Ranjith Muhandiram

SickKids, Toronto, Ontario, Canada

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Publications (11)39.08 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Intrinsically disordered proteins play crucial roles in regulatory processes and often function as protein interaction hubs. Here, we present a detailed characterization of a full-length disordered hub protein region involved in multiple dynamic complexes. We performed NMR, CD, and fluorescence binding studies on the nonphosphorylated and highly PKA-phosphorylated human cystic fibrosis transmembrane conductance regulator (CFTR) regulatory region, a ∼200-residue disordered segment involved in phosphorylation-dependent regulation of channel trafficking and gating. Our data provide evidence for dynamic, phosphorylation-dependent, multisite interactions of various segments of the regulatory region for its intra- and intermolecular partners, including the CFTR nucleotide binding domains 1 and 2, a 42-residue peptide from the C terminus of CFTR, the SLC26A3 sulphate transporter and antisigma factor antagonist (STAS) domain, and 14-3-3β. Because of its large number of binding partners, multivalent binding of individually weak sites facilitates rapid exchange between free and bound states to allow the regulatory region to engage with different partners and generate a graded or rheostat-like response to phosphorylation. Our results enrich the understanding of how disordered binding segments interact with multiple targets. We present structural models consistent with our data that illustrate this dynamic aspect of phospho-regulation of CFTR by the disordered regulatory region.
    Proceedings of the National Academy of Sciences 11/2013; · 9.74 Impact Factor
  • Ranjith Muhandiram, Lewis E. Kay
    03/2007; , ISBN: 9780470034590
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    ABSTRACT: The N terminus of the c-Myc oncoprotein interacts with Bin1, a ubiquitously expressed nucleocytoplasmic protein with features of a tumor suppressor. The c-Myc/Bin1 interaction is dependent on the highly conserved Myc Box 1 (MB1) sequence of c-Myc. The c-Myc/Bin1 interaction has potential regulatory significance as c-Myc-mediated transformation and apoptosis can be modulated by the expression of Bin1. Multiple splicing of the Bin1 transcript results in ubiquitous, tissue-specific and tumor-specific populations of Bin1 proteins in vivo. We report on the structural features of the interaction between c-Myc and Bin1, and describe two mechanisms by which the binding of different Bin1 isoforms to c-Myc may be regulated in cells. Our findings identify a consensus class II SH3-binding motif in c-Myc and the C-terminal SH3 domain of Bin1 as the primary structure determinants of their interaction. We present biochemical and structural evidence that tumor-specific isoforms of Bin1 are precluded from interaction with c-Myc through an intramolecular polyproline-SH3 domain interaction that inhibits the Bin1 SH3 domain from binding to c-Myc. Furthermore, c-Myc/Bin1 interaction can be inhibited by phosphorylation of c-Myc at Ser62, a functionally important residue found within the c-Myc SH3-binding motif. Our data provide a structure-based model of the c-Myc/Bin1 interaction and suggest a mode of regulation that may be important for c-Myc function as a regulator of gene transcription.
    Journal of Molecular Biology 09/2005; 351(1):182-94. · 3.91 Impact Factor
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    ABSTRACT: The signal transduction protein phospholipase C-gamma1 (PLC-gamma1) is activated when its C-terminal SH2 domain (PLCC) binds the phosphorylated Tyr-1021 site (pTyr-1021) in the beta-platelet-derived growth factor receptor (PDGFR). To better understand the contributions that dynamics make to binding, we have used NMR relaxation experiments to investigate the motional properties of backbone amide and side chain methyl groups in a peptide derived from the pTyr-1021 site of PDGFR, both free and in complex with the PLCC SH2 domain. The free peptide has relaxation properties that are typical for a small, unstructured polymer, while the backbone of the bound peptide is least flexible for residues in the central portion of the binding site with the amplitude of pico- to nanosecond time scale motions increasing toward the C-terminus of the peptide. The increase in large amplitude motion toward the end of the pY1021 peptide is consistent with the bound peptide existing as an ensemble of states with C-terminal residues having the broadest distribution of backbone conformations, while residues in the central binding site are the most restricted. Deuterium spin relaxation experiments establish that the protein-peptide interface is highly dynamic, and this mobility may play an important role in modulating the affinity of the interaction.
    Biochemistry 02/2005; 44(2):694-703. · 3.38 Impact Factor
  • Patrick J Finerty, Ranjith Muhandiram, Julie D Forman-Kay
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    ABSTRACT: X-linked lymphoproliferative disease is caused by mutations in the protein SAP, which consists almost entirely of a single SH2 domain. SAP interacts with the Tyr281 site of the T<-->B cell signaling protein SLAM via its SH2 domain. Interestingly, binding is not dependent on phosphorylation but does involve interactions with residues N-terminal to the Tyr. We have used 15N and 2H NMR relaxation experiments to investigate the motional properties of the SAP SH2 domain backbone amides and side-chain methyl groups in the free protein and complexes with phosphorylated and non-phosphorylated peptides derived from the Tyr281 site of SLAM. The most mobile methyl groups are in side-chains with large RMSD values between the three crystal structures of SAP, suggesting that fast time-scale dynamics in side-chains is associated with conformational plasticity. The backbone amides of two residues which interact with the C-terminal part of the peptides experience fast time-scale motions in the free SH2 domain that are quenched upon binding of either the phosphorylated or non-phosphorylated peptide. Of most importance, the mobility of methyl groups in and around the binding site for residues in the N-terminus of the peptide is significantly restricted in the complexes, underscoring the dominance of this interaction with SAP and demonstrating a correlation between changes in rapid side-chain motion upon binding with local binding energy.
    Journal of Molecular Biology 09/2002; 322(3):605-20. · 3.91 Impact Factor
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    Vitali Tugarinov, Ranjith Muhandiram, Ayeda Ayed, Lewis E Kay
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    ABSTRACT: A four-dimensional (4-D) NMR study of Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa), is described. Virtually complete backbone (1)HN, (15)N, (13)C, and (13)C(beta) chemical shift assignments of this largely alpha-helical protein are reported. The assignment strategy follows from our previously described approach based on TROSY triple resonance 4-D NMR spectroscopy [Yang, D.; Kay, L. E. J. Am. Chem. Soc. 1999, 121, 2571-2575. Konrat, R; Yang, D; Kay, L. E. J. Biomol. NMR 1999, 15, 309-313] with a number of modifications necessitated by the large size of the protein. A protocol for refolding deuterated MSG in vitro was developed to protonate the amides deeply buried in the protein core. Of interest, during the course of the assignment, an isoaspartyl linkage in the protein sequence was unambiguously identified. Chemical shift assignments of this system are a first step in the study of how the domains of the protein change in response to ligand binding and for characterizing the dynamical properties of the enzyme that are likely important for function.
    Journal of the American Chemical Society 09/2002; 124(34):10025-35. · 10.68 Impact Factor
  • Toshio Yamazaki, Ranjith Muhandiram, Lewis E. Kay
    04/2002;
  • Vitali Tugarinov, Ranjith Muhandiram, Ayeda Ayed, Lewis E. Kay
    Journal of The American Chemical Society - J AM CHEM SOC. 01/2002; 124(34):10025-10035.
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    ABSTRACT: Dietary tannins are polyphenols that are effectively precipitated by salivary histatins (Hsts), a novel family of tannin binding proteins. Epigallocatechin gallate (EGCG), a flavan-3-ol ester related to condensed tannins (polymerized products of flavan-3-ols), and pentagalloyl glucose (PGG), a hydrolyzable tannin, were used to evaluate the molecular nature of Hst-polyphenol interaction. NMR demonstrated that Hst5, a representative Hst, bound to EGCG in a hydrophobic manner via basic and aromatic residues. In contrast, proline plays a dominant role in polyphenol binding to other tannin precipitating proteins. The role of basic and aromatic amino acids in EGCG binding was investigated using a series of modified Hsts in each of which one type of amino acid was substituted by Ala. EGCG bound to all modified Hsts, but the binding was diminished. Optimal EGCG binding also depended on the primary structure, as a polypeptide with randomised Hst5 sequence showed significantly diminished interaction with EGCG. Soluble EGCG/Hst5 complexes containing up to seven molecules of EGCG per mol of Hst5 had a 1-mM dissociation constant. In contrast to EGCG, PGG formed small soluble complexes with Hst5 consisting of only one molecule each of PGG and Hst5, as demonstrated by analytical ultracentrifugation. These complexes became insoluble upon binding of additional molecules of PGG. Diminished PGG binding was seen to a peptide with a Hst5 randomized sequence showing the importance of the primary structure. Hsts may serve to form insoluble complexes with tannins thereby preventing their absorption from the intestines and potentially harmful biological effects. In contrast the much weaker interaction with EGCG may allow its uptake into the organism and exploitation of its antioxidant effect.
    European Journal of Biochemistry 09/2001; 268(16):4384-97. · 3.58 Impact Factor
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    ABSTRACT: †Dietary tannins are polyphenols that are effectively precipitated by salivary histatins (Hsts), a novel family of tannin binding proteins. Epigallocatechin gallate (EGCG), a flavan-3-ol ester related to condensed tannins (polymerized products of flavan-3-ols), and pentagalloyl glucose (PGG), a hydrolyzable tannin, were used to evaluate the molecular nature of Hst–polyphenol interaction. NMR demonstrated that Hst5, a representative Hst, bound to EGCG in a hydrophobic manner via basic and aromatic residues. In contrast, proline plays a dominant role in polyphenol binding to other tannin precipitating proteins. The role of basic and aromatic amino acids in EGCG binding was investigated using a series of modified Hsts in each of which one type of amino acid was substituted by Ala. EGCG bound to all modified Hsts, but the binding was diminished. Optimal EGCG binding also depended on the primary structure, as a polypeptide with randomised Hst5 sequence showed significantly diminished interaction with EGCG. Soluble EGCG/Hst5 complexes containing up to seven molecules of EGCG per mol of Hst5 had a 1-mm dissociation constant. In contrast to EGCG, PGG formed small soluble complexes with Hst5 consisting of only one molecule each of PGG and Hst5, as demonstrated by analytical ultracentrifugation. These complexes became insoluble upon binding of additional molecules of PGG. Diminished PGG binding was seen to a peptide with a Hst5 randomized sequence showing the importance of the primary structure. Hsts may serve to form insoluble complexes with tannins thereby preventing their absorption from the intestines and potentially harmful biological effects. In contrast the much weaker interaction with EGCG may allow its uptake into the organism and exploitation of its antioxidant effect.
    European Journal of Biochemistry. 08/2001; 268(16):4384 - 4397.
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    ABSTRACT: Solution NMR studies on the physiologically relevant ligand-free and maltotriose-bound states of maltodextrin-binding protein (MBP) are presented. Together with existing data on MBP in complex with beta-cyclodextrin (non-physiological, inactive ligand), these new results provide valuable information on changes in local structure, dynamics and global fold that occur upon ligand binding to this two-domain protein. By measuring a large number of different one-bond residual dipolar couplings, the domain conformations, critical for biological function, were investigated for all three states of MBP. Structural models of the solution conformation of MBP in a number of different forms were generated from the experimental dipolar coupling data and X-ray crystal structures using a quasi-rigid-body domain orientation algorithm implemented in the structure calculation program CNS. Excellent agreement between relative domain orientations in ligand-free and maltotriose-bound solution conformations and the corresponding crystal structures is observed. These results are in contrast to those obtained for the MBP/beta-cyclodextrin complex where the solution state is found to be approximately 10 degrees more closed than the crystalline state. The present study highlights the utility of residual dipolar couplings for orienting protein domains or macromolecules with respect to each other.
    Journal of Molecular Biology 07/2001; 309(4):961-74. · 3.91 Impact Factor