Michael Caffrey

Molecular structure of cytochrome c2 isolated from Rhodobacter capsulatus determined at 2·5 Å resolution

The molecular structure of the cytochrome c2, isolated from the purple photosynthetic bacterium Rhodobacter capsulatus, has been solved to a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 16.8% for all observed X-ray data. Crystals used for this investigation belong to the space group R32 with two molecules in the asymmetric unit and unit cell dimensions of a = b = 100.03 A, c = 162.10 A as expressed in the hexagonal setting. An interpretable electron density map calculated at 2.5 A resolution was obtained by the combination of multiple isomorphous replacement with four heavy atom derivatives, molecular averaging and solvent flattening. At this stage of the structural analysis the electron densities corresponding to the side-chains are well ordered except for several surface lysine, glutamate and aspartate residues. Like other c-type cytochromes, the secondary structure of the protein consists of five alpha-helices forming a basket around the heme prosthetic group with one heme edge exposed to the solvent. The overall alpha-carbon trace of the molecule is very similar to that observed for the bacterial cytochrome c2, isolated from Rhodospirillum rubrum, with the exception of a loop, delineated by amino acid residues 21 to 32, that forms a two stranded beta-sheet-like motif in the Rb. capsulatus protein. As observed in the eukaryotic cytochrome c proteins, but not in the cytochrome c2 from Rsp. rubrum, there are two evolutionarily conserved solvent molecules buried within the heme binding pocket.

Model for the structure of the HIV gp41 ectodomain: insight into the intermolecular interactions of the gp41 loop.

In human immunodeficiency virus (HIV) the viral envelope proteins gp41 and gp120 form a non-covalent complex, which is a potential target for AIDS therapies. In addition gp41 plays a possible role in HIV infection of B cells via the complement system. In an effort to better understand the molecular interactions of gp41, the structure of the HIV gp41 ectodomain has been modeled using the NMR restraints of the simian immunodeficiency virus (SIV) gp41 ectodomain (M. Caffrey, M. Cai, J. Kaufman, S.J. Stahl, P.T. Wingfield, A.M. Gronenborn, G.M. Clore, Solution structure of the 44 kDa ectodomain of SIV gp41, EMBO J. 17 (1998) 4572--4584). The resulting model presents the first structural information for the HIV gp41 loop, which has been implicated to play a direct role in binding to gp120 and C1q of the complement system.

HIV envelope: challenges and opportunities for development of entry inhibitors

The HIV envelope proteins glycoprotein 120 (gp120) and glycoprotein 41 (gp41) play crucial roles in HIV entry, therefore they are of extreme interest in the development of novel therapeutics. Studies using diverse methods, including structural biology and mutagenesis, have resulted in a detailed model for envelope-mediated entry, which consists of multiple conformations, each a potential target for therapeutic intervention. In this review, the challenges, strategies and progress to date for developing novel entry inhibitors directed at disrupting HIV gp120 and gp41 function are discussed.

Heparin binding by the HIV-1 tat protein transduction domain

The protein transduction domain from the HIV‐1 tat protein (termed PTD‐tat) has been fused to the C‐terminus of a model cargo protein, the IgG binding domain of streptococcal protein G. We demonstrate that PG‐Ctat (PTD‐tat fused to the C‐terminus of protein G) binds to a heparin affinity column. PG‐Ctat binds with relatively high affinity, as shown by its elution at 1.6 M NaCl. The heparin binding properties of PTD‐tat are consistent with the idea that heparan sulfate, an analog of heparin found at the cell surface, plays a role in the translocation of PTD‐tat fusions. We suggest that the heparin‐binding properties of PTD‐tat can be exploited for purification of PTD‐tat fusions in the absence of affinity tags.

The Src Homology 3 Domain Is Required for Junctional Adhesion Molecule Binding to the Third PDZ Domain of the Scaffolding Protein ZO-1.

Background: ZO-1 is a scaffolding protein implicated in the assembly of tight junctions. Results: Structures of core PDZ-SH3-GUK, plus and minus JAM-A peptide, and isolated PDZ are presented. Conclusion: The SH3 domain is required for JAM-A binding to PDZ3. Significance: This is the first demonstration for the role of an adjacent domain to the binding of ligands to PDZ domains in the MAGUK family. Tight junctions are cell-cell contacts that regulate the paracellular flux of solutes and prevent pathogen entry across cell layers. The assembly and permeability of this barrier are dependent on the zonula occludens (ZO) membrane-associated guanylate kinase (MAGUK) proteins ZO-1, -2, and -3. MAGUK proteins are characterized by a core motif of protein-binding domains that include a PDZ domain, a Src homology 3 (SH3) domain, and a region of homology to guanylate kinase (GUK); the structure of this core motif has never been determined for any MAGUK. To better understand how ZO proteins organize the assembly of protein complexes we have crystallized the entire PDZ3-SH3-GUK core motif of ZO-1. We have also crystallized this core motif in complex with the cytoplasmic tail of the ZO-1 PDZ3 ligand, junctional adhesion molecule A (JAM-A) to determine how the activity of different domains is coordinated. Our study shows a new feature for PDZ class II ligand binding that implicates the two highly conserved Phe−2 and Ser−3 residues of JAM. Our x-ray structures and NMR experiments also show for the first time a role for adjacent domains in the binding of ligands to PDZ domains in the MAGUK proteins family.

Analysis of hemagglutinin-mediated entry tropism of H5N1 avian influenza

BackgroundAvian influenza virus H5N1 is a major concern as a potential global pandemic. It is thought that multiple key events must take place before efficient human-to-human transmission of the virus occurs. The first step in overcoming host restriction is viral entry which is mediated by HA, responsible for both viral attachment and viral/host membrane fusion. HA binds to glycans-containing receptors with terminal sialic acid (SA). It has been shown that avian influenza viruses preferentially bind to α2,3-linked SAs, while human influenza A viruses exhibit a preference for α2,6-linked SAs. Thus it is believed the precise linkage of SAs on the target cells dictate host tropism of the viruses.ResultsWe demonstrate that H5N1 HA/HIV pseudovirus can efficiently transduce several human cell lines including human lung cells. Interestingly, using a lectin binding assay we show that the presence of both α2,6-linked and α2,3-linked SAs on the target cells does not always correlate with efficient transduction. Further, HA substitutions of the residues implicated in switching SA-binding between avian and human species did not drastically affect HA-mediated transduction of the target cells or target cell binding.ConclusionOur results suggest that a host factor(s), which is yet to be identified, is required for H5N1 entry in the host cells.

Identification of a Broad-Spectrum Antiviral Small Molecule against Severe Acute Respiratory Syndrome Coronavirus and Ebola, Hendra, and Nipah Viruses by Using a Novel High-Throughput Screening Assay

ABSTRACT Severe acute respiratory syndrome coronavirus (SARS-CoV) and Ebola, Hendra, and Nipah viruses are members of different viral families and are known causative agents of fatal viral diseases. These viruses depend on cathepsin L for entry into their target cells. The viral glycoproteins need to be primed by protease cleavage, rendering them active for fusion with the host cell membrane. In this study, we developed a novel high-throughput screening assay based on peptides, derived from the glycoproteins of the aforementioned viruses, which contain the cathepsin L cleavage site. We screened a library of 5,000 small molecules and discovered a small molecule that can inhibit the cathepsin L cleavage of all viral peptides with minimal inhibition of cleavage of a host protein-derived peptide (pro-neuropeptide Y). The small molecule inhibited the entry of all pseudotyped viruses in vitro and the cleavage of SARS-CoV spike glycoprotein in an in vitro cleavage assay. In addition, the Hendra and Nipah virus fusion glycoproteins were not cleaved in the presence of the small molecule in a cell-based cleavage assay. Furthermore, we demonstrate that the small molecule is a mixed inhibitor of cathepsin L. Our broad-spectrum antiviral small molecule appears to be an ideal candidate for future optimization and development into a potent antiviral against SARS-CoV and Ebola, Hendra, and Nipah viruses. IMPORTANCE We developed a novel high-throughput screening assay to identify small molecules that can prevent cathepsin L cleavage of viral glycoproteins derived from SARS-CoV and Ebola, Hendra, and Nipah viruses that are required for their entry into the host cell. We identified a novel broad-spectrum small molecule that could block cathepsin L-mediated cleavage and thus inhibit the entry of pseudotypes bearing the glycoprotein derived from SARS-CoV or Ebola, Hendra, or Nipah virus. The small molecule can be further optimized and developed into a potent broad-spectrum antiviral drug.

ARF Directly Binds DP1: Interaction with DP1 Coincides with the G1 Arrest Function of ARF

ABSTRACT The tumor suppressor ARF inhibits cell growth in response to oncogenic stress in a p53-dependent manner. Also, there is an increasing appreciation of ARFs ability to inhibit cell growth via multiple p53-independent mechanisms, including its ability to regulate the E2F pathway. We have investigated the interaction between the tumor suppressor ARF and DP1, the DNA binding partner of the E2F family of factors (E2Fs). We show that ARF directly binds to DP1. Interestingly, binding of ARF to DP1 results in an inhibition of the interaction between DP1 and E2F1. Moreover, ARF regulates the association of DP1 with its target gene, as evidenced by a chromatin immunoprecipitation assay with the dhfr promoter. By analyzing a series of ARF mutants, we demonstrate a strong correlation between ARFs ability to regulate DP1 and its ability to cause cell cycle arrest. S-phase inhibition by ARF is preceded by an inhibition of the E2F-activated genes. Moreover, we provide evidence that ARF inhibits the E2F-activated genes independently of p53 and Mdm2. Also, the interaction between ARF and DP1 is enhanced during oncogenic stress and “culture shock.” Taken together, our results show that DP1 is a critical direct target of ARF.

SITE-SPECIFIC MUTAGENESIS STUDIES OF CYTOCHROMES C

Abstract Cytochromes c are among the best characterized proteins, which consequently make them attractive candidates for study by mutagenesis. Site-specific mutagenesis studies have been reported for five species of cytochromes c , including those from Rhodobacter capsulatus, Saccharomyces cerevisiae, Drosophila melanogaster , rat and horse. The effect of mutations to highly conserved residues on redox potential indicates that substitutions of the M80 axial heme ligand result in the greatest effect (i.e., > 200 mV ) while other mutations generally have a small negative effect (i.e., mV ). Denaturation of the mutants suggests that conformational stability is generally decreased upon substitution of conserved residues, with the largest observed destabilization being approx. 4 kcal/mol. As judged by the p K for the alkaline transition of the mutants, the stability of the Fe-S bond can be increased or decreased by approx. 1 kcal/mol with the largest effects occurring when the mutated group is proximal to the heme. Electron transfer reactions between cytochrome c mutants and various physiological partners are generally not affected to a large degree, an observation that is consistent with their function in vivo. Structural characterization of several mutants by X-ray crystallography suggests that site-specific substitutions generally do not disrupt the overall conformation but result in small local and remote structural perturbations. NMR characterization of several mutants supports the lack of large structural changes but suggests that changes in the dynamic properties of mutants often occur. Taken together, these observations suggest that comprehensive study of equivalent mutations in a number of species is necessary to understand the determinants of cytochrome c structure and function as well as the determinants of evolutionary conservation.

Structural and dynamic properties of the HIV-1 tat transduction domain in the free and heparin-bound states.

An 11-residue basic domain of the HIV-1 tat protein, termed the tat transduction domain (TTD), has been shown to mediate transfer of biomolecules across biological membranes. The mechanism of TTD-mediated membrane translocation is currently unknown but thought to involve binding to heparan sulfate, which is found in proteoglycans that are ubiquitously present on cell surfaces. To study the mechanism of TTD-mediated membrane translocation, the TTD was fused to the C-terminus of a model cargo protein, the IgG binding domain of streptococcal protein G (PG) to form PG-TTD. NMR studies of PG-TTD in the free state indicated that the structure of the PG moiety of PG-TTD was not perturbed by the presence of the TTD and that the TTD moiety is in an extended conformation. Heteronuclear relaxation measurements of PG-TTD in the free state show that the TTD moiety of PG-TTD is relatively mobile (e.g., the average S(2) value of the TTD and PG core are approximately 0.54 and approximately 0.84, respectively). PG-TTD has been shown to bind to heparin by isothermal titration calorimetry (K(D) = 0.37 microM, Delta H = -12 kcal/mol, Delta S = -11 cal/mol/T). NMR spectroscopy demonstrated that heparin binds to the TTD moiety of PG-TTD. The heteronuclear relaxation measurements of PG-TTD in complex with heparin show that the TTD becomes less dynamic when bound to heparin (average S(2) value of the TTD is 0.69 in the presence of heparin). A model for the first step of TTD-mediated entry into cells is presented.