 14Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 15Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1305) Google Scholar, 16Soskic V. Gorlach M. Poznanovic S. Boehmer F.D. Godovac-Zimmermann J. Biochemistry. 1999; 38: 1757-1764Crossref PubMed Scopus (176) Google Scholar). Residue Thr308/Thr309/Thr305 is phosphorylated by the 3-phosphoinositide-dependent kinase 1, whereas the mechanism responsible for the phosphorylation of Ser473/Ser474/Ser472 has not been resolved (8Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1040) Google Scholar, 17Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (876) Google Scholar). The PH domain of PKB (PKB-PH) has been shown to be essential for mediating the targeting and co-activation of PKB by proteins of the T-cell leukemia-1 (TCL1) family (18Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 19Pekarsky Y. Koval A. Hallas C. Bichi R. Tresini M. Malstrom S. Russo G. Tsichlis P. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3028-3033Crossref PubMed Scopus (314) Google Scholar). Normally, the cellular expression of TCL1 family genes (TCL1, TCL1b, and MTCP1) is mainly restricted to the lymphoid cell lineage and to the early stages of embryogenesis (20Narducci M.G. Fiorenza M.T. Kang S.M. Bevilacqua A. Di Giacomo M. Remotti D. Picchio M.C. Fidanza V. Cooper M.D. Croce C.M. Mangia F. Russo G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11712-11717Crossref PubMed Scopus (81) Google Scholar) where they co-activate PKB, possibly to promote a growth advantage during development through PKB-stimulated cell survival (3Pekarsky Y. Hallas C. Croce C.M. Oncogene. 2001; 20: 5638-5643Crossref PubMed Scopus (80) Google Scholar). However, in certain T-cell malignancies, chromosomal translocations lead to changes in the expression patterns of TCL1 family genes. Thus the TCL1 oncogene was identified because of characteristic chromosomal translocations and inversions at 14q32.1 in clonal T-cell proliferations and malignancies (21Virgilio L. Narducci M.G. Isobe M. Billips L.G. Cooper M.D. Croce C.M. Russo G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12530-12534Crossref PubMed Scopus (230) Google Scholar). Repositioning of T-cell receptor α/δ or β-chain control sequences next to the TCL1 coding region yields deregulated T-cell-specific expression. The product of the TCL1 gene is a 14-kDa protein (p14TCL1) that has been shown to localize in the cytoplasm and nucleus of expressing cells (22Fu T.B. Virgilio L. Narducci M.G. Facchiano A. Russo G. Croce C.M. Cancer Res. 1994; 54: 6297-6301PubMed Google Scholar). Crystallographic studies indicated that p14TCL1 exhibits a novel β-barrel structure (23Hoh F. Yang Y.S. Guignard L. Padilla A. Stern M.H. Lhoste J.M. van Tilbeurgh H. Structure. 1998; 6: 147-155Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). A similar structure was found for the 13-kDa product of the MTCP1 gene (p13MTCP1) (23Hoh F. Yang Y.S. Guignard L. Padilla A. Stern M.H. Lhoste J.M. van Tilbeurgh H. Structure. 1998; 6: 147-155Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 24Guignard L. Padilla A. Mispelter J. Yang Y.S. Stern M.H. Lhoste J.M. Roumestand C. J. Biomol. NMR. 2000; 17: 215-230Crossref PubMed Scopus (22) Google Scholar, 25Fu Z.Q. Du Bois G.C. Song S.P. Kulikovskaya I. Virgilio L. Rothstein J.L. Croce C.M. Weber I.T. Harrison R.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3413-3418Crossref PubMed Scopus (33) Google Scholar). The MTCP1 gene, located in the Xq28 chromosomal region, was the first gene to be identified in the heterogeneous group of uncommon T-cell leukemias presenting a mature phenotype (26Stern M.H. Soulier J. Rosenzwajg M. Nakahara K. Canki-Klain N. Aurias A. Sigaux F. Kirsch I.R. Oncogene. 1993; 8: 2475-2483PubMed Google Scholar). It is involved in the translocation t(X;14)(q28;q11), recurrently associated with this type of T-cell proliferations. In addition to structural similarity, p13MTCP1 exhibits high sequence homology (40% identity, 61% similarity) with p14TCL1 and with p14TCL1b (36% identity, 63% similarity), the product of the newly identified TCL1b oncogene (27Pekarsky Y. Hallas C. Isobe M. Russo G. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2949-2951Crossref PubMed Scopus (94) Google Scholar). Evidence confirming a tumorigenic role for aberrant TCL1 and MTCP1 expression has been obtained from transgenic mice (28Gritti C. Dastot H. Soulier J. Janin A. Daniel M.T. Madani A. Grimber G. Briand P. Sigaux F. Stern M.H. Blood. 1998; 92: 368-373Crossref PubMed Google Scholar, 29Virgilio L. Lazzeri C. Bichi R. Nibu K. Narducci M.G. Russo G. Rothstein J.L. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3885-3889Crossref PubMed Scopus (131) Google Scholar), suggesting that these proteins are the first identified members of a novel family of proto-oncoproteins. Functionally, the association of TCL1 proteins with PKB, which is mediated by its PH domain, enhances the phosphorylation of PKB on Thr308/Thr309/Thr305 and Ser473/Ser474/Ser472, increases PKB-mediated phosphorylation of its substrates glycogen synthase kinase-3β and Bc12-antagonist cell of death, and allows PKB to enter the nucleus (18Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 19Pekarsky Y. Koval A. Hallas C. Bichi R. Tresini M. Malstrom S. Russo G. Tsichlis P. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3028-3033Crossref PubMed Scopus (314) Google Scholar). The underlying molecular mechanisms remain controversial. It was suggested that TCL1 proteins mimic PtdIns-P binding to PKB-PH (30Brazil D.P. Park J. Hemmings B.A. Cell. 2002; 111: 293-303Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar), facilitate PKB trans-phosphorylation by the formation of heterotrimeric complexes with PKB (18Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar), or serve as adaptor proteins to link PKB with factors containing nuclear localization signals (19Pekarsky Y. Koval A. Hallas C. Bichi R. Tresini M. Malstrom S. Russo G. Tsichlis P. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3028-3033Crossref PubMed Scopus (314) Google Scholar). Identification of TCL1 and PKB-PH binding surfaces is an important step in deciphering these complex mechanisms. A putative site of interaction at the TCL1 surface was previously proposed from a mutational analysis of p14TCL1 and molecular modeling (31Kunstle G. Laine J. Pierron G. Kagami Si S. Nakajima H. Hoh F. Roumestand C. Stern M.H. Noguchi M. Mol. Cell. Biol. 2002; 22: 1513-1525Crossref PubMed Scopus (83) Google Scholar, 32French S.W. Shen R.R. Koh P.J. Malone C.S. Mallick P. Teitell M.A. Biochemistry. 2002; 41: 6376-6382Crossref PubMed Scopus (34) Google Scholar). However, the region(s) on PKB-PH that contribute to the interaction are unknown. Advances toward identifying this interaction site were provided recently by the resolution of the three-dimensional structure of the PKB-PH domain (α-and β-isoforms) and the delineation of the binding site of the PtdIns-P (33Thomas C.C. Deak M. Alessi D.R. van Aalten D.M. Curr. Biol. 2002; 12: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 34Auguin D. Barthe P. Auge-Senegas M.T. Stern M.H. Noguchi M. Roumestand C. J. Biomol. NMR. 2004; 28: 137-155Crossref PubMed Scopus (45) Google Scholar). Here, we used a combination of biophysical techniques (fluorescence anisotropy, NMR, and small angle x-ray scattering (SAXS)) to determine the structural basis of the interaction between PKBβ-PH and p13MTCP1/p14TCL1. Based on our experimental data, a molecular model of the complex formed between PKBβ-PH and p14TCL1 is proposed. Our analysis gives insights into how TCL1 family proteins promote their array of cellular effects. Protein Preparation—PKBβ-PH, p13MTCP1, and p14TCL1 were produced, purified, and, in the case of PKBβ-PH and p13MTCP1, 15N-labeled as described previously (23Hoh F. Yang Y.S. Guignard L. Padilla A. Stern M.H. Lhoste J.M. van Tilbeurgh H. Structure. 1998; 6: 147-155Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 35Auguin D. Barthe P. Auge-Senegas M.T. Hoh F. Noguchi M. Roumestand C. J. Biomol. NMR. 2003; 27: 287-288Crossref PubMed Scopus (3) Google Scholar, 36Yang Y.S. Guignard L. Padilla A. Hoh F. Strub M.P. Stern M.H. Lhoste J.M. Roumestand C. J. Biomol. NMR. 1998; 11: 337-354Crossref PubMed Scopus (19) Google Scholar). NMR Experiments—Chemical shift assignments of PKBβ-PH and p13MTCP1 were described previously (24Guignard L. Padilla A. Mispelter J. Yang Y.S. Stern M.H. Lhoste J.M. Roumestand C. J. Biomol. NMR. 2000; 17: 215-230Crossref PubMed Scopus (22) Google Scholar, 35Auguin D. Barthe P. Auge-Senegas M.T. Hoh F. Noguchi M. Roumestand C. J. Biomol. NMR. 2003; 27: 287-288Crossref PubMed Scopus (3) Google Scholar). All of the NMR experiments were carried out at 10 °C on a 500-MHz BRUKER AVANCE spectrometer equipped with a 5-mm z-gradient 1H-13C-15N triple-resonance cryoprobe. Protein samples were dissolved in 300 μl of buffer (10 mm Tris-HCl, 300 mm NaCl, pH 7.4) in Shigemi cells. In all of the experiments, the 1H carrier was centered on the water resonance and a WATERGATE (37Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3527) Google Scholar, 38Sklenar V. J. Magn. Reson. 1995; A114: 132-135Crossref Scopus (144) Google Scholar) sequence was incorporated to suppress the solvent resonance. All of the NMR spectra were acquired in the phase-sensitive mode with Digital Quadrature Detection in the F2 dimension and the hypercomplex States-TPPI (Time Proportional Phase Incrementation) method in F1 dimension (39Marion D. Ikura M. Tschudin R. Bax A. J. Magn. Reson. 1989; 85: 393-399Google Scholar) and processed with Gifa (version 4.22) (40Pons J. Malliavin T. Delsuc M. J. Biomol. NMR. 1996; 8: 445-452Crossref PubMed Scopus (228) Google Scholar) software utility. Titration Experiments—For each titration experiment, eleven aliquots were prepared where the concentration of 15N-labeled PKBβ-PH (or 15N-labeled p13MTCP1) was kept constant (60 μm) and the concentration of unlabeled p13MTCP1 (or PKBβ-PH) and p14TCL1 was increased from 5 to 700 μm and from 2.5 to 200 μm, respectively. [1H,15N]-HSQC (41Bax A. Ikura M. Kay L.E. Torchia D.A. Tschudin R. J. Magn. Reson. 1990; 86: 304-318Google Scholar, 42Bodenhausen G. Ruben D.J. Chem. Phys. Lett. 1980; 69: 185-189Crossref Scopus (2430) Google Scholar) spectra were recorded for each sample using a time domain data size of 64 t1 × 2K t2 complex points and 32 transients/complex t1 increment. The dissociation constants (KD) per residue were measured from the fit of the decreased intensities of all non-overlapping cross-peaks with Equation 1 (43Phizicky E.M. Fields S. Microbiol. Rev. 1995; 59: 94-123Crossref PubMed Google Scholar),y=Imax−(Imax−Imin)×([Pt]+[Lt]+KD)−([Pt]+[Lt]+KD)2−4[Pt][Lt]2[Pt](Eq. 1) where [Pt] and [Lt] are the total concentration of 15N-labeled and unlabeled proteins, respectively. T2-mapping Experiments—Transverse relaxation rates (R2) were measured and analyzed following a standard protocol previously reported in details for the backbone dynamics analysis of 15N-p13MTCP1 (24Guignard L. Padilla A. Mispelter J. Yang Y.S. Stern M.H. Lhoste J.M. Roumestand C. J. Biomol. NMR. 2000; 17: 215-230Crossref PubMed Scopus (22) Google Scholar) and 15N-PKBβ-PH (34Auguin D. Barthe P. Auge-Senegas M.T. Stern M.H. Noguchi M. Roumestand C. J. Biomol. NMR. 2004; 28: 137-155Crossref PubMed Scopus (45) Google Scholar). In this particular case, the single Hahn echo experiment was preferred to the classical CPMG (Carr-Purcell-Meiboom-Gill) experiment for R2 measurements because of its increased sensitivity to exchange contributions (44Wang L. Pang Y. Holder T. Brender J.R. Kurochkin A.V. Zuiderweg E.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7684-7689Crossref PubMed Scopus (107) Google Scholar). R2 were measured for 15N-p13MTCP1 and 15N-PKBβ-PH “free” in solution and compared with the ones measured for the same protein in the presence of a predetermined amount from titration experiments of unlabeled partner (15N-p13MTCP1 ·PKBβ-PH (1:1), 15N-PKBβ-PH ·p13MTCP1 (1:1), and 15N-PKBβ-PH ·p13MTCP1 (5:1)). In these experiments, the total (labeled plus unlabeled) protein concentration was fixed to 0.5 mm for all of the samples in order to avoid any parasitic contribution from viscosity changes. Fluorescence Anisotropy Experiments—Proteins were labeled at pH 8.0 at 4 °C for 2 h, conditions under which labeling ratios are far from unity and that favor unique labeling of the amino terminus. The labeling ratio for the PKBβ-PH proteins was 16%, and that for the p13MTCP1 protein was 10%. Concentrations for the target protein were calculated for total protein, such that the concentration of fluorophore was in both cases considerably lower. Binding assays were performed with a Beacon 2000 polarization instrument (Panvera Corp., Madison, WI) at 4 °C using either Alexa488-labeled PKBβ-PH at a total concentration of 32 nm or Alexa488-labeled p13MTCP1 at a total concentration of 100 nm. The buffer used in the assay was 10 mm Tris-HCl, pH 7.4, and 300 mm NaCl. Anisotropy profiles were obtained in the dilution format. In this approach, data points were taken at equilibrium starting from 240 μl of a 0.1 mm solution of p14TCL1 containing 32.5 nm Alexa488-PKBβ-PH or 0.5 mm solution of PKBβ-PH containing 100 nm Alexa488-labeled p13MTCP1. For each subsequent measurement, 60-μl aliquots of solution were removed from the initial solution and replaced by 60 μl containing 32.5 nm Alexa488-PKBβ-PH or 100 nm Alexa488-labeled p13MTCP1 only. Anisotropy values were recorded as a function of time, and the final five or six values after stabilization were averaged. Anisotropy data were fit using a 1:1 association mass action law in a 99% confidence interval using a GraphPad Prism Fitter. SAXS—SAXS data were recorded on beamline D24 at LURE (Orsay, France) at a wavelength of 1.49 Å using a linear 512 channel detector and sample-detector distances of 1.872 and 0.880 m. For data acquisition, the samples were kept in a quartz capillary of ∼1.5 mm in diameter maintained at a temperature of 10 °C. Protein samples were dialyzed into 10 mm Tris-HCl and 300 mm NaCl at pH 7.4 and filtered through 0.22-μm pores prior to data recording. Sample concentrations were adjusted to 0.6 mm. For each detector distance, eight frames of 200 s each were averaged for both the protein samples and corresponding buffer. The individual frames did not show signs of x-ray damage and were averaged and scaled to transmitted intensity. The buffer contribution was subtracted from the protein-scattering curve. The data were merged and processed using the programs Primus and Gnom (45Svergun D.I. Koch M.H. Curr. Opin. Struct. Biol. 2002; 12: 654-660Crossref PubMed Scopus (168) Google Scholar). Dammin and Gasbor were used for ab initio form determination (45Svergun D.I. Koch M.H. Curr. Opin. Struct. Biol. 2002; 12: 654-660Crossref PubMed Scopus (168) Google Scholar). SAXS envelopes determined by these programs showed P2 symmetry. This symmetry constraint was imposed for subsequent ab initio shape determinations. Ten of the obtained ab initio models were averaged to obtain the most likely molecular envelope (using Damaver (45Svergun D.I. Koch M.H. Curr. Opin. Struct. Biol. 2002; 12: 654-660Crossref PubMed Scopus (168) Google Scholar)). The χ2 values of atomic models against SAXS data were calculated with Crysol (45Svergun D.I. Koch M.H. Curr. Opin. Struct. Biol. 2002; 12: 654-660Crossref PubMed Scopus (168) Google Scholar) using default parameters. Molecular Modeling—Manual modeling of the PKBβ-PH ·p14TCL1 complex was carried out using the program O (46Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar) for visualization of atomic models and SAXS envelopes. Computational docking was carried out with the program FTDock (47Gabb H.A. Jackson R.M. Sternberg M.J. J. Mol. Biol. 1997; 272: 106-120Crossref PubMed Scopus (672) Google Scholar). A total of 37,000 possible orientations of PH relative to p14TCL1 or p13MTCP1 were tested by FTDock for combinations of the x-ray structure of PKBα-PH and NMR structure of PKBβ-PH with x-ray structures of p14TCL1 and p13MTCP1 and an NMR structure of p13MTCP1. This initial set of orientations was filtered to suffice the constraint that residues of the interface of both partners are <6 Å away from the other molecule. The best-scored models from FTDock were submitted to rigid body and side chain refinement by the program MultiDock (48Jackson R.M. Gabb H.A. Sternberg M.J. J. Mol. Biol. 1998; 276: 265-285Crossref PubMed Scopus (213) Google Scholar). An alternative approach using the HADDOCK (High Ambiguity Driven DOCKing) (49Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2188) Google Scholar) program provided us with the means to dock the TCL1 proteins separately onto PKBβ-PH. The chemical shift perturbation data resulting from NMR-mapping experiments were used as ambiguous interaction restraints to drive the docking process. An ambiguous interaction restraint is defined as an ambiguous distance among all of the residues shown to be involved in the interaction. According to established criteria of the HADDOCK program, the “active” residues are those that have been shown to alter the HSQC spectra and also have a high solvent accessibility. The “passive” residues correspond to the residues that are surface neighbors of the active residues and also have a high solvent accessibility calculated with NACCESS program. Therefore, PKBβ-PH residues Lys64, Glu66, and Arg67 in strand β5 and Met100, Arg101, Gln104, Met105, Asn108, and Ser109 in the COOH-terminal helix are considered to be active residues. Five neighboring PKBβ-PH residues, Leu62, Met63, Pro70, Gly97, and Lys111, are considered to be passive residues. For the p14TCL1 protein, residues Asp16, Arg17, Trp19, Glu29, Lys30, Gln31, Ile74, Gln77, and Ser87 are considered active residues when the Asp13, His32, Leu58, Pro61, Pro64, Tyr79, and Asp88 are passive residues. The default HADDOCK parameters were used with the except that only the 300 initial complex structures were generated, the best 50 solutions in terms of intermolecular energies then were refined in water, and the 20 final structures were analyzed. All of the molecular images were produced with Aesop 2M. E. M. Noble, unpublished data. or PyMOL (50DeLano W.L. PyMOL Reference Manual. DeLano Scientific LLC, San Carlos, CA2003Google Scholar). Affinities of the PKB-PH-p13MTCP1/p14TCL1 Interactions— The affinity of PKBβ-PH for p14TCL1 was determined using fluorescence anisotropy. In this experiment, the PKBβ-PH protein was labeled with an amine-reactive fluorescent dye (Alexa488) under conditions that favor the selective labeling of the amino terminus. A solution of this labeled protein at a concentration of 32 nm in the presence of a high concentration of unlabeled p14TCL1 was sequentially diluted with a solution containing only the labeled protein and at each point the fluorescence anisotropy was measured (see “Materials and Methods”). The total change in the anisotropy was 50 millianisotropy units from a value of 185 at very low concentrations of p14TCL1 into 235 millianisotropy units at saturating p14TCL1. The raw data were fit to a model of simple binding of one dimer of p14TCL1 to PKBβ-PH, and the normalized change in anisotropy is plotted along with the normalized fit of the raw data (Fig. 2, squares). The fit was satisfactory as evidenced by a low relative value of χ2 and random residuals and yielded a KD value of 5.7 μm. We note that, since the total concentration of the labeled PKB-PH protein was quite low in order to ensure true equilibrium binding, the probability of populating a complex containing more than one PKB-PH protein per p14TCL1 dimer was infinitely small. The binding of PKB-PH to p13MTCP1 was also measured by fluorescence anisotropy using a p13MTCP1 sample labeled in the same manner by Alexa488 to maximize the total anisotropy change. The labeled p13MTCP1 at a concentration of 100 nm was titrated by unlabeled PKB-PH (Fig. 2, triangles). The total change in anisotropy was from a value of 110 millianisotropy units at low concentrations of PKBβ-PH to a value of 210 millianisotropy units at the highest concentration available, which was determined by the limit of solubility of the used PKBβ-PH construct. The data were fit to the same simple binding model, and the normalized data and fit are plotted also in Fig. 2. It is clear from the lack of high concentration plateau that the affinity of the complex between PKB-PH and p13MTCP1 is significantly lower than that formed by PKB-PH and p14TCL1. The fit of the raw data allowing the undetermined plateau value to float yields a KD value of 537 μm, ∼100-fold of that found for p14TCL1. We also carried out NMR titration experiments of PKB-PH (isoform β; PKBβ-PH) by p13MTCP1 or p14TCL1. These measurements are not redundant with fluorescence anisotropy measurements, because they are indicative of the exchange regime between the free and bound states with regard to the NMR time scale under the conditions of the NMR study. This information is mandatory for the choice of the NMR methods to be used for delineating the binding interfaces. Of course, the NMR titrations are expected to yield less accurate KD values than fluorescence anisotropy because of the relatively high concentration of the labeled target and especially because different mechanisms can participate to the line broadening of resonances belonging to residues located on the binding interface (see below), leading to an underestimation of the apparent KD. Nevertheless, only a few residues are expected to belong to this category, such that only a small bias should be observed in the average result over all of the residues. Adding increasing amounts of unlabeled p13MTCP1 or p14TCL1 to a solution of 15N-labeled PKBβ-PH causes a progressive line broadening in the [1H-15N]HSQC spectrum of PKBβ-PH, indicative of an intermediate-to-slow-exchange process. Under such limiting conditions, it has been shown that a dissociation constant can be estimated from progressively disappearing resonances (51Zuiderweg E.R. Hamers L.F. Rollema H.S. de Bruin S.H. Hilbers C.W. Eur. J. Biochem. 1981; 118: 95-104Crossref PubMed Scopus (28) Google Scholar, 52" @default.
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• W2078230526 title "Structural Basis for the Co-activation of Protein Kinase B by T-cell Leukemia-1 (TCL1) Family Proto-oncoproteins" @default.
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