The structure of Mengo virus, a representative member of the cardio picornaviruses, is substantially different from the structures of rhino- and polioviruses. The structure of Mengo virus was solved with the use of human rhinovirus 14 as an 8 A resolution structural approximation. Phase information was then extended to 3 A resolution by use of the icosahedral symmetry. This procedure gives promise that many other virus structures also can be determined without the use of the isomorphous replacement technique. Although the organization of the major capsid proteins VP1, VP2, and VP3 of Mengo virus is essentially the same as in rhino- and polioviruses, large insertions and deletions, mostly in VP1, radically alter the surface features. In particular, the putative receptor binding "canyon" of human rhinovirus 14 becomes a deep "pit" in Mengo virus because of polypeptide insertions in VP1 that fill part of the canyon. The minor capsid peptide, VP4, is completely internal in Mengo virus, but its association with the other capsid proteins is substantially different from that in rhino- or poliovirus. However, its carboxyl terminus is located at a position similar to that in human rhinovirus 14 and poliovirus, suggesting the same autocatalytic cleavage of VP0 to VP4 and VP2 takes place during assembly in all these picornaviruses.

The covalently closed terminal hairpins of the linear duplex-DNA genomes of the orthopoxvirus vaccinia and the leporipoxvirus Shope fibroma virus (SFV) have been cloned as imperfect palindromes within circular plasmids in yeast cells and recombination-deficient Escherichia coli. The viral telomeres inserted within these recombinant plasmids are equivalent to the inverted-repeat structures detected as telomeric replicative intermediates during poxvirus replication in vivo. Although the telomeres of vaccinia and SFV show little sequence homology, the termini from both viral genomes exist as AT-rich terminal hairpins with extrahelical bases and alternate "flip-flop" configurations. Using an in vivo replication assay in which circular plasmid DNA was transfected into poxvirus-infected cells, we demonstrated the efficient replication and resolution of the cloned imperfect palindromes to bona fide hairpin termini. The resulting linear minichromosomes, which were readily purified from transfected cells, were shown by restriction enzyme mapping and by electron microscopy to have intact covalently closed hairpin termini at both ends. In addition, staggered unidirectional deletion derivatives of both the cloned vaccinia and SFV telomeric palindromes localized an approximately 200-basepair DNA region in which the sequence organization was highly conserved and which was necessary for the resolution event. These data suggest a conserved mechanism of the resolution of poxvirus telomeres.

Fhospho~ipase C (PL-C) digestion of human low density lipoprotein (LDL) results in llydro~ytic cleavage of the phosph~hoiine head group of phosphatidylchoiine, thereby generating diacylglyceroi. Loss of amphiphillic surface lipids and/or accumulation of diacylgiycerol causes LDL samples to develop turbidity. Examination of PL-C treated LDL by electron microscopy revealed a progressive aggregation of LDL as a function of phosphatidylcholine hydrolysis: fused particles, clusters, and multiple stacked aggregates were observed. Lipid analysis of untreated and aggregated LDL confirmed that the phosphatidylcholille content of the latter had decreased with a corresponding increase in diacylglycerol. It is likely that phospholipolysis created hydrophobic gaps within the surFace monolayer of LDL, thereby inducing LDL fusion and aggregation. When amphipathic a-helix-containing apolipoproteins, such as human apoA-I or Mana'uca se.ua apohpophorin III (apoLp-III) were present, PL-C treated LDL did not aggregate. Compositional analysis of apolipoprotein-containing PL-C LDL showed that phospholipolysis was not affected by the presence of apolipoproteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of lipoproteins re-isolated following incubation with PL-C revealed an association of apoA-I or apolp-III with PL-C digested LDL. Electron microscopy showed no major morphological differences between native LDL and apoprotein stabilized PL-C treated LDL and the average particle diameter of apoA-I stabilized PL-C LDL was 22.5 ?r 2.2 nm versus 22.8 + 1.6 nm for control LDL. Incubation of tritium-labeled apolp-Ill with LDL and PL-C demonstrated that association of apolp-III with PL-C LDL correlated with the extent of phosphohpid hydrolysis, the apohpoproteins apparently being recruited to compensate for the increased hydrophobic surface created by conversion of phosphatidylcholine into diacylglycerol. The results suggest that transient association of amphipathic apolipoproteins with damaged or unstable LDL may provide a mechanism to obviate formation of atherogenic LDL aggregates in vivo.

Apolipophorin III (apoLp-III) from the Sphinx moth, Manduca sexta, is an exchangeable, amphipathic apolipoprotein that alternately exists in water-soluble and lipid-bound forms. It is organized as a five-helix bundle in solution, which has been postulated to open at putative hinge domains to expose the hydrophobic interior, thereby facilitating interaction with the lipoprotein surface (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603-608). To test this hypothesis, we engineered two cysteine residues in apoLp-III, which otherwise lacks cysteine, by site-directed mutagenesis at Asn-40 and Leu-90. Under oxidizing conditions the two cysteines spontaneously form a disulfide bond, which should tether the helix bundle and thereby prevent opening and concomitant lipid interaction. N40C/L90C apoLp-III was overexpressed in Escherichia coli and characterized for disulfide bond formation, secondary structure content, and stability, under both oxidizing and reducing conditions. Functional characterization was carried out by comparing the abilities of the oxidized and reduced protein to associate with modified lipoproteins in vitro. While the reduced form behaved like wild type apoLp-III, the oxidized form was unable to associate with lipoproteins. These results suggest that opening of the helix bundle is required for interaction with lipoproteins and provide a molecular basis for the dual existence of water-soluble and lipid-bound forms of apoLp-III. However, in phospholipid bilayer association assays, wild type, reduced, and oxidized N40C/L90C apoLp-III exhibited similar abilities to transform dimyristoylphosphatidylcholine multilamellar vesicles to disclike complexes, as judged by electron microscopy. These data emphasize that underlying differences exist in initiating or maintaining a stable interaction of apoLp-III with phospholipid disc complexes versus spherical lipoprotein surfaces.Exchangeable apolipoproteins belong to a class of amphipathic ␣-helical proteins that regulate the metabolism and dynamics of lipoprotein interconversions. These proteins reversibly associate with the surface of lipoprotein particles in response to hydrophobic surface availability or their intrinsic ability to displace pre-existing apolipoproteins. This functional property implies an ability to exist in both lipid-free and lipidbound forms in plasma, and it has been proposed that a dramatic conformational change is required for initiation and maintenance of interaction with lipid surfaces (Breiter et al., 1991;Weisgraber, 1994). The sole exchangeable apolipoprotein found in insect hemolymph, apolipophorin III (apoLp-III), 1 provides an excellent model to study lipid association-induced conformational changes of amphipathic exchangeable apolipoproteins. ApoLp-III is well characterized in terms of physicochemical and functional properties (see Van der Horst, 1990;Blacklock and Ryan, 1994; Soulages and Wells, 1994, for reviews). Structural informa...

Dimethyl sulfoxide reductase is a trimeric, membrane-bound, iron-sulfur molybdoenzyme induced in Escherichia coli under anaerobic growth conditions. The enzyme catalyzes the reduction of dimethyl sulfoxide, trimethylamine N-oxide, and a variety of S-and N-oxide compounds. The topology of dimethyl sulfoxide reductase subunits was probed by a combination of techniques. Immunoblot analysis of the periplasmic proteins from the osmotic shock and chloroform wash fluids indicated that the subunits were not free in the periplasm. The reductase was susceptible to proteases in everted membrane vesicles, but the enzyme in outer membrane-permeabilized cells became protease sensitive only after detergent solubilization of the E. coli plasma membrane. Lactoperoxidase catalyzed the iodination of each of the three subunits in an everted membrane vesicle preparation. Antibodies to dimethyl sulfoxide reductase and fumarate reductase specifically agglutinated the everted membrane vesicles. No TnphoA fusions could be found in the dmsA or -B genes, indicating that these subunits were not translocated to the periplasm. Immunogold electron microscopy of everted membrane vesicles and thin sections by using antibodies to the DmsABC, DmsA, or DmsB subunits resulted in specific labeling of the cytoplasmic surface of the inner membrane. These results show that the DmsA (catalytic subunit) and DmsB (electron transfer subunit) are membrane-extrinsic subunits facing the cytoplasmic side of the plasma membrane.Dimethyl sulfoxide (DMSO) reductase of Escherichia coli (5) is a terminal enzyme expressed constitutively under anaerobic conditions and is capable of reducing DMSO and trimethylamine-N-oxide (TMAO). We have reported that the E. coli DMSO reductase is a membrane-bound iron-sulfur molybdoenzyme genetically distinct from the single or dual subunit forms of TMAO reductase (40). The structural genes encoding the enzyme have been cloned, and their DNA sequences have been determined (4, 7). The enzyme has been purified to homogeneity and shown to be composed of three nonidentical subunits: a catalytic subunit (DmsA; 82,600 daltons), an iron-sulfur subunit (DmsB; 23,600 daltons), and a membrane anchor subunit (DmsC; 22,700 daltons).The presence of one constitutive and three inducible forms of TMAO reductase have been demonstrated (32,36,42); however, the significance and precise localization of these multiple forms of TMAO reductase in the energy transduction mechanisms of the cell are not clear. The enzymes responsible for TMAO and DMSO reduction in certain marine organisms and nonphotosynthetic bacteria have been characterized in some detail (2). A single enzyme in Rhodobacter sp. has been found to be responsible for the reduction of both DMSO and TMAO (23). The reductases in these organisms were found to be localized in the periplasmic compartment.