Andrew J. Fisher

Crystal structure of baculovirus P35: role of a novel reactive site loop in apoptotic caspase inhibition

The aspartate‐specific caspases are critical protease effectors of programmed cell death and consequently represent important targets for apoptotic intervention. Baculovirus P35 is a potent substrate inhibitor of metazoan caspases, a property that accounts for its unique effectiveness in preventing apoptosis in phylogenetically diverse organisms. Here we report the 2.2 Å resolution crystal structure of P35, the first structure of a protein inhibitor of the death caspases. The P35 monomer possesses a solvent‐exposed loop that projects from the proteins main β‐sheet core and positions the requisite aspartate cleavage site at the loops apex. Distortion or destabilization of this reactive site loop by site‐directed mutagenesis converted P35 to an efficient substrate which, unlike wild‐type P35, failed to interact stably with the target caspase or block protease activity. Thus, cleavage alone is insufficient for caspase inhibition. These data are consistent with a new model wherein the P35 reactive site loop participates in a unique multi‐step mechanism in which the spatial orientation of the loop with respect to the P35 core determines post‐cleavage association and stoichiometric inhibition of target caspases.

Structure of bacterial luciferase.

The generation of light by living organisms such as fireflies, glow-worms, mushrooms, fish, or bacteria growing on decaying materials has been a subject of fascination throughout the ages, partly because it occurs without the need for high temperatures. The chemistry behind the numerous bioluminescent systems is quite varied, and the enzymes that catalyze the reactions, the luciferases, are a large and evolutionarily diverse group. The structure of the best understood of these intriguing enzymes, bacterial luciferase, has recently been determined, allowing discussion of features of the protein in structural terms for the first time.

Allosteric Inhibition via R-State Destabilization in ATP Sulfurylase from Penicillium chrysogenum

The structure of the cooperative hexameric enzyme ATP sulfurylase from Penicillium chrysogenum bound to its allosteric inhibitor, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), was determined to 2.6 Å resolution. This structure represents the low substrate-affinity T-state conformation of the enzyme. Comparison with the high substrate-affinity R-state structure reveals that a large rotational rearrangement of domains occurs as a result of the R-to-T transition. The rearrangement is accompanied by the 17 Å movement of a 10-residue loop out of the active site region, resulting in an open, product release-like structure of the catalytic domain. Binding of PAPS is proposed to induce the allosteric transition by destabilizing an R-state-specific salt linkage between Asp 111 in an N-terminal domain of one subunit and Arg 515 in the allosteric domain of a trans-triad subunit. Disrupting this salt linkage by site-directed mutagenesis induces cooperative inhibition behavior in the absence of an allosteric effector, confirming the role of these two residues.

Comparative studies of T = 3 and T = 4 icosahedral RNA insect viruses

Crystallographic and molecular biological studies of T = 3 nodaviruses (180 identical subunits in the particle) and T = 4 tetraviruses (240 identical subunits in the particle) have revealed similarity in both the architecture of the particles and the strategy for maturation. The comparative studies provide a novel opportunity to examine an apparent evolution of particle size, from smaller (T = 3) to larger (T = 4), with both particles based on similar subunits. The BBV and FHV nodavirus structures are refined at 2.8 A and 3 A respectively, while the N omega V structure is at 6 A resolution. Nevertheless, the detailed comparisons of the noda and tetravirus X-ray electron density maps show that the same type of switching in subunit twofold contacts is used in the T = 3 and T = 4 capsids, although differences must exist between quasi and icosahedral threefold contacts in the T = 4 particle that have not yet been detected. The analyses of primary and tertiary structures of noda and tetraviruses show that N omega V subunits undergo a post assembly cleavage like that observed in nodaviruses and that the cleaved 76 C-terminal residues remain associated with the particle.

Crystallization and preliminary data analysis of Flock House virus.

Flock House virus, purified from infected cultured Drosophila cells, crystallizes into three different forms under identical growth conditions. Two crystal forms grow in the trigonal space group R3, both with equivalent cell constants a = 323.6 A, alpha = 61.7 degrees. The difference between the two trigonal crystal forms is 1.1 degrees in the orientation of the virus particle as determined from the rotation function. Early crystal setups grew in one form, while recent crystals grew in the other form. The third space group, which accounts for 5% of the observed crystals and grows with both trigonal forms, is orthorhombic I222 with cell parameters a = 416.7, b = 332.1, c = 351.2 A. The trigonal crystal forms contain one virion per unit cell and the orthorhombic form contains two particles per cell. All three crystal forms diffract X-rays to 2.8 A resolution.

Production and crystallization of virus-like particles assembled in a heterologous protein expression system.

It is of considerable interest to separate the processes of viral infectivity and virion assembly. Until recently this has only been possible with viruses that could be disassembled and reassembled in vitro. Even in these cases it was difficult to establish the authenticity of reassembled capsid protein because of possible irreversible damage that may have occurred to the protein during disassembly. An ideal method for the study of virus assembly is a protein expression system in which conditions are appropriate for spontaneous particle formation from freshly synthesized polypeptides. The baculovirus expression system has proven to be an excellent means to this end. Recently, this approach has been used to study the T = 3 Flock House insect virus and it has been demonstrated that subunits with the wild-type protein sequence, and with site-specific mutations that prevent particle maturation, will assemble and crystallize. This same approach has now been used at Purdue to study the T = 4 Nudaurelia omega capensis insect virus. There is no cell culture system currently available for the study of NomegaV, thus the expression system provides the first opportunity to study assembly under controlled conditions.