Characterization of the Ca Binding Affinity and Coordination Site of the LIN-12/Notch-Repeat 2+ Christina Hao, Didem Vardar-Ulu Wellesley College Chemistry Department Wellesley, Massachusetts Introduction Results • Notch receptors are transmembrane glycoproteins that regulate cell fate in multicellular organisms via a highly conserved signaling pathway (Figure A). Ca2+ dependency during folding of hN1 LNRA, glucose transferase and hN4LNRA Representations of overall structures and calcium binding sites for (A) hN1LNRA (B) Glucose Transferase LNRA (C) hN4LNRA (A)NMR Structure. (B) and (C) homology Modeling • Deregulation of notch signaling pathway in all four identified notch homologues (Notch1 – Notch4) has been implicated in numerous disease phenotypes. A. hN1 LNRA • Three conserved Lin12/Notch Repeat (LNRA, LNRB, and LNRC) modules are located in tandem in the extracellular region of the notch receptors. They maintain the receptor in a resting conformation prior to ligand-induced activation. (Figure B). B. Glucose Transferase LNRA C. hN4 LNRA • Each LNR module in the Notch receptor consists of three characteristic disulfide bonds and a Ca 2+ ion, essential for structural integrity (Figure C). Domain Organization of the Notch Receptors and the Notch Signaling Pathway Crystal Structure of LNR and HD Domain of human Notch21 Figure B NMR Structure of hN1LNRA2 Figure C C 4 N15 C9 C27 Figure A Nterm Key: C22 D30 • Calcium binding sites (7 angstroms from the Ca2+): red and green ribbons Red sticks: Aspartates - Green sticks: other residues besides aspartates - Blue sphere: calcium ion Ca2+ C34 • Non-binding sites: silver ribbons D33 S19 C18 C-term Representative ITC data on the calorimetric titrations of hN1LNRA with Ca2+,Zn2+ ,Tb3+ Distances and distribution of coordinating residues from Ca 2+ Objectives • Quantify calcium binding affinity and specificity of LNR homologues across different proteins using ITC • Determine calcium dependency of different LNR homologues for autonomous folding • Understand the molecular basis of calcium binding in LNR using computer modeling Tb Zn2 Ca 3+ + 2+ Key: Red residues: residues that coordinate calcium with both side chain and backbone oxygen moiety Sequence Alignment of LNR homologues investigated in this study: Distances ranges highlighted yellow: aspartate is present in this distance range Conclusions • HN1 LNRA exclusively binds to Ca2+ in an exothermic reaction with a dissociation constant of 22.05 +/3.27 µM and a stoichiometry of 1:1 at pH 7.0. Representative ITC data on the calorimetric titrations of glucose transferase and hN4LNRA with Ca2+ Material and Methods Glucose transferase LNRA hN1 LNR A hN4 LNR A Protein Acquisition: • Human N1LNRA recombinantly expressed in Escherichia coli. Human Notch 4 LNRA and glucose transferase LNRA were synthesized by EZ Biolabs. • All proteins were folded and purified as follows: • Folding: 6-8 hours dialysis against refolding buffer: 2.5mM cysteine, 0.5mM cystine, 100mM NaCl, 200mM sucrose, 10mM CaCl2 and 20mM Tris pH 8. • Purification: Elution through reverse phase high pressure liquid chromatography (RPHPLC) using 0.1% formic acid in acetonitrile based buffer systems. Peaks corresponding to the folded species were collected and lyophilized. Folding Experiments: • Denatured proteins were refolded in redox solution containing 5:1 cysteine/cystine ratio, 100mM NaCl and 20mM Tris at pH 8 and with/without 10mM CaCl2. Proteins were folded under partial nitrogen atmosphere for six hours and promptly analyzed on RP-HPLC. Isothermal titration calorimetry (ITC) Experiments: • Lyophilized protein of appropriate concentration was demetalized with sigma chelex beads and suspended in 35mM Hepes pH7, 100mM NaCl buffer . • Stock metal solution of 0.2 – 1mM CaCl2 was used • Isothermal titration calorimetry experiments (ITC), were carried out using a high-precision VP-ITC titration calorimetry instrument (Microcal Inc., Northampton, MA) where the metal solution was titrated in 5µL increments into the protein solution at 20°C. Computer Modeling Software used: • Clustal W: sequence alignment • Modeller: homology modeling • Pymol: visualization • Glucose transferase LNRA display strong binding to calcium in a non-stoichiometric manner • HN4 LNRA does not bind to calcium • Calcium is necessary for the folding of HN1 LNRA and glucose transferase LNRA but not for HN4 LNRA • Homology modeling suggests differences in distribution of aspartic acids lead to distinct calcium binding behaviors of the LNR repeats. Future Directions • Determine precise roles of disulphide bonds and aspartic acids in calcium binding affinity mutational studies. through • Correlate calcium binding affinity and specificity with structural stability to gain insight into the biological significance of calcium binding by the LNRs in vivo. • Design of calcium binding peptides through de novo experiments based on understanding of the LNRs Summary of thermodynamic parameters associated with the binding of Ca2+ to Zn2+ and Tb3+ presaturated hN1LNRA Constructs hN1LNRA Metal Calcium N 0.9600.005 Kd (µM) 22.053.27 Zinc Calcium hN4LNRA Calcium -9.140.25 No binding Terbium GlucTran H (kcal/mol) Undefined mode of binding 0.07 9 -4.64 E4  1.36E4 No binding References 1) Gordon, W. R.;* Vardar-Ulu, D.;* Histen, G.; Sanchez-Irizarry, C.; Aster, J. C.; Blacklow, S. C. “Structural basis for autoinhibition of Notch” Nat Struct Mol Biol. 2007, 14, 295–300.2. 2) Vardar, D.; North, C. L.; Sanchez-Irizarry, C.; Aster, J. C.; Blacklow, S. C. “NMR Structure of a Prototype LNR Module from Human Notch1” Biochemistry 2003, 42, 7061–7067. 3) N. Eswar, M.A. Marti-Renom, b. Webb, m.S. Madhusudhan, D. Eramian, M. Shen, U. Pieper, A. Sali, Comparative Protein Structure Modeling with MODELLER. Current Protocols in Bioinformatics, John Wilery & Sons, Inc., Supplement 15, 5.6. 1-5.6.30, 2000 4) Cheng G, Baker D and Samudrala R. A Novel Small Molecule Crystal Structure Derived Potential Function To Predict Protein Metal Ion Binding Site, Affinity and Specificity From Structure. xxxx.YYYY,aa-bb,2007 http://protinfo.compbio.washington.edu/soak/
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What is Notch?  Transmembrane protein receptors of 300-350kDa  Highly conserved  Regulates cell growth, differentiation, and cell death in a vast array of tissues through Notch signaling pathway  Deregulation of Notch signaling pathway is associated wi th diseases, eg. Cancer  Four mammalian Notch homologs identified (Notch 1-4)
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Figure 7.29 Radius and ulna of the right forearm. Olecranon Head Neck Coronoid process Radial notch of the ulna Head Neck Radial tuberosity Radial tuberosity Olecranon Trochlear notch Coronoid process Proximal radioulnar joint Head of radius Neck of radius Interosseous membrane Ulna Ulna Radius Radius Ulnar notch of the radius Radius Head of ulna Ulnar styloid process Radial styloid process Photo, anterior view © 2016 Pearson Education, Inc. Radial styloid process Ulnar styloid process Distal radioulnar joint Illustration, anterior view Radial styloid process r Illustration, posterior view
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Figure 7.29 Radius and ulna of the right forearm (continued). Ulnar notch of radius Olecranon View Articulation for lunate Trochlear notch Articulation for scaphoid Coronoid process Radial styloid process Radial notch View Proximal portion of ulna, lateral view © 2016 Pearson Education, Inc. Head of ulna Ulnar styloid process Distal ends of the radius and ulna at the wrist
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Radial notch of the ulna Head Neck Radial tuberosity Olecranon process Trochlear notch Coronoid process Proximal radioulnar joint Interosseous membrane Ulna Head of radius Neck of radius Radius Ulnar notch Radius of the radius Head of ulna Styloid Styloid process of ulna process Distal radioulnar Styloid process of radius joint of radius (a) Anterior view (b) Posterior view Copyright © 2010 Pearson Education, Inc. Figure 7.27a-b
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Olecranon process Trochlear notch View Coronoid process Radial notch (c) Proximal portion of ulna, lateral view Ulnar notch of radius Articulation for lunate Articulation for scaphoid Styloid process View Copyright © 2010 Pearson Education, Inc. Styloid Head of ulna process (d) Distal ends of the radius and ulna at the wrist Figure 7.27c-d
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Angiogenic Sprouting -Tip cells induce expression of Notch (DLL-4) and its release -Notch binds to its receptor on neighboring cells -Notch Receptor Intracellular domain (NICD) is released and is transported to the nucleus to repress VEGFR-2 and turns on VEGFR-1
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NOTCH PROTEINS • Transmembrane receptor protein • Found in many different animals – from worms to humans. – mammals have four Notch homologs: Notch1-4 • Function through highly conserved Notch signaling pathway – 3 cleavages at sites: S1, S2 & S3 • S1: Processed on way to cell surface • S2, S3: Upon ligand binding
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aling ell Notch Signaling Pathway Negative regulatory region (NRR) Ligand A B C HD-N ICN HD-C Ligand-binding Region Notch Activation LNR Domain I. Ligand binding II. Regulated cleavages HD Domain III. Release of intracellular notch/ Regulation of gene transcription S1 S2 S3 Nucleus
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References  Gordon, Vardar-Ulu, Histen, Sanchez-Iriarry, Aster, Blacklow (2007) Structur al basis for autoinhibition of Notch. Nature: Structural and molecular biology  Sjolund, Manetopoulos, Stockhausen, Axelson (2005). Review: The Notch pathway in cancer: Differentiation gone awry. European Journal of Cancer 4 1: 2620-2629 Sorensen, Mortensen (2004) Advanced genetic strategies for recombinant p rotein expression in Escherichia coli. Journal of Biotechnology (115) 2:113128 Vardar, North, Sanchez-Irizarry, Aster, Blacklow (2003) Nuclear Magnetic Re sonance Structure of a Prototype Lin12-Notch repeat Modules from Human Notch1. American Chemical Society (42)7061-7067 http://www.emdbiosciences.com/product/69661 http://wolfson.huji.ac.il/expression/Bacterial_Strains.htm#strains-exp    
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sternal notch clavicular notch sternal angle manubrium body intercostal space xiphoid process STERNUM
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trochlear notch coronoid process radial notch olecranon process ULNA head styloid process
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iliac crest Posterior superior iliac spine greater sciatic notch anterior superior iliac spine ischial spine lesser sciatic notch ischial tuberosity HIP HIP (COXAL) (COXAL) BONE BONE
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Cope Cuts When the tops of beams are to be flushed, a notch must be cut into the connecting beam to prevent interference with the flange of the other beam. Such a notch is called a cope, block, or cut. It is the practice of the structural detailer to draw the depth dimension to scale so that the relation of the detail is correct and so the fabricator can interpret the relation of the holes to bolts or other holes more readily. Q1 = k distance This is a minimum dimension. Q1 can never be smaller than the k-distance Q2 = ((bf - tw)/ 2) + 1/2 The Q2 dimension is measured from the back of the connection angle The Q2 dimension is rounded upward to the nearest 1/4"
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Why the Mandible? • In modern H. sapiens, the ascending mandibular ramus are in two processes, the coronoid and condylar process, and separated by a deep notch. • In Neanderthals, the coronoid appears larger and more elevated than the condylar process, with a shallow notch in between.
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Forearm (cont.) • Ulna – Medial bone in forearm – Forms major portion of elbow joint with humerus – Bone features • Olecranon and coronoid processes: grip trochlea of humerus, forming hinge joint – Processes separated by trochlear notch • Radial notch: articulates with head of radius • Ulnar head: knoblike distal portion • Ulnar styloid process: ligament attachment © 2016 Pearson Education, Inc.
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