research advances

Protein pathways

SBKB [doi:10.1038/fa_sbkb.2010.41]

Structural characterization can reveal interaction interfaces between the proteins in a pathway and give insight into potential therapeutics.

The Bacillus anthracis AsbF enzyme active site, a potential drug target studied by the PSI. (PDB 3DX5)

Building pathways of complex biochemical reactions is a key feature of the Protein Structure Initiative (PSI). High-resolution three-dimensional (3D) structures are required to determine the binding interfaces and structural changes that are involved in sequential protein-protein interactions. Recent papers highlight the progress made in defining molecular structures, helping to characterize novel molecules and relating structure to function.

Most enzymes utilize small molecules, such as metals and cofactors, to aid catalysis and expand the range of enzymatic functions. Two studies have provided some insights into the mechanisms of biosynthesis of one such cofactor, coenzyme F420. F420 has unique electron-transfer properties and plays an important role in various metabolic pathways in archea and bacteria, such as methanogenesis, antibiotic synthesis and DNA repair.

F420 is composed of a flavin-like Fo ring, a phospholactate and two (or more) glutamates. F420 synthesis involves six steps which are catalyzed by five enzymes — CofA to CofE. In the fourth step, catalyzed by CofD (2-phospho-L-lactate transferase), the Fo ring is attached to the phospholactate molecule. The first crystal structures of CofD have recently been identified by researchers in the PSI NESG consortium, revealing the binding sites of the Fo and GDP molecules 1 .

One of the goals of structural genomics is to discover proteins with new folding patterns. The last two steps of F420 synthesis involve the GTP-dependent addition of two successive L-glutamate residues and are catalyzed by the enzyme F420-0: γ-glutamyl ligase (CofE). Crystal analysis of CofE by MCSG researchers reveals a novel protein fold containing the putative active site; two-domain monomers form tight butterfly-like dimers. GTP-binding and the active site have been predicted computationally and confirmed experimentally. CofE represents the first member of a new structural family of non-ribosomal peptide synthases 2 .

Structural characterization of pathway molecules is not only revealing novel structures but is driving the field of pathway engineering. New therapeutic agents can be derived from naturally occurring molecules. One group of molecules, the enediynes, are highly efficient at damaging DNA/RNA and are proving to be potent antitumour agents.

The two different groups of enediynes, 9- and 10-membered, are derived from the same core structure. In addition, most enediynes contain additional constituents of polyketide origin, for example, the calicheamicin (CLM) orsellinic acid moiety. For the first time, the crystal structure of the putative CLM orsellinic acid P450 oxidase (CalO2) has been determined and the downstream modification of the polyketide products further characterized. The P450 molecule was shown to contain unique ligand-binding sites compared to other P450 oxidases 3 .

Biosynthetic pathway modifications of CLM have also been analyzed. CLM is targeted to DNA by a novel aryltetrasaccharide comprising an aromatic unit and four unusual carbohydrates. The CLM glycosyltransferases (GTs), CalG1 and CalG4, can produce more than 70 differentially glycosylated CLM variants. The remaining two GTs, CalG2 and CalG3, have now been structurally and biochemically characterized, showing that the biosynthesis of CLM involves a sequential glycosylation pathway. All four CLM GTs share the structural fold that is common to naturally occurring GT structures. This provides a blueprint for engineering novel glycosylation catalysts with optimized therapeutic properties 4 .

New potential drug targets are being discovered by the PSI investigators. Petrobactin is a virulence-associated siderophore produced by Bacillus anthracis and other Bacillus species and is essential for full virulence within a mammalian host. Petrobactin is synthesized by six enzymes encoded in the asb operon. The MCSG researchers have expressed all asb-encoded proteins. The structure of AsbF protein in complex with 3,4-dihydroxybenzoic acid (3,4-DHBA) revealed a 3-DHS dehydratase that converts 3-dehydroshikimate to 3,4-DHBA, a rare isomer of dihydroxybenzoic acid 5 . Most catechol siderophores use 2,3-DHBA as a biosynthetic subunit. The unusual 3,4-DHBA moiety enables the molecule to escape sequestration by the mammalian immune protein siderocalin. Because petrobactin is essential for full virulence, the enzyme is a potential drug target.

In a complementary study, the first structure of a Gram-positive siderophore receptor, YclQ, was determined at the MCSG 6 . The YclQ receptor binds selectively and with high affinity to iron-free and ferric petrobactin, and the petrobactin precursor, 3,4-DHBA. Isogenic disruption mutants in the yclNOPQ transporter, including permease YclN, ATPase YclP, and a substrate-binding protein YclQ, are unable to use either petrobactin or the photoproduct of FePB for iron delivery and growth, in contrast to the wild-type Bacillus subtilis. Complementation of the mutations with the copies of the respective genes restores this capability. Therefore, orthologs of the B. subtilis petrobactin-transporter YclNOPQ in petrobactin-producing Bacillus species are likely contributors to the pathogenicity of these species and provide a potential target for antibacterial strategies.

The complexity of many biochemical pathways has been recognized by those working in the field of human cancer. Although multiple databases exist that provide data on structure, protein-protein interactions or signaling pathways, there has been no integration of these different aspects of cancer biology with structural data in one database. Now, the PSI NESG has constructed a Human Cancer Pathway Protein Interaction Network (HCPIN) by analyzing several classic cancer-associated signaling pathways and presenting these as networks with experimental structures and homology models 7 . The HCPIN network is used to target proteins for structure determination, resulting in more than 50 new human protein structures in the last few years. By providing biochemical and structural information, as well as NMR assignments and key reagents, through a single website, the HCPIN is expanding the applications of structural genomics to biochemical pathways.

Catherine Whitlock

References:
  1. F. Forouhar, M. Abashidze, H. Xu, L. L. Grochowski, J. Seetharaman et al. Molecular insights into the biosynthesis of the F420 coenzyme.

    J. Biol. Chem. 238, 11832-11840 (2008). doi:10.1074/jbc.M710352200

  2. B. Nocek, E. Evdokimova, M. Proudfoot, M. Kudritska, L. L. Grochowski et al. Structure of an amide bond forming F420:gamma-glutamyl ligase from Archaeoglobus fulgidus: a member of a new family of non-ribosomal peptide synthases.

    J. Mol. Biol. 372, 456-469 (2007). doi:10.1016/j.jmb.2007.06.063

  3. J. G. McCoy, H. Johnson, S. Singh, C. A. Bingman, I.-K. Lei, J. S. Thorson et al. Structural characterization of CalO2: a putative orsellinic acid P450 oxidase in the calicheamicin biosynthetic pathway.

    Proteins 74, 50-60 (2009). doi:10.1002/prot.22131

  4. C. Zhang, E. Bitto, R. D. Goff, S. Singh, C. A. Bingman et al. Biochemical and structural insights of the early glycosylation steps in calicheamicin biosnythesis.

    Chem. Biol. 15, 842-853 (2008). doi:10.1016/j.chembiol.2008.06.011

  5. B. F. Pfleger, T. D. Nusca, Y.-c. Kim, J. Y. Lee, C. M. Rath et al. Structural and functional analysis of AsbF: origin of the stealth 3, 4-dihydroxybenzoic acid subunit for petrobactin biosynthesis in Bacillus anthracis.

    Proc. Natl Acad. Sci. USA 105, 17133-17138 (2008). doi:10.1073/pnas.0808118105

  6. A. M. Zawadzka, Y. C. Kim, N. Maltseva, R. Nichiporuk, Y. Fan et al. Characterization of a Bacillus subtilis transporter for petrobactin, an anthrax stealth siderophore.

    Proc. Natl Acad. Sci. USA 106, 21854-21859 (2009). doi:10.1073/pnas.0904793106

  7. Y. J. Huang, D. Hang, L. J. Lu, L. Tong, M. B. Gerstein & G. T. Montelione Targeting the human cancer pathway protein interaction network by structural genomics.

    Mol. Cell Proteomics 10, 2048-2060 (2008). doi:10.1074/mcp.M700550-MCP200

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