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    William E Royer PhD

    TitleProfessor
    InstitutionUniversity of Massachusetts Medical School
    DepartmentBiochemistry and Molecular Pharmacology
    AddressUniversity of Massachusetts Medical School
    364 Plantation Street, LRB
    Worcester MA 01605
    Phone508-856-6912
      Other Positions
      InstitutionUMMS - Graduate School of Biomedical Sciences
      DepartmentBiochemistry and Molecular Pharmacology

      InstitutionUMMS - Graduate School of Biomedical Sciences
      DepartmentMD/PhD Program

      InstitutionUMMS - Graduate School of Biomedical Sciences
      DepartmentTranslational Science

        Overview 
        Narrative

        Academic Background

        William E. Royer, Jr. received his BS from Pennsylvania State University in 1976 and his PhD in biophysics from The Johns Hopkins University in 1984. He was a postdoctoral fellow in Dr. Wayne Hendrickson's laboratory at ColumbiaUniversity and became an associate of the Howard Hughes Medical Institute in 1986 and an associate research scientist at Columbia University in1988. He joined the faculty of the University of Massachusetts MedicalSchool in 1990. He was an established Investigator of the American Heart Association from 1994-1999.

         

        Photo: William E Royer

        Research Interests

        Our laboratory explores the structural basis by which intermolecular interactions regulate biological function in a number of systems.  One focus is how assembly of protein subunits regulates their function.  Two systems in this area of current interest are interferon regulatory factors, in which phosphorylation triggers dimeric assembly and nuclear translocation, and a dimeric hemoglobin, for which assembly results in substantial cooperative ligand binding.  A second focus is the structure-based development of inhibitors for a cancer target, CtBP.  These three research areas are described below.

        Structural regulation of interferon regulatory factors (IRFs) in the innate immune response

        IRF family members play important roles in innate immunity, inflammation and apoptosis.  Their clinical importance is evidenced by a strong link between IRF5 and autoimmune diseases, particularly lupus.  Our goal is to obtain a structural understanding of activation that will permit insight into approaches for inactivation that could inform development of future therapies in autoimmune disease.  The first step in IRF activation is triggered by phosphorylation of Ser/Thr residues in a C-terminal autoinhibitory region. Phosphylation stimulates dimerization, translocation into the nucleus and assembly with the coactivator CBP/p300 and other proteins to stimulate transcription of type I interferons and other target genes. In collaboration with Dr. Celia Schiffer (BMP) and Dr. Kate Fitzgerald (Medicine), we are continuing work on the structural basis for activation of IRFs that was pioneered by our extraordinary colleague, Dr. Kai Lin, prior his tragic death. Our crystal structure of dimeric pseudophosphorylated IRF-5, in comparison with structures of monomeric IRF-3 determined by Dr. Lin, has revealed how phosphorylation triggers a striking conformational rearrangement of the C-terminal region converting it from an autoinhibitory to a dimerization role. Activated dimers are then translocated into the nucleus, where they assemble with transcriptional coactivators to activate transcription.

        Figure 1 legend: The crystal structure of the IRF-5 dimer (PDB id code 3DSH) is shown as a ribbon diagram with one subunit in green and one in blue, except for the C-terminal autoinhibition/dimerization region, which is shown in purple for both subunits.  Likely phosphorylation sites are shown as small yellow spheres at their alpha-carbon positions.  Comparison of the IRF-5 structure with that of monomeric autoinhibited IRF-3 strongly suggests a common mechanism for activation of the interfereon regulatory factors in which phosphorylation triggers a dramatic structural transition of the C-terminal region, resulting in IRF dimerization and exposure of the CBP binding site.  These two effects are key steps leading to the transcriptional activation of type 1 interferons and other target genes (see Chen et al. (2008) NSMB 15, 1213-1220).

         

        Structure-based development of inhibitors against CtBP, a cancer target

        The paralogous transcription coregulators C-terminal Binding Proteins (CtBP) 1 and 2 are critical modulators of numerous cellular processes.  CtBP 1 and 2 have been shown to be overexpressed in many human cancers where they act to inhibit apoptosis and promote metastasis.  Unusual among transcription factors, CtBP harbors an essential D-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) catalytic domain that enables CtBP to be a redox sensor and provides an attractive target for small molecule inhibition. High (mM) concentrations of the CtBP substrate MTOB have been shown by our collaborator, Dr. Steven Grossman at Virginia Commonwealth University, to be able to inhibit tumor growth in cell culture and mouse studies providing proof of principle that CtBP could be an excellent cancer target.  By determining the crystal structures of CtBP 1 and 2 in complex with MTOB, we elucidated unique active site features and then used these to identify compounds with substantially higher affinity.  Among these features is a water filled hydrophilic cavity, shown below, that is not shared by other D2-HDH family members.  We are working to exploit such structural features to design potentially therapeutically useful inhibitors that bind specifically to CtBP with high affinity.

        Figure 2 legend: The active site hydrophilic cavity in CtBP1, with the protein cavity surface shown in gray.  Four crystallographically observed water molecules, W1-W4, link substrate MTOB (cyan carbon atoms) with an NAD+ (yellow) phosphate.  This cavity is not present in other members of the D2-HDH family.  Therefore, creating inhibitors with functional groups that bind in this cavity should both gain affinity and specificity for CtBP. (See Hilbert et al. (2014) FEBS Lett. 588, 1743-1748)

        Time-resolved crystallographic analysis of cooperative ligand binding

        Invertebrate hemoglobins provide a number of useful model systems for exploring how protein subunits communicate.  We have investigated systems ranging from the simplest possible allosteric systems to much more complex protein assemblages.  As part of our efforts, we determined crystal structures of respiratory proteins with molecular masses of 3.6 x 106 Da comprising 180 different subunits.  (Our crystal structure of Lumbricus erythrocruorin was highlighted as the March, 2013 molecule of the month at the protein data bank – see http://www.rcsb.org/pdb/101/motm.do?momID=159.) Our current efforts are focused on applying sub-ns time-resolved crystallographic analysis to a dimeric hemoglobin (Scapharca HbI) to dissect the time-dependent ligand-linked structural changes that underlie cooperative ligand binding.  These experiments are being carried out at BioCARS (Advanced Photon Source) in collaboration with Dr. Vukica Srajer and Dr. Zhong Ren.  They have already led to a new understanding of the role of water molecules in intersubunit communication as well as permitting the development of a novel dynamic model that incorporates a direct linkage between two active sites.  In collaboration with Dr. Francesca Massi (BMP), we are combining the crystallographic results with NMR experiments that investigate the role of interface dynamics in cooperative behavior.

         



        Rotation Projects

        Potential Rotation Projects

        Project #1: Probing the structural basis for cooperativity in a dimeric hemoglobin: Scapharca dimeric hemoglobin is an elegantly simple model system for exploring the structural basis for intersubunit communication. Our analysis to date has established a new paradigm for cooperativity in which tightly bound water molecules are used as sensors for ligand state.

        In this project, the student will first mutate the gene for the native hemoglobin at a residue that is suspected of playing a role in the intersubunit communication. The mutated protein will then be expressed in E. coli and purified and subjected to functional analysis of oxygen binding. Using conditions already established for the native protein, the mutant hemoglobin will then be crystallized and subjected to preliminary x-ray analysis.

        This project will provide the student with an introduction to two of the most powerful techniques for investigating the structure and function of proteins: site-directed mutagenesis and x-ray crystallography. Additionally, valuable experience in protein purification will be obtained.

        Project #2: Structural analysis of the polymerization of sickle-cell hemoglobin Sickle cell disease results from the pathological polymerization of deoxygenated hemoglobin S (ß6E->V) within erythrocytes. This polymerization depends upon a complicated interplay of multiple interactions between tetramers. Our high resolution crystal structure of this molecule has elucidated details of some of the important interactions. Several projects are available for rotation students. These include crystallization of sickle-cell hemoglobin in the presence of an inhibitor to identify its binding site, site-directed mutagenesis of hemoglobin S and crystallization of mutants that show altered polymerization characteristics.



        Bibliographic 
        selected publications
        List All   |   Timeline
        1. Hilbert BJ, Grossman SR, Schiffer CA, Royer WE. Crystal structures of human CtBP in complex with substrate MTOB reveal active site features useful for inhibitor design. FEBS Lett. 2014 May 2; 588(9):1743-8.
          View in: PubMed
        2. Ren Z, Chan PW, Moffat K, Pai EF, Royer WE, Šrajer V, Yang X. Resolution of structural heterogeneity in dynamic crystallography. Acta Crystallogr D Biol Crystallogr. 2013 Jun; 69(Pt 6):946-59.
          View in: PubMed
        3. Ronda L, Bettati S, Henry ER, Kashav T, Sanders JM, Royer WE, Mozzarelli A. Tertiary and Quaternary Allostery in Tetrameric Hemoglobin from Scapharca inaequivalvis. Biochemistry. 2013 Mar 26; 52(12):2108-17.
          View in: PubMed
        4. Ren Z, Srajer V, Knapp JE, Royer WE. Cooperative macromolecular device revealed by meta-analysis of static and time-resolved structures. Proc Natl Acad Sci U S A. 2012 Jan 3; 109(1):107-12.
          View in: PubMed
        5. Romano KP, Ali A, Royer WE, Schiffer CA. Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding. Proc Natl Acad Sci U S A. 2010 Dec 7; 107(49):20986-91.
          View in: PubMed
        6. Choi J, Muniyappan S, Wallis JT, Royer WE, Ihee H. Protein conformational dynamics of homodimeric hemoglobin revealed by combined time-resolved spectroscopic probes. Chemphyschem. 2010 Jan 18; 11(1):109-14.
          View in: PubMed
        7. Shandilya SM, Nalam MN, Nalivaika EA, Gross PJ, Valesano JC, Shindo K, Li M, Munson M, Royer WE, Harjes E, Kono T, Matsuo H, Harris RS, Somasundaran M, Schiffer CA. Crystal structure of the APOBEC3G catalytic domain reveals potential oligomerization interfaces. Structure. 2010 Jan 13; 18(1):28-38.
          View in: PubMed
        8. Chen W, Royer WE. Structural insights into interferon regulatory factor activation. Cell Signal. 2010 Jun; 22(6):883-7.
          View in: PubMed
        9. Knapp JE, Pahl R, Cohen J, Nichols JC, Schulten K, Gibson QH, Srajer V, Royer WE. Ligand migration and cavities within Scapharca Dimeric HbI: studies by time-resolved crystallo-graphy, Xe binding, and computational analysis. Structure. 2009 Nov 11; 17(11):1494-504.
          View in: PubMed
        10. Chen W, Lam SS, Srinath H, Jiang Z, Correia JJ, Schiffer CA, Fitzgerald KA, Lin K, Royer WE. Insights into interferon regulatory factor activation from the crystal structure of dimeric IRF5. Nat Struct Mol Biol. 2008 Nov; 15(11):1213-20.
          View in: PubMed
        11. Chen W, Srinath H, Lam SS, Schiffer CA, Royer WE, Lin K. Contribution of Ser386 and Ser396 to activation of interferon regulatory factor 3. J Mol Biol. 2008 May 30; 379(2):251-60.
          View in: PubMed
        12. Srajer V, Royer WE. Time-resolved x-ray crystallography of heme proteins. Methods Enzymol. 2008; 437:379-95.
          View in: PubMed
        13. Nienhaus K, Knapp JE, Palladino P, Royer WE, Nienhaus GU. Ligand migration and binding in the dimeric hemoglobin of Scapharca inaequivalvis. Biochemistry. 2007 Dec 11; 46(49):14018-31.
          View in: PubMed
        14. Chen W, Lam SS, Srinath H, Schiffer CA, Royer WE, Lin K. Competition between Ski and CREB-binding protein for binding to Smad proteins in transforming growth factor-beta signaling. J Biol Chem. 2007 Apr 13; 282(15):11365-76.
          View in: PubMed
        15. Nichols JC, Royer WE, Gibson QH. An optical signal correlated with the allosteric transition in Scapharca inaequivalvis HbI. Biochemistry. 2006 Dec 26; 45(51):15748-55.
          View in: PubMed
        16. Royer WE, Omartian MN, Knapp JE. Low resolution crystal structure of Arenicola erythrocruorin: influence of coiled coils on the architecture of a megadalton respiratory protein. J Mol Biol. 2007 Jan 5; 365(1):226-36.
          View in: PubMed
        17. Royer WE, Sharma H, Strand K, Knapp JE, Bhyravbhatla B. Lumbricus erythrocruorin at 3.5 A resolution: architecture of a megadalton respiratory complex. Structure. 2006 Jul; 14(7):1167-77.
          View in: PubMed
        18. Knapp JE, Pahl R, Srajer V, Royer WE. Allosteric action in real time: time-resolved crystallographic studies of a cooperative dimeric hemoglobin. Proc Natl Acad Sci U S A. 2006 May 16; 103(20):7649-54.
          View in: PubMed
        19. Knapp JE, Bonham MA, Gibson QH, Nichols JC, Royer WE. Residue F4 plays a key role in modulating oxygen affinity and cooperativity in Scapharca dimeric hemoglobin. Biochemistry. 2005 Nov 8; 44(44):14419-30.
          View in: PubMed
        20. Royer WE, Zhu H, Gorr TA, Flores JF, Knapp JE. Allosteric hemoglobin assembly: diversity and similarity. J Biol Chem. 2005 Jul 29; 280(30):27477-80.
          View in: PubMed
        21. Flores JF, Fisher CR, Carney SL, Green BN, Freytag JK, Schaeffer SW, Royer WE. Sulfide binding is mediated by zinc ions discovered in the crystal structure of a hydrothermal vent tubeworm hemoglobin. Proc Natl Acad Sci U S A. 2005 Feb 22; 102(8):2713-8.
          View in: PubMed
        22. Strand K, Knapp JE, Bhyravbhatla B, Royer WE. Crystal structure of the hemoglobin dodecamer from Lumbricus erythrocruorin: allosteric core of giant annelid respiratory complexes. J Mol Biol. 2004 Nov 12; 344(1):119-34.
          View in: PubMed
        23. Knapp JE, Srajer V, Pahl R, Royer WE. Immobilization of Scapharca HbI crystals improves data quality in time-resolved crystallographic experiments. Micron. 2004; 35(1-2):107-8.
          View in: PubMed
        24. Bennett EJ, Bjerregaard J, Knapp JE, Chavous DA, Friedman AM, Royer WE, O'Connor CM. Catalytic implications from the Drosophila protein L-isoaspartyl methyltransferase structure and site-directed mutagenesis. Biochemistry. 2003 Nov 11; 42(44):12844-53.
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        25. Knapp JE, Royer WE. Ligand-linked structural transitions in crystals of a cooperative dimeric hemoglobin. Biochemistry. 2003 Apr 29; 42(16):4640-7.
          View in: PubMed
        26. Knapp JE, Gibson QH, Cushing L, Royer WE. Restricting the ligand-linked heme movement in Scapharca dimeric hemoglobin reveals tight coupling between distal and proximal contributions to cooperativity. Biochemistry. 2001 Dec 11; 40(49):14795-805.
          View in: PubMed
        27. Heaslet HA, Royer WE. Crystalline ligand transitions in lamprey hemoglobin. Structural evidence for the regulation of oxygen affinity. J Biol Chem. 2001 Jul 13; 276(28):26230-6.
          View in: PubMed
        28. Royer WE, Knapp JE, Strand K, Heaslet HA. Cooperative hemoglobins: conserved fold, diverse quaternary assemblies and allosteric mechanisms. Trends Biochem Sci. 2001 May; 26(5):297-304.
          View in: PubMed
        29. Royer WE, Strand K, van Heel M, Hendrickson WA. Structural hierarchy in erythrocruorin, the giant respiratory assemblage of annelids. Proc Natl Acad Sci U S A. 2000 Jun 20; 97(13):7107-11.
          View in: PubMed
        30. Richardson RC, King NM, Harrington DJ, Sun H, Royer WE, Nelson DJ. X-Ray crystal structure and molecular dynamics simulations of silver hake parvalbumin (Isoform B). Protein Sci. 2000 Jan; 9(1):73-82.
          View in: PubMed
        31. Heaslet HA, Royer WE. The 2.7 A crystal structure of deoxygenated hemoglobin from the sea lamprey (Petromyzon marinus): structural basis for a lowered oxygen affinity and Bohr effect. Structure. 1999 May; 7(5):517-26.
          View in: PubMed
        32. Pardanani A, Gambacurta A, Ascoli F, Royer WE. Mutational destabilization of the critical interface water cluster in Scapharca dimeric hemoglobin: structural basis for altered allosteric activity. J Mol Biol. 1998 Dec 4; 284(3):729-39.
          View in: PubMed
        33. Harrington DJ, Adachi K, Royer WE. Crystal structure of deoxy-human hemoglobin beta6 Glu --> Trp. Implications for the structure and formation of the sickle cell fiber. J Biol Chem. 1998 Dec 4; 273(49):32690-6.
          View in: PubMed
        34. Harrington DJ, Adachi K, Royer WE. The high resolution crystal structure of deoxyhemoglobin S. J Mol Biol. 1997 Sep 26; 272(3):398-407.
          View in: PubMed
        35. Pardanani A, Gibson QH, Colotti G, Royer WE. Mutation of residue Phe97 to Leu disrupts the central allosteric pathway in Scapharca dimeric hemoglobin. J Biol Chem. 1997 May 16; 272(20):13171-9.
          View in: PubMed
        36. Royer WE, Fox RA, Smith FR, Zhu D, Braswell EH. Ligand linked assembly of Scapharca dimeric hemoglobin. J Biol Chem. 1997 Feb 28; 272(9):5689-94.
          View in: PubMed
        37. Royer WE, Pardanani A, Gibson QH, Peterson ES, Friedman JM. Ordered water molecules as key allosteric mediators in a cooperative dimeric hemoglobin. Proc Natl Acad Sci U S A. 1996 Dec 10; 93(25):14526-31.
          View in: PubMed
        38. Royer WE, Heard KS, Harrington DJ, Chiancone E. The 2.0 A crystal structure of Scapharca tetrameric hemoglobin: cooperative dimers within an allosteric tetramer. J Mol Biol. 1995 Oct 13; 253(1):168-86.
          View in: PubMed
        39. Summerford CM, Pardanani A, Betts AH, Poteete AR, Colotti G, Royer WE. Bacterial expression of Scapharca dimeric hemoglobin: a simple model system for investigating protein cooperatively. Protein Eng. 1995 Jun; 8(6):593-9.
          View in: PubMed
        40. Condon PJ, Royer WE. Crystal structure of oxygenated Scapharca dimeric hemoglobin at 1.7-A resolution. J Biol Chem. 1994 Oct 14; 269(41):25259-67.
          View in: PubMed
        41. Aronson HE, Royer WE, Hendrickson WA. Quantification of tertiary structural conservation despite primary sequence drift in the globin fold. Protein Sci. 1994 Oct; 3(10):1706-11.
          View in: PubMed
        42. Royer WE. High-resolution crystallographic analysis of a co-operative dimeric hemoglobin. J Mol Biol. 1994 Jan 14; 235(2):657-81.
          View in: PubMed
        43. Chiancone E, Elber R, Royer WE, Regan R, Gibson QH. Ligand binding and conformation change in the dimeric hemoglobin of the clam Scapharca inaequivalvis. J Biol Chem. 1993 Mar 15; 268(8):5711-8.
          View in: PubMed
        44. Chiancone E, Verzili D, Boffi A, Royer WE, Hendrickson WA. A cooperative hemoglobin with directly communicating hemes. The Scapharca inaequivalvis homodimer. Biophys Chem. 1990 Aug 31; 37(1-3):287-92.
          View in: PubMed
        45. Royer WE, Hendrickson WA, Chiancone E. Structural transitions upon ligand binding in a cooperative dimeric hemoglobin. Science. 1990 Aug 3; 249(4968):518-21.
          View in: PubMed
        46. Royer WE, Hendrickson WA, Chiancone E. The 2.4-A crystal structure of Scapharca dimeric hemoglobin. Cooperativity based on directly communicating hemes at a novel subunit interface. J Biol Chem. 1989 Dec 15; 264(35):21052-61.
          View in: PubMed
        47. Royer WE, Hendrickson WA. Molecular symmetry of Lumbricus erythrocruorin. J Biol Chem. 1988 Sep 25; 263(27):13762-5.
          View in: PubMed
        48. Royer WE, Hendrickson WA, Love WE. Crystals of Lumbricus erythrocruorin. J Mol Biol. 1987 Sep 5; 197(1):149-53.
          View in: PubMed
        49. Hendrickson WA, Royer WE. Principles in the assembly of annelid erythrocruorins. Biophys J. 1986 Jan; 49(1):177-89.
          View in: PubMed
        50. Royer WE, Love WE, Fenderson FF. Cooperative dimeric and tetrameric clam haemoglobins are novel assemblages of myoglobin folds. Nature. 1985 Jul 18-24; 316(6025):277-80.
          View in: PubMed
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