William E. Royer

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding.

Hepatitis C virus infects an estimated 180 million people worldwide, prompting enormous efforts to develop inhibitors targeting the essential NS3/4A protease. Resistance against the most promising protease inhibitors, telaprevir, boceprevir, and ITMN-191, has emerged in clinical trials. In this study, crystal structures of the NS3/4A protease domain reveal that viral substrates bind to the protease active site in a conserved manner defining a consensus volume, or substrate envelope. Mutations that confer the most severe resistance in the clinic occur where the inhibitors protrude from the substrate envelope, as these changes selectively weaken inhibitor binding without compromising the binding of substrates. These findings suggest a general model for predicting the susceptibility of protease inhibitors to resistance: drugs designed to fit within the substrate envelope will be less susceptible to resistance, as mutations affecting inhibitor binding would simultaneously interfere with the recognition of viral substrates.

Crystal Structure of the APOBEC3G Catalytic Domain Reveals Potential Oligomerization Interfaces

APOBEC3G is a DNA cytidine deaminase that has antiviral activity against HIV-1 and other pathogenic viruses. In this study the crystal structure of the catalytically active C-terminal domain was determined to 2.25 A. This structure corroborates features previously observed in nuclear magnetic resonance (NMR) studies, a bulge in the second beta strand and a lengthening of the second alpha helix. Oligomerization is postulated to be critical for the function of APOBEC3G. In this structure, four extensive intermolecular interfaces are observed, suggesting potential models for APOBEC3G oligomerization. The structural and functional significance of these interfaces was probed by solution NMR and disruptive variants were designed and tested for DNA deaminase and anti-HIV activities. The variant designed to disrupt the most extensive interface lost both activities. NMR solution data provides evidence that another interface, which coordinates a novel zinc site, also exists. Thus, the observed crystallographic interfaces of APOBEC3G may be important for both oligomerization and function.

Cooperative hemoglobins: conserved fold, diverse quaternary assemblies and allosteric mechanisms

Assembly of hemoglobin subunits into cooperative complexes produces a remarkable variety of architectures, ranging in oligomeric state from dimers to complexes containing 144 hemoglobin subunits. Diverse stereochemical mechanisms for modulating ligand affinity through intersubunit interactions have been revealed from studies of three distinct hemoglobin assemblages. This mechanistic diversity, which occurs between assemblies of subunits that have the same fold, provides insight into the range of regulatory strategies that are available to protein molecules.

Insights into interferon regulatory factor activation from the crystal structure of dimeric IRF5.

Interferon regulatory factors (IRFs) are essential in the innate immune response and other physiological processes. Activation of these proteins in the cytoplasm is triggered by phosphorylation of serine and threonine residues in a C-terminal autoinhibitory region, which stimulates dimerization, transport into the nucleus, assembly with the coactivator CBP/p300 and initiation of transcription. The crystal structure of the transactivation domain of pseudophosphorylated human IRF5 strikingly reveals a dimer in which the bulk of intersubunit interactions involve a highly extended C-terminal region. The corresponding region has previously been shown to block CBP/p300 binding to unphosphorylated IRF3. Mutation of key interface residues supports the observed dimer as the physiologically activated state of IRF5 and IRF3. Thus, phosphorylation is likely to activate IRF5 and other family members by triggering conformational rearrangements that switch the C-terminal segment from an autoinihibitory to a dimerization role.

Allosteric action in real time: Time-resolved crystallographic studies of a cooperative dimeric hemoglobin

Protein allostery provides mechanisms for regulation of biological function at the molecular level. We present here an investigation of global, ligand-induced allosteric transition in a protein by time-resolved x-ray diffraction. The study provides a view of structural changes in single crystals of Scapharca dimeric hemoglobin as they proceed in real time, from 5 ns to 80 μs after ligand photodissociation. A tertiary intermediate structure forms rapidly (<5 ns) as the protein responds to the presence of an unliganded heme within each R-state protein subunit, with key structural changes observed in the heme groups, neighboring residues, and interface water molecules. This intermediate lays a foundation for the concerted tertiary and quaternary structural changes that occur on a microsecond time scale and are associated with the transition to a low-affinity T-state structure. Reversal of these changes shows a considerable lag as a T-like structure persists well after ligand rebinding, suggesting a slow T-to-R transition.

Allosteric hemoglobin assembly: diversity and similarity

From the ‡Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, ¶Hematology Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, Institute of Veterinary Physiology, Vetsuisse Faculty of the University of Zurich, CH-8057 Zurich, Switzerland, and **Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802

Structural insights into interferon regulatory factor activation

The interferon regulatory factors (IRFs) play important roles in development of the immune system and host defense. Recent crystallographic and biochemical studies have provided insights into the mechanism of activation of IRFs by phosphorylation. The activation of a latent closed conformation of IRF in the cytoplasm is triggered by phosphorylation of Ser/Thr residues in a C-terminal region. Phosphorylation stimulates the C-terminal autoinhibitory domain to attain a highly extended conformation triggering dimerization through extensive contacts to a second subunit. Dimers are then transported into the nucleus and assemble with the coactivator CBP/p300 to activate transcription of type I interferons and other target genes. The advances made in understanding the release of inhibition after IRF dimerization have generated a detailed structural model of how IRFs signaling pathways are activated.

Ligand migration and cavities within Scapharca Dimeric HbI: studies by time-resolved crystallo-graphy, Xe binding, and computational analysis.

As in many other hemoglobins, no direct route for migration of ligands between solvent and active site is evident from crystal structures of Scapharca inaequivalvis dimeric HbI. Xenon (Xe) and organic halide binding experiments, along with computational analysis presented here, reveal protein cavities as potential ligand migration routes. Time-resolved crystallographic experiments show that photodissociated carbon monoxide (CO) docks within 5 ns at the distal pocket B site and at more remote Xe4 and Xe2 cavities. CO rebinding is not affected by the presence of dichloroethane within the major Xe4 protein cavity, demonstrating that this cavity is not on the major exit pathway. The crystal lattice has a substantial influence on ligand migration, suggesting that significant conformational rearrangements may be required for ligand exit. Taken together, these results are consistent with a distal histidine gate as one important ligand entry and exit route, despite its participation in the dimeric interface.

Contribution of Ser386 and Ser396 to Activation of Interferon Regulatory Factor 3

IRF-3, a member of the interferon regulatory factor (IRF) family of transcription factors, functions in innate immune defense against viral infection. Upon infection, host cell IRF-3 is activated by phosphorylation at its seven C-terminal Ser/Thr residues: (385)SSLENTVDLHISNSHPLSLTS(405). This phosphoactivation triggers IRF-3 to react with the coactivators, CREB-binding protein (CBP)/p300, to form a complex that activates target genes in the nucleus. However, the role of each phosphorylation site for IRF-3 phosphoactivation remains unresolved. To address this issue, all seven Ser/Thr potential phosphorylation sites were screened by mutational studies, size-exclusion chromatography, and isothermal titration calorimetry. Using purified proteins, we show that CBP (amino acid residues 2067-2112) interacts directly with IRF-3 (173-427) and six of its single-site mutants to form heterodimers, but when CBP interacts with IRF-3 S396D, oligomerization is evident. CBP also interacts in vitro with IRF-3 double-site mutants to form different levels of oligomerization. Among all the single-site mutants, IRF-3 S396D showed the strongest binding to CBP. Although IRF-3 S386D alone did not interact as strongly with CBP as did other mutants, it strengthened the interaction and oligomerization of IRF-3 S396D with CBP. In contrast, IRF-3 S385D weakened the interaction and oligomerization of IRF-3 S396D and S386/396D with CBP. Thus, it appears that Ser385 and Ser386 serve antagonistic functions in regulating IRF-3 phosphoactivation. These results indicate that Ser386 and Ser396 are critical for IRF-3 activation, and support a phosphorylation-oligomerization model for IRF-3 activation.

Cooperative macromolecular device revealed by meta-analysis of static and time-resolved structures.

Here we present a meta-analysis of a large collection of static structures of a protein in the Protein Data Bank in order to extract the progression of structural events during protein function. We apply this strategy to the homodimeric hemoglobin HbI from Scapharca inaequivalvis. We derive a simple dynamic model describing how binding of the first ligand in one of the two chemically identical subunits facilitates a second binding event in the other partner subunit. The results of our ultrafast time-resolved crystallographic studies support this model. We demonstrate that HbI functions like a homodimeric mechanical device, such as pliers or scissors. Ligand-induced motion originating in one subunit is transmitted to the other via conserved pivot points, where the E and F′ helices from two partner subunits are “bolted” together to form a stable dimer interface permitting slight relative rotation but preventing sliding.