Virus particles are stable yet exhibit highly dynamic character given the events that shape their life cycle. Isolated from their hosts, the nucleoprotein particles are macromolecules that can be crystallized and studied by x-ray diffraction. During assembly, maturation and entry, however, they are highly dynamic and display remarkable plasticity. These dynamic properties can only be inferred from the x-ray structure and must be studied by methods that are sensitive to mobility. We have used matrix-assisted laser desorption/ionization mass spectrometry combined with time resolved, limited proteolysis (Cohen, S. L., Ferre-D'Amare, A. R., Burley, S. K., and Chait, B. T. (1995) Protein Sci. 4, 1088 -1099; Kriwacki, R. W., Wu, J., Tennant, T., Wright, P. E., and Siuzdak, G. (1997) J. Chromatogr. 777, 23-30; Kriwacki, R. W., Wu, J., Siuzdak, G., and Wright, P. E. (1996) J. Am. Chem. Soc. 118, 5320 -5321) to examine the viral capsid of flock house virus. Employing less than 10 g of virus, time course digestion products were assigned to polypeptides of the subunit. Although surface regions in the three-dimensional structure were susceptible to cleavage on extended exposure to the protease, the first digestion products were invariably from parts of the subunit that are internal to the x-ray structure. Regions in the N-and C-terminal portions of the subunit, located within the shell in the x-ray structure, but implicated in RNA neutralization and RNA release and delivery, respectively, were the most susceptible to cleavage demonstrating transient exposure of these polypeptides to the viral surface.The protein capsid of virions is a noncovalent association of protein subunits that is responsible for an array of functions, including cell attachment, cell entry, and RNA release. Mobile regions associated with these events can only be inferred from inherently static methods such as x-ray crystallography (4) and cryo-electron microscopy ( 5). To better understand the dynamic nature of the viral capsid it is necessary to develop methods that are sensitive to functional mobility. One recently developed method has combined limited proteolysis with mass spectrometry (1-3) to explore protein/DNA (1) and protein/ protein interactions (2, 3). While previous applications of mass spectrometry to viruses have either focused on characterizing protein and DNA structure ( 6 -11) or the intact virus (12), here we have applied the proteolysis method with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) 1 to study the surface-accessible regions of the viral particle.MALDI-MS typically provides picomole sensitivity and accuracy on the order of 0.05% (i.e. Ϯ 0.5 Da on a 1000-Da peptide) and therefore offers a useful method for identifying the proteolysis products of the virus. The limited proteolysis/MALDI-MS experiments were performed on flock house virus (FHV), a non-enveloped, icosahedral, RNA animal virus ( Fig. 1) with dimensions similar to the rhino and polio viruses (ϳ300 Å). Its protein coat or capsid is compose...

The crystal structure of the terminal component of the cumene dioxygenase multicomponent enzyme system of Pseudomonas fluorescens IP01 (CumDO) was determined at a resolution of 2.2 Å by means of molecular replacement by using the crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NphDO). The ligation of the two catalytic centers of CumDO (i.e., the nonheme iron and Rieske [2Fe-2S] centers) and the bridging between them in neighboring catalytic subunits by hydrogen bonds through a single amino acid residue, Asp231, are similar to those of NphDO. An unidentified external ligand, possibly dioxygen, was bound at the active site nonheme iron. The entrance to the active site of CumDO is different from the entrance to the active site of NphDO, as the two loops forming the lid exhibit great deviation. On the basis of the complex structure of NphDO, a biphenyl substrate was modeled in the substrate-binding pocket of CumDO. The residues surrounding the modeled biphenyl molecule include residues that have already been shown to be important for its substrate specificity by a number of engineering studies of biphenyl dioxygenases.Aromatic hydrocarbons are common contaminants of soil and groundwater (18). One of the most attractive means of removal of these compounds from the environment is the use of microorganisms (34). Dihydroxylation of the aromatic ring by a bacterial aromatic hydrocarbon dioxygenase is a prerequisite for subsequent oxidation of the aromatic nucleus by a ring fission dioxygenase (6). Aromatic hydrocarbon dioxygenases belong to a large family named the Rieske nonheme iron oxygenases (11). Werlen et al. delineated four dioxygenase subfamilies in this large family (the toluene/biphenyl, naphthalene, benzoate, and phthalate subfamilies) based on sequence alignment of the catalytic components (␣ subunits) (37). The toluene/biphenyl subfamily includes enzymes for the degradation of toluene, benzene, cumene (isopropylbenzene), biphenyl, and polychlorinated biphenyls (PCBs). The naphthalene subfamily consists of enzymes for the degradation of naphthalene and phenanthrene. The Rieske dioxygenases involved in bacterial hydrocarbon degradation comprise multicomponent enzyme systems (36) in which reduced pyridine nucleotide is used as the initial source of two electrons for dioxygen activation. The electrons pass through a flavin cofactor and Rieske [2Fe-2S] centers into the mononuclear iron center of the terminal Rieske nonheme iron dioxygenase component.The crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. NCIB 9816-4 (NphDO) has been reported previously (4,15,17), and the structure-function relationship of this enzyme has been well studied (28). NphDO is an ␣ 3 ␤ 3 hexamer, and each ␣ subunit contains a Rieske [2Fe-2S] cluster and nonheme iron coordinated by His208, His213, and Asp362. The active site iron center of one of the ␣ subunits is directly connected by hydrogen bonds through a singl...

The capsid of flock house virus is composed of 180 copies of a single type of coat protein which forms a T=3 icosahedral shell. High-resolution structural analysis has shown that the protein subunits, although chemically identical, form different contacts across the twofold axes of the virus particle. Subunits that are related by icosahedral twofold symmetry form flat contacts, whereas subunits that are related by quasi-twofold symmetry form bent contacts. The flat contacts are due to the presence of ordered genomic RNA and an ordered peptide arm which is inserted in the groove between the subunits and prevents them from forming the dihedral angle observed at the bent quasi-twofold contacts. We hypothesized that by deleting the residues that constitute the ordered peptide arm, formation of flat contacts should be impossible and therefore result in assembly of particles with only bent contacts. Such particles would have T=1 symmetry. To test this hypothesis we generated two deletion mutants in which either 50 or 31 residues were eliminated from the N terminus of the coat protein. We found that in the absence of residues 1 to 50, assembly was completely inhibited, presumably because the mutation removed a cluster of positively charged amino acids required for neutralization of encapsidated RNA. When the deletion was restricted to residues 1 to 31, assembly occurred, but the products were highly heterogeneous. Small bacilliform-like structures and irregular structures as well as wild-type-like T=3 particles were detected. The anticipated T=1 particles, on the other hand, were not observed. We conclude that residues 20 to 30 are not critical for formation of flat protein contacts and formation of T=3 particles. However, the N terminus of the coat protein appears to play an essential role in regulating assembly such that only one product, T=3 particles, is synthesized.

Leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase) is an N-end rule pathway enzyme, which catalyzes the transfer of Leu and Phe from aminoacyl-tRNAs to exposed N-terminal Arg or Lys residues of acceptor proteins. Here, we report the 1.6 Å resolution crystal structure of L/F-transferase (JW0868) from Escherichia coli, the first three-dimensional structure of an L/F-transferase. The L/Ftransferase adopts a monomeric structure consisting of two domains that form a bilobate molecule. The N-terminal domain forms a small lobe with a novel fold. The large C-terminal domain has a highly conserved fold, which is observed in the GCN5-related N-acetyltransferase (GNAT) family. Most of the conserved residues of L/F-transferase reside in the central cavity, which exists at the interface between the N-terminal and C-terminal domains. A comparison of the structures of L/F-transferase and the bacterial peptidoglycan synthase FemX, indicated a structural homology in the C-terminal domain, and a similar domain interface region. Although the peptidyltransferase function is shared between the two proteins, the enzymatic mechanism would differ. The conserved residues in the central cavity of L/F-transferase suggest that this region is important for the enzyme catalysis.

The pseudouridine synthase (Psi synthase) TruA catalyzes the conversion of uridine to pseudouridine at positions 38, 39 and/or 40 in the anticodon stem-loop (ASL) of tRNA. We have determined the crystal structure of TruA from Thermus thermophilus HB8 at 2.25 A resolution. TruA and the other (Psi synthases have a completely conserved active site aspartate, which suggests that the members of this enzyme family share a common catalytic mechanism. The T. thermophilus TruA structure reveals the remarkably flexible structural features in the tRNA-binding cleft, which may be responsible for the primary tRNA interaction. In addition, the charged residues occupying the intermediate positions in the cleft may lead the tRNA to the active site for catalysis. Based on the TruB-tRNA complex structure, the T. thermophilus TruA structure reveals that the tRNA probably makes the melting base pairs move into the cleft, and suggests that a conformational change of the substrate tRNA is necessary to facilitate access to the active site aspartate residue, deep within the cleft.