Response of SCP-2L domain of human MFE-2 to ligand removal: binding site closure and burial of peroxisomal targeting signal

In the study of the structure and function relationship of human MFE-2, we have investigated the dynamics of human MFE-2SCP-2L (hSCP-2L) and its response to ligand removal. A comparison was made with homologous rabbit SCP-2. Breathing and a closing motion are found, identifiable with an adjustment in size and a closing off of the binding pocket. Crucial residues for structural integrity have been identified. Particularly mobile areas of the protein are loop 1 that is connecting helices A and C in space, and helix D, next to the entrance of the pocket. In hSCP-2L, the binding pocket gets occupied by Phe93, which is making a tight hydrophobic contact with Trp36. In addition, it is found that the C-terminal peroxisomal targeting signal (PTS1) that is solvent exposed in the complexed structure becomes buried when no ligand is present. Moreover, an anti-correlation exists between burial of PTS1 and the size of the binding pocket. The results are in accordance with plant nsLTPs, where a similar accommodation of binding pocket size was found after ligand binding/removal. Furthermore, the calculations support the suggestion of a ligand-assisted targeting mechanism.     

Localization of a potyvirus and the viral genome-linked protein in wild potato leaves at an early stage of systemic infection

The upper noninoculated 'sink' leaves of the wild potato species, Solanum commersonii, were studied for distribution of Potato virus A (PVA) at an early stage of systemic infection. Viral RNA was detected by in situ hybridization, and five viral proteins were localized using immunohistochemical staining in leaf sections. Initial systemic infection foci were found at the vicinity of major and minor veins. In these infection foci, the viral coat protein, cylindrical inclusion protein, and helper component-proteinase colocalized with viral RNA in parenchyma and mesophyll cells, but none of these were detected in companion cells (CC). In contrast, VPg, which is the N-proximal half of the NIa protein (separated from the C-terminal proteinase domain, NIapro, by an autocatalytic cleavage) and acts as a viral genome-linked protein, was detected in CC in the infection foci, but only at an early stage of virus unloading. Outside the infection foci, conspicuous signals for VPg were readily and exclusively detected in CC of many veins in all vein classes in the absence of signals for NIapro, other viral proteins, and viral RNA. Taken together, our data indicate that both major and minor veins may unload PVA in the sink leaves of potato. The data suggest that VPg is translocated from inoculated source leaves to the sink leaves, where it accumulates in CC at an early stage of systemic infection. These findings suggest that VPg may be a 'phloem protein' that specifically acts in CC in the sink leaves to facilitate virus unloading.     

Ultrastructural changes in skeletal muscle of the tail of the lizard Hemidactylus mabouia immediately following autotomy

Araújo, T.H., Faria, F.P., Katchburian, E. and Freymüller, E. (2009). Ultrastructural changes in skeletal muscle of the tail of the lizard Hemidactylus mabouia immediately following autotomy. —Acta Zoologica (Stockholm) 91 : 440–446. Although autotomy and subsequent regeneration of lizard tails has been extensively studied, there is little information available on ultrastructural changes that occur to the muscle fibers at the site of severance. Thus, in the present study, we examine the ultrastructure of the musculature of the remaining tail stump of the lizard Hemidactylus mabouia immediately after autotomy. Our results show that exposed portions of the skeletal muscle fibers of the stump that are unprotected by connective tissue bulge to produce large mushroom‐like protrusions. These exposed portions show abnormal structure but suffer no leakage of cytoplasmic contents. Many small and large vesicular structures appeared between myofibrils in the interface at this disarranged region (distal) and the other portion of the fibers that remain unchanged (proximal). These vesicles coalesce, creating a gap that leads to the release of the mushroom‐like protrusion. So, our results showed that after the macroscopic act of autotomy the muscular fibers release part of the sarcoplasm as if a second and microscopic set of autotomic events takes place immediately following the macroscopic act of autotomy. Presumably these changes pave the way for the formation of a blastema and the beginning of regeneration.     

Crystal structure of 2-enoyl-CoA hydratase 2 from human peroxisomal multifunctional enzyme type 2

2-Enoyl-CoA hydratase 2 is the middle part of the mammalian peroxisomal multifunctional enzyme type 2 (MFE-2), which is known to be important in the beta-oxidation of very-long-chain and alpha-methyl-branched fatty acids as well as in the synthesis of bile acids. Here, we present the crystal structure of the hydratase 2 from the human MFE-2 to 3A resolution. The three-dimensional structure resembles the recently solved crystal structure of hydratase 2 from the yeast, Candida tropicalis, MFE-2 having a two-domain subunit structure with a C-domain complete hot-dog fold housing the active site, and an N-domain incomplete hot-dog fold housing the cavity for the aliphatic acyl part of the substrate molecule. The ability of human hydratase 2 to utilize such bulky compounds which are not physiological substrates for the fungal ortholog, e.g. CoA esters of C26 fatty acids, pristanic acid and di/trihydroxycholestanoic acids, is explained by a large hydrophobic cavity formed upon the movements of the extremely mobile loops I-III in the N-domain. In the unliganded form of human hydratase 2, however, the loop I blocks the entrance of fatty enoyl-CoAs with chain-length >C8. Therefore, we expect that upon binding of substrates bulkier than C8, the loop I gives way, contemporaneously causing a secondary effect in the CoA-binding pocket and/or active site required for efficient hydration reaction. This structural feature would explain the inactivity of human hydratase 2 towards short-chain substrates. The solved structure is also used as a tool for analyzing the various inactivating mutations, identified among others in MFE-2-deficient patients. Since hydratase 2 is the last functional unit of mammalian MFE-2 whose structure has been solved, the organization of the functional units in the biologically active full-length enzyme is also discussed.     

Harpin of Pseudomonas syringae pv. phaseolicola harbors a protein binding site

Harpin HrpZ of plant-pathogenic bacterium Pseudomonas syringae elicits a hypersensitive response (HR) in some nonhost plants, but its function in the pathogenesis process is still obscure. HrpZ-interacting proteins were identified by screening a phage-display library of random peptides. HrpZ of the bean pathogen P. syringae pv. phaseolicola (HrpZPph) shows affinity to peptides with a consensus amino acid motif W(L)ARWLL(G/L). To localize the peptide-binding site, the hrpZPph gene was mutagenized with randomly placed 15-bp insertions, and the mutant proteins were screened for the peptide-binding ability. Mutations that inhibited peptide-binding localized to the central region of hrpZPph, which is separate from the previously determined HR-inducing region. Antiserum raised against one of the hrpZPph-binding peptides recognized small proteins in bean, tomato, parsley, and Arabidopsis thaliana but none in tobacco. On native protein blots, hrpZPph bound to a bean protein with similar pI as the protein recognized by the peptide antiserum. The result suggests a protein-protein interaction between the harpin and a host plant protein, possibly involved in the bacterial pathogenesis.     

Biology and epidemics of Candidatus Liberibacter species,psyllid‐transmitted plant‐pathogenic bacteria

Candidatus Liberibacter species are Gram‐negative bacteria that live as phloem‐limited obligate parasites in plants, and are associated with several plant diseases. These bacteria are transmitted by insects called psyllids, or jumping plant lice, which feed on plant phloem sap. Citrus huanglongbing (yellow shoot) or citrus greening disease is associated with three different species of Ca. Liberibacter – Ca. L. asiaticus, Ca. L. africanus and Ca. L. americanus – all originally found on different continents. Ca. L. asiaticus is the most severe pathogen, spread by Asian citrus psyllid Diaphorina citri and causing devastating epidemics in several countries. Ca. L. africanus occurs in Africa where it is spread by the African citrus psyllid Trioza erytreae. Ca. Liberibacter solanacearum is associated with diseases in several solanaceous plants, and transmitted by potato psyllid Bactericera cockerelli. Zebra chip disease is causing large damage in potato crops in North America. In Europe Ca. Liberibacter solanacearum is associated with diseases of the Apiaceae family of plants, carrot and celery, and transmitted by psyllids Trioza apicalis and Bactericera trigonica. When Ca. Liberibacter is suspected as the disease agent, the diagnosis is confirmed by DNA‐based detection methods. Ca. Liberibacter‐associated plant diseases can be controlled by using healthy plant propagation material, eradicating symptomatic plants, and by controlling the psyllid populations spreading the disease.     

The thiolase superfamily: condensing enzymes with diverse reaction specificities

The formation of a carbon-carbon bond is an essential step in the biosynthetic pathways by which fatty acids and polyketides are made. The thiolase superfamily enzymes catalyse this carbon-carbon-bond formation via a thioester-dependent Claisen-condensation-reaction mechanism. In this way, fatty-acid chains and polyketides are made by sequentially adding simple building blocks, such as acetate units, to the growing molecule. A common feature of these enzymes is a reactive cysteine residue that is transiently acylated in the catalytic cycle. The wide catalytic diversity of the thiolase superfamily enzymes is of great interest. In particular, the type-III polyketide synthases make complicated compounds of great biological importance using multiple, subsequent condensation reactions, which are all catalysed in the same active-site cavity. The crucial metabolic importance of the bacterial fatty-acid-synthesizing enzymes stimulates in-depth studies that aim to develop efficient anti-bacterial drugs.     

Crystal structure of yeast peroxisomal multifunctional enzyme: structural basis for substrate specificity of (3R)-hydroxyacyl-CoA dehydrogenase units

(3R)-hydroxyacyl-CoA dehydrogenase is part of multifunctional enzyme type 2 (MFE-2) of peroxisomal fatty acid beta-oxidation. The MFE-2 protein from yeasts contains in the same polypeptide chain two dehydrogenases (A and B), which possess difference in substrate specificity. The crystal structure of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase AB heterodimer, consisting of dehydrogenase A and B, determined at the resolution of 2.2A, shows overall similarity with the prototypic counterpart from rat, but also important differences that explain the substrate specificity differences observed. Docking studies suggest that dehydrogenase A binds the hydrophobic fatty acyl chain of a medium-chain-length ((3R)-OH-C10) substrate as bent into the binding pocket, whereas the short-chain substrates are dislocated by two mechanisms: (i) a short-chain-length 3-hydroxyacyl group ((3R)-OH-C4) does not reach the hydrophobic contacts needed for anchoring the substrate into the active site; and (ii) Leu44 in the loop above the NAD(+) cofactor attracts short-chain-length substrates away from the active site. Dehydrogenase B, which can use a (3R)-OH-C4 substrate, has a more shallow binding pocket and the substrate is correctly placed for catalysis. Based on the current structure, and together with the structure of the 2-enoyl-CoA hydratase 2 unit of yeast MFE-2 it becomes obvious that in yeast and mammalian MFE-2s, despite basically identical functional domains, the assembly of these domains into a mature, dimeric multifunctional enzyme is very different.