@article{stephenson_taggart_xu_fowler_wu_suo_2023, title={The inhibitor of & kappa;B kinase & beta; (IKK & beta;) phosphorylates I & kappa;B & alpha; twice in a single binding event through a sequential mechanism}, volume={299}, ISSN={["1083-351X"]}, DOI={10.1016/j.jbc.2022.102796}, abstractNote={Phosphorylation of Inhibitor of κB (IκB) proteins by IκB Kinase β (IKKβ) leads to IκB degradation and subsequent activation of nuclear factor κB transcription factors. Of particular interest is the IKKβ-catalyzed phosphorylation of IκBα residues Ser32 and Ser36 within a conserved destruction box motif. To investigate the catalytic mechanism of IKKβ, we performed pre-steady-state kinetic analysis of the phosphorylation of IκBα protein substrates catalyzed by constitutively active, human IKKβ. Phosphorylation of full-length IκBα catalyzed by IKKβ was characterized by a fast exponential phase followed by a slower linear phase. The maximum observed rate (kp) of IKKβ-catalyzed phosphorylation of IκBα was 0.32 s-1 and the binding affinity of ATP for the IKKβ•IκBα complex (Kd) was 12 μM. Substitution of either Ser32 or Ser36 with Ala, Asp, or Cys reduced the amplitude of the exponential phase by approximately 2-fold. Thus, the exponential phase was attributed to phosphorylation of IκBα at Ser32 and Ser36, whereas the slower linear phase was attributed to phosphorylation of other residues. Interestingly, the exponential rate of phosphorylation of the IκBα(S32D) phosphomimetic amino acid substitution mutant was nearly twice that of WT IκBα and 4-fold faster than any of the other IκBα amino acid substitution mutants, suggesting that phosphorylation of Ser32 increases the phosphorylation rate of Ser36. These conclusions were supported by parallel experiments using GST-IκBα(1-54) fusion protein substrates bearing the first 54 residues of IκBα. Our data suggest a model wherein, IKKβ phosphorylates IκBα at Ser32 followed by Ser36 within a single binding event.}, number={1}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Stephenson, Anthony A. and Taggart, David J. and Xu, Guozhou and Fowler, Jason D. and Wu, Hao and Suo, Zucai}, year={2023}, month={Jan} }
 @article{pan_kaur_barnes_detwiler_sanford_liu_xu_mahn_tang_hao_et al._2021, title={Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA}, volume={297}, ISSN={["1083-351X"]}, DOI={10.1016/j.jbc.2021.101080}, abstractNote={TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation. TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation. Telomeres are nucleoprotein structures that prevent the degradation or fusion of the ends of linear chromosomes, which are threatened by at least seven distinct DNA damage response (DDR) pathways (1Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1344) Google Scholar, 2Muraki K. Nyhan K. Han L. Murnane J.P. Mechanisms of telomere loss and their consequences for chromosome instability.Front. Oncol. 2012; 2: 135Crossref PubMed Google Scholar, 3de Lange T. Shelterin-mediated telomere protection.Annu. Rev. Genet. 2018; 52: 223-247Crossref PubMed Scopus (280) Google Scholar). 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A shared docking motif in TRF1 and TRF2 used for differential recruitment of telomeric proteins.Science. 2008; 319: 1092-1096Crossref PubMed Scopus (187) Google Scholar). TIN2 stabilizes both TRF1 and TRF2 at telomeres (31Kim S.H. Han S. You Y.H. Chen D.J. Campisi J. The human telomere-associated protein TIN2 stimulates interactions between telomeric DNA tracts in vitro.EMBO Rep. 2003; 4: 685-691Crossref PubMed Scopus (42) Google Scholar, 32Ye J.Z. Donigian J.R. van Overbeek M. Loayza D. Luo Y. Krutchinsky A.N. Chait B.T. de Lange T. TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres.J. Biol. Chem. 2004; 279: 47264-47271Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The loss of the TRF1 or TRF2-binding domains in TIN2 triggers a DNA damage response (33Takai K.K. Kibe T. Donigian J.R. Frescas D. de Lange T. Telomere protection by TPP1/POT1 requires tethering to TIN2.Mol. Cell. 2011; 44: 647-659Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Binding of TPP1 to TIN2 is required for POT1-mediated telomere protection (34Frescas D. de Lange T. Binding of TPP1 protein to TIN2 protein is required for POT1a,b protein-mediated telomere protection.J. Biol. Chem. 2014; 289: 24180-24187Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). As an integral component of the “TIN2/TPP1/POT1 processivity complex,” TIN2 functions together with TPP1/POT1 to stimulate telomerase processivity (35Pike A.M. Strong M.A. Ouyang J.P.T. Greider C.W. TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.Mol. Cell. Biol. 2019; 39e00593-18Crossref PubMed Scopus (22) Google Scholar). Furthermore, TIN2 directly interacts with the cohesin subunit SA1 and plays a key role in a distinct SA1-TRF1-TIN2-mediated sister telomere cohesion pathway that is largely independent of the cohesin ring subunits (8Canudas S. Houghtaling B.R. Kim J.Y. Dynek J.N. Chang W.G. Smith S. Protein requirements for sister telomere association in human cells.EMBO J. 2007; 26: 4867-4878Crossref PubMed Scopus (80) Google Scholar, 36Canudas S. Smith S. Differential regulation of telomere and centromere cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells.J. Cell Biol. 2009; 187: 165-173Crossref PubMed Scopus (122) Google Scholar). Binding of TRF1-TIN2 to telomeres is regulated by the poly(ADP-ribose) polymerase Tankyrase 1 (37Smith S. Giriat I. Schmitt A. de Lange T. Tankyrase, a poly(ADP-ribose) polymerase at human telomeres.Science. 1998; 282: 1484-1487Crossref PubMed Scopus (891) Google Scholar). ADP-ribosylation of TRF1 by Tankyrase 1 reduces its binding to telomeric DNA in vitro, and the depletion of Tankyrase 1 using siRNA leads to mitotic arrest and persistent telomere cohesion that can be rescued by depletion of TIN2 (8Canudas S. Houghtaling B.R. Kim J.Y. Dynek J.N. Chang W.G. Smith S. Protein requirements for sister telomere association in human cells.EMBO J. 2007; 26: 4867-4878Crossref PubMed Scopus (80) Google Scholar, 36Canudas S. Smith S. Differential regulation of telomere and centromere cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells.J. Cell Biol. 2009; 187: 165-173Crossref PubMed Scopus (122) Google Scholar, 38Dynek J.N. Smith S. Resolution of sister telomere association is required for progression through mitosis.Science. 2004; 304: 97-100Crossref PubMed Scopus (216) Google Scholar). Three distinct TIN2 isoforms have been identified in human cell lines (35Pike A.M. Strong M.A. Ouyang J.P.T. Greider C.W. TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.Mol. Cell. Biol. 2019; 39e00593-18Crossref PubMed Scopus (22) Google Scholar, 39Kaminker P.G. Kim S.H. Desprez P.Y. Campisi J. A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix.Cell Cycle. 2009; 8: 931-939Crossref PubMed Scopus (35) Google Scholar, 40Smith S. The long and short of it: A new isoform of TIN2 in the nuclear matrix.Cell Cycle. 2009; 8: 797-798Crossref PubMed Scopus (2) Google Scholar) that include TIN2S (354 AAs), TIN2L (451 AAs), and TIN2M (TIN2 medium, 420 AAs). TIN2S, TIN2L, and TIN2M share the same TRF1, TRF2, and TPP1-binding domains and localize to telomeres (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar, 35Pike A.M. Strong M.A. Ouyang J.P.T. Greider C.W. TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.Mol. Cell. Biol. 2019; 39e00593-18Crossref PubMed Scopus (22) Google Scholar, 39Kaminker P.G. Kim S.H. Desprez P.Y. Campisi J. A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix.Cell Cycle. 2009; 8: 931-939Crossref PubMed Scopus (35) Google Scholar). Consistent with its key role in telomere maintenance, germline inactivation of TIN2 in mice is embryonic lethal (41Chiang Y.J. Kim S.H. Tessarollo L. Campisi J. Hodes R.J. Telomere-associated protein TIN2 is essential for early embryonic development through a telomerase-independent pathway.Mol. Cell. Biol. 2004; 24: 6631-6634Crossref PubMed Scopus (61) Google Scholar). Removal of TIN2 leads to the formation of telomere dysfunction-induced foci (TIFs). Importantly, clinical studies further highlight the biological significance of TIN2 in telomere protection (42Savage S.A. Giri N. Baerlocher G.M. Orr N. Lansdorp P.M. Alter B.P. TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita.Am. J. Hum. 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However, since TIN2 itself does not directly bind to DNA and instead serves as a "mediator/enhancer" for shelterin and telomerase activities, defining TIN2's distinct function at the molecular level has been challenging. The bottleneck for studying TIN2 lies in the fact that results from bulk biochemical assays do not fully reveal the heterogeneity and dynamics of the protein–protein and protein–DNA interactions. Furthermore, cell-based assays only provide information on the outcomes from downstream effectors after the knocking down of TIN2 that also removes TRF1 and TRF2 from telomeres. These approaches do not allow us to investigate the molecular structures and dynamics in which TIN2 directly participates. In vivo, the amount of TIN2 is sufficient for binding every TRF1 and TRF2 molecule (44Takai K.K. Hooper S. Blackwood S. Gandhi R. de Lange T. In vivo stoichiometry of shelterin components.J. Biol. Chem. 2010; 285: 1457-1467Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), while TPP1 and POT1 are ~10-fold less than TRF1 and TIN2. Thus, it is important to study the DNA-binding properties of TRF1-TIN2 complexes. To fill this important knowledge gap, we applied complementary single-molecule imaging platforms, including atomic force microscopy (AFM) (45Yang Y. Wang H. Erie D.A. Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy.Methods. 2003; 29: 175-187Crossref PubMed Scopus (88) Google Scholar, 46Wang H. Nora G.J. Ghodke H. Opresko P.L. Single molecule studies of physiologically relevant telomeric tails reveal POT1 mechanism for promoting G-quadruplex unfolding.J. Biol. Chem. 2011; 286: 7479-7489Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 47Kaur P. Wu D. Lin J. Countryman P. Bradford K.C. Erie D.A. Riehn R. Opresko P.L. Wang H. Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2.Sci. Rep. 2016; 6: 20513Crossref PubMed Scopus (20) Google Scholar), total internal reflection fluorescence microscopy (TIRFM) (48Erie D.A. Weninger K.R. Single molecule studies of DNA mismatch repair.DNA Repair. 2014; 20: 71-81Crossref PubMed Scopus (46) Google Scholar), and the DNA tightrope assay to monitor TRF1-TIN2-mediated DNA compaction and DNA-DNA bridging (49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (18) Google Scholar, 50Countryman P. Fan Y. Gorthi A. Pan H. Strickland J. Kaur P. Wang X. Lin J. Lei X. White C. You C. Wirth N. Tessmer I. Piehler J. Riehn R. et al.Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates.J. Biol. Chem. 2018; 293: 1054-1069Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 51Pan H. Jin M. Ghadiyaram A. Kaur P. Miller H.E. Ta H.M. Liu M. Fan Y. Mahn C. Gorthi A. You C. Piehler J. Riehn R. Bishop A.J.R. Tao Y.J. et al.Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA.Nucleic Acids Res. 2020; 48: 5639-5655Crossref PubMed Google Scholar). Through using DNA substrates on different length scales (6 and 270 TAAGGG repeats), these imaging platforms provide complementary results demonstrating that both TIN2S and TIN2L facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. In some cases, TRF1-TIN2 is capable of mediating the bridging of multiple copies of telomeric DNA fragments. Importantly, our results demonstrate that TIN2 protects the disassembly of TRF1-TIN2-mediated DNA-DNA bridging by Tankyrase 1. In addition, the N-terminal domain of TPP1 inhibits TRF1-TIN2-mediated DNA-DNA bridging. In summary, this study uncovered the unique biophysical function of TIN2 as a telomeric architectural protein, acting together with TRF1 to mediate interactions between distant telomeric sequences. Tankyrase 1 and TPP1 regulate TRF1-TIN2-mediated DNA-DNA bridging. Furthermore, this work establishes a unique combination of single-molecule imaging platforms for future examination of TIN2 disease variants and provides a new direction for investigating molecular mechanisms underlying diverse TIN2 functions. A previous study suggested that TIN2 modulates the bridging of telomeric DNA by TRF1 (31Kim S.H. Han S. You Y.H. Chen D.J. Campisi J. The human telomere-associated protein TIN2 stimulates interactions between telomeric DNA tracts in vitro.EMBO Rep. 2003; 4: 685-691Crossref PubMed Scopus (42) Google Scholar). However, the bulk biochemical assays using short telomeric DNA (six telomeric repeats) did not provide information regarding the structure and dynamics of the TRF1-TIN2-DNA complex. To investigate the molecular function of TIN2, we applied AFM imaging to investigate how TIN2 affects the telomeric DNA-DNA pairing mediated by TRF1 at the single-molecule level on longer telomeric DNA substrates (270 TTAGGG repeats). We purified TRF1 (Fig. S1A) and obtained TIN2S (1–354 amino acids, 39.4 kDa) and TIN2L (1–451 amino acids, 50.0 kDa) proteins purified from insect cells (Fig. 1A and Fig. S1D). Previously, we established an AFM imaging-based calibration method to investigate the oligomeric states and protein–protein interactions by correlating AFM volumes of proteins and their molecular weights (45Yang Y. Wang H. Erie D.A. Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy.Methods. 2003; 29: 175-187Crossref PubMed Scopus (88) Google Scholar, 47Kaur P. Wu D. Lin J. Countryman P. Bradford K.C. Erie D.A. Riehn R. Opresko P.L. Wang H. Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2.Sci. Rep. 2016; 6: 20513Crossref PubMed Scopus (20) Google Scholar, 52Wang H. Yang Y. Erie D.A. Characterization of protein-protein interactions using atomic force microscopy.in: Schuck P. Protein Interactions Biophysical approaches for the Study of Complex Reversible Systems. Springer Science+Business Media, LLC, Berlin, Germany2007: 39-78Crossref Google Scholar). AFM volumes of TRF1 alone in solution showed two distinct peaks, which were consistent with TRF1 monomers (51 KDa) and dimers (102 KDa, Fig. S1B). In addition, based on the population of TRF1 under the monomer and dimer peaks (53Wang H. DellaVecchia M.J. Skorvaga M. Croteau D.L. Erie D.A. Van Houten B. UvrB domain 4, an autoinhibitory gate for regulation of DNA binding and ATPase activity.J. Biol. Chem. 2006; 281: 15227-15237Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), the estimated TRF1 dimer equilibrium dissociation constant (Kd) is 18.4 nM (Fig. S1C). Meanwhile, AFM volumes of purified TIN2S at 41.3 nm3 (±28.3 nm3) and TIN2L at 41.9 nm3 (±12.8 nm3) were consistent with the notion that TIN2 does not interact with itself (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar), and TIN2 exists in a monomeric state in solution (Fig. S1D). Furthermore, we conducted size-exclusive chromatography using TRF1 and TIN2S and confirmed the presence of TRF1 dimers, TIN2 monomers, as well as the interaction between TRF1 and TIN2S in solution (Fig. S2). To further validate the activities of TIN2, we used electrophoresis mobility shift assays (EMSAs) to verify the interaction of TIN2 with TRF1 on a double-stranded telomeric DNA substrate (48 bp containing three TTAGGG repeats, Fig. S3, A–C). Consistent with previous studies (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar), EMSA experiments showed that TIN2S and TIN2L did not directly bind to telomeric dsDNA (Fig. S3A). Both TRF1-TIN2S and TRF1-TIN2L induced a clear supershift of the telomeric DNA substrate compared with TRF1 alone (Complex III in Fig. S3, B and C), indicating the formation of stable TRF1-TIN2-telomeric DNA complexes. Next, to study TRF1-TIN2 DNA binding at the single-molecule level, we used the linear DNA substrate (5.4 kb) that contains 1.6 kb (270 TTAGGG) telomeric repeats in the middle region that is 35%–50% from DNA ends (T270 DNA, Experimental procedures, Fig. 1A) (21Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. Gibson G. You C. Watkins S.C. Piehler J. Opresko P.L. Kad N.M. Wang H. TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (44) Google Scholar, 49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (18) Google Scholar). Previously, AFM and electron microscopy imaging–based studies established that TRF1 specifically binds to the telomeric region and mediates DNA-DNA pairing (21Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. Gibson G. You C. Watkins S.C. Piehler J. Opresko P.L. Kad N.M. Wang H. TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (44) Google Scholar, 22Bianchi A. Stansel R.M. Fairall L. Griffith J.D. Rhodes D. de Lange T. TRF1 binds a bipartite telomeric site with extreme spatial flexibility.EMBO J. 1999; 18: 5735-5744Crossref PubMed Scopus (163) Google Scholar, 49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (18) Google Scholar). To study the function of TIN2, we preincubated TRF1 without or with TIN2 (either TIN2S or TIN2L), followed by the addition}, number={3}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Pan, Hai and Kaur, Parminder and Barnes, Ryan and Detwiler, Ariana C. and Sanford, Samantha Lynn and Liu, Ming and Xu, Pengning and Mahn, Chelsea and Tang, Qingyu and Hao, Pengyu and et al.}, year={2021}, month={Sep} }
 @article{trihemasava_chakraborty_blackburn_xu_2020, title={Expression, purification, and phylogenetic analysis of MDIS1-INTERACTING RECEPTOR-LIKE KINASE1 (MIK1)}, volume={39}, ISSN={["1573-4943"]}, DOI={10.1007/s10930-020-09926-9}, abstractNote={An abundance of protein structures has been solved in the last six decades that are paramount in defining the function of such proteins. For unsolved protein structures, however, predictions based on sequence and phylogenetic similarity can be useful for identifying key domains of interaction. Here, we describe expression and purification of a recombinant plant LRR-RLK ectodomain MIK1 using a modified baculovirus-mediated expression system with subsequent N-linked glycosylation analysis using LC-MS/MS and computational sequence-based analyses. Though highly ubiquitous, glycosylation site specificity and the degree of glycosylation influenced by genetic and exogenous factors are still largely unknown. Our experimental analysis of N-glycans on MIK1 identified clusters of glycosylation that may explicate the regions involved in MIK1 ectodomain binding. Whether these glycans are necessary for function is yet to be determined. Phylogenetic comparison using multiple sequence alignment between MIK1 and other LRR-RLKs, namely TDR in Arabidopsis thaliana, revealed conserved structural motifs that are known to play functional roles in ligand and receptor binding.}, number={5}, journal={PROTEIN JOURNAL}, author={Trihemasava, Krittin and Chakraborty, Sayan and Blackburn, Kevin and Xu, Guozhou}, year={2020}, month={Oct}, pages={461–471} }
 @misc{chakraborty_nguyen_wasti_xu_2019, title={Plant Leucine-Rich Repeat Receptor Kinase (LRR-RK): Structure, Ligand Perception, and Activation Mechanism}, volume={24}, ISSN={["1420-3049"]}, DOI={10.3390/molecules24173081}, abstractNote={In recent years, secreted peptides have been recognized as essential mediators of intercellular communication which governs plant growth, development, environmental interactions, and other mediated biological responses, such as stem cell homeostasis, cell proliferation, wound healing, hormone sensation, immune defense, and symbiosis, among others. Many of the known secreted peptide ligand receptors belong to the leucine-rich repeat receptor kinase (LRR-RK) family of membrane integral receptors, which contain more than 200 members within Arabidopsis making it the largest family of plant receptor kinases (RKs). Genetic and biochemical studies have provided valuable data regarding peptide ligands and LRR-RKs, however, visualization of ligand/LRR-RK complex structures at the atomic level is vital to understand the functions of LRR-RKs and their mediated biological processes. The structures of many plant LRR-RK receptors in complex with corresponding ligands have been solved by X-ray crystallography, revealing new mechanisms of ligand-induced receptor kinase activation. In this review, we briefly elaborate the peptide ligands, and aim to detail the structures and mechanisms of LRR-RK activation as induced by secreted peptide ligands within plants.}, number={17}, journal={MOLECULES}, author={Chakraborty, Sayan and Nguyen, Brian and Wasti, Syed Danyal and Xu, Guozhou}, year={2019}, month={Sep} }
 @article{tang_chakraborty_xu_2018, title={Mechanism of vaccinia viral protein B14–mediated inhibition of IκB kinase β activation}, volume={293}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/JBC.RA118.002817}, DOI={10.1074/JBC.RA118.002817}, abstractNote={Activation of IκB kinase β (IKKβ) is a central event in the NF-κB–mediated canonical pro-inflammatory pathway. Numerous studies have reported that oligomerization-mediated trans autophosphorylation of IKKβ is indispensable for its phosphorylation, leading to its activation and IKKβ-mediated phosphorylation of substrates such as IκB proteins. Moreover, IKKβ's interaction with the NF-κB essential modifier (NEMO) is necessary for IKKβ activation. Interestingly, some viruses encode virulence factors that target IKKβ to inhibit NF-κB–mediated antiviral immune responses. One of these factors is the vaccinia viral protein B14, which directly interacts with and inhibits IKKβ. Here we mapped the interaction interface on the B14 and IKKβ proteins. We observed that B14 binds to the junction of the kinase domain (KD) and scaffold and dimerization domain (SDD) of IKKβ. Molecular docking analyses identified key interface residues in both IKKβ and B14 that were further confirmed by mutational studies to promote binding of the two proteins. During trans autophosphorylation of protein kinases in the IKK complex, the activation segments of neighboring kinases need to transiently interact with each other's active sites, and we found that the B14–IKKβ interaction sterically hinders direct contact between the kinase domains of IKKβ in the IKK complex, containing IKKβ, IKKα, and NEMO in human cells. We conclude that binding of B14 to IKKβ prevents IKKβ trans autophosphorylation and activation, thereby inhibiting NF-κB signaling. Our study provides critical structural and mechanistic information for the design of potential therapeutic agents to target IKKβ activation for the management of inflammatory disorders.}, number={26}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Tang, Qingyu and Chakraborty, Sayan and Xu, Guozhou}, year={2018}, month={May}, pages={10344–10352} }
 @article{chakraborty_trihemasava_xu_2018, title={Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells}, ISSN={["1940-087X"]}, DOI={10.3791/58283}, abstractNote={It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.}, number={138}, journal={JOVE-JOURNAL OF VISUALIZED EXPERIMENTS}, author={Chakraborty, Sayan and Trihemasava, Krittin and Xu, Guozhou}, year={2018}, month={Aug} }
 @article{chakraborty_pan_tang_woolard_xu_2018, title={The Extracellular Domain of Pollen Receptor Kinase 3 is structurally similar to the SERK family of co-receptors}, volume={8}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-018-21218-y}, abstractNote={Abstract}, journal={SCIENTIFIC REPORTS}, author={Chakraborty, Sayan and Pan, Haiyun and Tang, Qingyu and Woolard, Colin and Xu, Guozhou}, year={2018}, month={Feb} }
 @article{li_chakraborty_xu_2017, title={Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions}, volume={12}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0175317}, abstractNote={Tracheary Element Differentiation Inhibitory Factor (TDIF) belongs to the family of post-translationally modified CLE (CLAVATA3/embryo surrounding region (ESR)-related) peptide hormones that control root growth and define the delicate balance between stem cell proliferation and differentiation in SAM (shoot apical meristem) or RAM (root apical meristem). In Arabidopsis, Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and its ligand TDIF signaling pathway is involved in the regulation of procambial cell proliferation and inhibiting its differentiation into xylem cells. Here we present the crystal structures of the extracellular domains (ECD) of TDR alone and in complex with its ligand TDIF resolved at 2.65 Ǻ and 2.75 Ǻ respectively. These structures provide insights about the ligand perception and specific interactions between the CLE peptides and their cognate receptors. Our in vitro biochemical studies indicate that the interactions between the ligands and the receptors at the C-terminal anchoring site provide conserved binding. While the binding interactions occurring at the N-terminal anchoring site dictate differential binding specificities between different ligands and receptors. Our studies will open different unknown avenues of TDR-TDIF signaling pathways that will enhance our knowledge in this field highlighting the receptor ligand interaction, receptor activation, signaling network, modes of action and will serve as a structure function relationship model between the ligand and the receptor for various similar leucine-rich repeat receptor-like kinases (LRR-RLKs).}, number={4}, journal={PLOS ONE}, author={Li, Zhijie and Chakraborty, Sayan and Xu, Guozhou}, year={2017}, month={Apr} }
 @article{hauenstein_xu_kabaleeswaran_wu_2017, title={Evidence for M1-Linked Polyubiquitin-Mediated Conformational Change in NEMO}, volume={429}, ISSN={["1089-8638"]}, DOI={10.1016/j.jmb.2017.10.026}, abstractNote={The NF-κB essential modulator (NEMO) is the scaffolding subunit of the inhibitor of κB kinase (IKK) holocomplex and is required for the activation of the catalytic IKK subunits, IKKα and IKKβ, during the canonical inflammatory response. Although structures of shorter constructs of NEMO have been solved, efforts to elucidate the full-length structure of NEMO have proved difficult due to its apparent high conformational plasticity. To better characterize the gross dimensions of full-length NEMO, we employed in-line size exclusion chromatography-small-angle X-ray scattering. We show that NEMO adopts a more compact conformation (Dmax=320Å) than predicted for a fully extended coiled-coil structure (>500Å). In addition, we map a region of NEMO (residues 112-150) in its coiled-coil 1 domain that impedes the binding of linear (M1-linked) di-ubiquitin to its coiled-coil 2-leucine zipper ubiquitin binding domain. This ubiquitin binding inhibition can be overcome by a longer chain of linear, but not K63-linked polyubiquitin. Collectively, these observations suggest that NEMO may be auto-inhibited in the resting state by intramolecular interactions and that during signaling, NEMO may be allosterically activated by binding to long M1-linked polyubiquitin chains.}, number={24}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Hauenstein, Arthur V. and Xu, Guozhou and Kabaleeswaran, Venkataraman and Wu, Hao}, year={2017}, month={Dec}, pages={3793–3800} }
 @article{li_chakraborty_xu_2016, title={X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa}, volume={72}, ISSN={["2053-230X"]}, DOI={10.1107/s2053230x16014278}, abstractNote={Does not respond to nucleotides 1 (DORN1) has recently been identified as the first membrane-integral plant ATP receptor, which is required for ATP-induced calcium response, mitogen-activated protein kinase activation and defense responses inArabidopsis thaliana. In order to understand DORN1-mediated ATP sensing and signal transduction, crystallization and preliminary X-ray studies were conducted on the extracellular domain of DORN1 (atDORN1-ECD) and that of an orthologous protein,Camelina sativalectin receptor kinase I.9 (csLecRK-I.9-ECD or csI.9-ECD). A variety of deglycosylation strategies were employed to optimize the glycosylated recombinant atDORN1-ECD for crystallization. In addition, the glycosylated csI.9-ECD protein was crystallized at 291 K. X-ray diffraction data were collected at 4.6 Å resolution from a single crystal. The crystal belonged to space groupC222 orC2221, with unit-cell parametersa= 94.7,b= 191.5,c= 302.8 Å. These preliminary studies have laid the foundation for structural determination of the DORN1 and I.9 receptor proteins, which will lead to a better understanding of the perception and function of extracellular ATP in plants.}, journal={ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS}, author={Li, Zhijie and Chakraborty, Sayan and Xu, Guozhou}, year={2016}, month={Oct}, pages={782–787} }