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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

        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 Institutein 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.


        Structural basis for assembly and functional regulation in macromolecular complexes

        Photo: William E Royer The focus of research in this laboratory is the investigation of the structural principles governing the assembly of protein molecules. We primarily use x-ray crystallography to obtain three-dimensional protein structures and complement this structural information with site-directed mutagenesis and biophysical techniques. Assembly of polypeptide chains can endow them with additional important functional properties. Classic among these are allosteric interactions in which the binding of small ligands act to regulate protein function. We are investigating this process using a number of invertebrate oxygen carrier molecules as models to understand the structural basis for intersubunit communication. Transmission of signals within cells often involves protein assembly. We are investigating such signaling in the interferon regulatory factors (IRFs) whose phosphorylation-induced assembly triggers activation of a number of target genes involved in host defense mechanisms.

        Invertebrate oxygen carriers

        These systems range from the simplest possible allosteric system, exemplified by a dimeric hemoglobin which we have shown to have a completely novel mechanism for cooperativity, to much more complex protein assemblages (up to several million Daltons ). The use of these systems will help to elucidate structural principles for allosteric regulation as well as provide information crucial for the design of blood substitutes. The dimeric hemoglobin from the blood clam, Scapharca inaequivalvis, is a particularly good model system for investigating allostery. The simplicity of this system has allowed us to elucidate the central role for ordered water molecules in the communication between subunits. In collaboration with Dr. Francesca Massi (BMP), we are using NMR to investigate the role of interface dynamics in the communication between subunits, for which the ligand-linked reorganization of interface water molecules may play a key role. In collaboration with Dr. Vukica Šrajer (University of Chicago ), we are using time-resolved crystallography to follow the allosteric transitions as they occur at sub nanosecond time resolution. One intriguing finding from this work is that movement of interface water molecules may facilitate the early nanosecond events in the transition between alternate states.

        Interferon regulatory factors (IRFs)

        IRF family members play important roles in innate immunity, inflammation and apoptosis. Activation of these proteins in the cytoplasm 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 to activate 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, before his tragic death from cancer. Our crystal structure of dimeric pseudophosphorylated IRF-5, in comparison with structures of monomeric IRF-3 determined by Dr. Lin, has revealed the structural basis for IRF activation. 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. Understanding IRF regulation, particularly that of IRF-5, is of potential clinical importance, as therapeutic agents that enhance activity could combat viral infection or tumor growth, whereas agents that attenuate activity could be used to minimize harmful inflammatory responses.

        Research Figures

        Interface water structure in deoxy and liganded Scapharca dimeric hemoglobin (HbI).

        Figure 1 Legend

        Interface water structure in deoxy and liganded Scapharca dimeric hemoglobin (HbI). This figure illustrates the striking disruption of the core interface water structure (depicted as blue spheres) that occurs upon binding ligand (either CO or oxygen). Our experiments have revealed that these water molecules play a key role in mediating intersubunit communication (Royer et al., 1996). Along with the water molecules, a ribbon trace (in red) of both subunits is shown with heme groups (in black) and side chains of Phe 97 and Thr 72 depicted by ball and stick representations (yellow balls for carbon and red balls for oxygen atoms). Upon ligation, by either CO or O2, Phe 97 is extruded from heme pocket into the interface, which disrupts the water cluster. (Coordinates are available from the Protein Data Bank as entry codes 3SDH (deoxy), 4SDH (CO-liganded) and 1HBI (oxygenated) )


        Stereo image of the interface water molecules in deoxy Scapharca dimeric hemoglobin.

        Figure 2 Legend

        Stereo image of the interface water molecules in deoxy Scapharca dimeric hemoglobin. The water cluster illustrated here plays a critical role in stabilizing the low affinity form Scapharca dimeric hemoglobin. Portions of the E helix (red) and F helix (blue) are shown for each of the two subunits along with bonds (black) for the heme groups and side chains of residues His 69 (distal His), Thr 72, Tyr 75, Asn 79, Lys 96, Phe 97 and His 101 (proximal His). Water molecules are depicted as small light-blue spheres, with likely hydrogen bonds illustrated as dotted lines. Note the multiple hydrogen bonds between water molecules that act to stabilize the water cluster.

        Two depictions of the molecular double strand found in deoxy sickle-cell hemoglobin crystals.

        Figure 3 Legend

        Two depictions of the molecular double strand found in deoxy sickle-cell hemoglobin crystals. In sickle-cell disease, mutation of the 6th residue of the beta (b) subunit from glutamate to valine results in deoxy hemoglobin polymerization into long fibers within the erythrocyte and numerous clinical manifestations. The double strand shown here has been shown by a variety of techniques to be the basic building block of the pathological sickle cell hemoglobin fiber. On the left, the strand is shown as a transparent molecular surface, with heme groups colored red and the mutant valine residues blue. In the representation on the right, the protein backbones are shown as white coils, again with hemes red and mutant valine residues blue. Axial contacts are located between molecules within a single strand in the vertical direction. Lateral contacts involving the blue mutant valine residues act to associate two single strands into a double strand. (PDB entry 2HBS, Harrington et al., 1997).

        Figure 4

        Figure 4 Legend

        Stereo diagram of the lateral contact. The backbone trace for the E and F helices from the acceptor beta (b) subunit of one tetramer is shown in magenta, along with a portion of the A helix (red) and H helix (blue) of the donor beta (b) subunit of the contacting tetramer. The mutant valine is shown in yellow and other important side-chains are shown in black. Likely hydrogen bonds are shown as dashed lines. The mutant valine packs in a hydrophobic pocket formed by Phe 85 (F85), Leu 88 (L88) and Ala 70 in the acceptor subunit. Additionally, a number of water mediated hydrogen bonds are formed at the contact periphery. These detailed interactions provide a template for the design of inhibitors to interfere with polymerization.



        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
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        1. Ren Z, Chan PW, Moffat K, Pai EF, Royer WE, Srajer 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
        2. 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
        3. 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
        4. 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.
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        5. 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.
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        6. Chen W, Royer WE. Structural insights into interferon regulatory factor activation. Cell Signal. 2010 Jun; 22(6):883-7.
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        7. 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
        8. 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.
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        9. 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.
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        10. Srajer V, Royer WE. Time-resolved x-ray crystallography of heme proteins. Methods Enzymol. 2008; 437:379-95.
          View in: PubMed
        11. 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.
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        12. 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.
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        13. 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.
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        14. 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
        15. 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.
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        16. 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
        17. 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.
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        18. 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.
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        19. 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.
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        20. 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.
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        21. 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.
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        22. 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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        23. Knapp JE, Royer WE. Ligand-linked structural transitions in crystals of a cooperative dimeric hemoglobin. Biochemistry. 2003 Apr 29; 42(16):4640-7.
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        24. 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.
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        25. 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.
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        26. 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.
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        27. 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.
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        28. 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
        29. 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.
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        30. 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.
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        31. 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.
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        32. Harrington DJ, Adachi K, Royer WE. The high resolution crystal structure of deoxyhemoglobin S. J Mol Biol. 1997 Sep 26; 272(3):398-407.
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        33. 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.
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        34. 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.
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        35. 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.
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        36. 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.
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        37. 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.
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        38. 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.
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        39. 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.
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        40. Royer WE. High-resolution crystallographic analysis of a co-operative dimeric hemoglobin. J Mol Biol. 1994 Jan 14; 235(2):657-81.
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        41. 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.
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        42. 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.
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        43. Royer WE, Hendrickson WA, Chiancone E. Structural transitions upon ligand binding in a cooperative dimeric hemoglobin. Science. 1990 Aug 3; 249(4968):518-21.
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        44. 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.
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        45. Royer WE, Hendrickson WA. Molecular symmetry of Lumbricus erythrocruorin. J Biol Chem. 1988 Sep 25; 263(27):13762-5.
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        46. Royer WE, Hendrickson WA, Love WE. Crystals of Lumbricus erythrocruorin. J Mol Biol. 1987 Sep 5; 197(1):149-53.
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        47. Hendrickson WA, Royer WE. Principles in the assembly of annelid erythrocruorins. Biophys J. 1986 Jan; 49(1):177-89.
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        48. 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.
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