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 Columbia University and became an associate of the Howard Hughes Medical Institute in 1986 and an associate research scientist at Columbia University in 1988. He joined the faculty of the University of Massachusetts Medical School 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

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

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) )

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.

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 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.