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Research Themes Protein design

Retrospective: 7,000 Structures Closer to Understanding Biology

SBKB [doi:10.1038/sbkb.2015.23]
Featured Article - July 2015
Short description: As PSI:Biology comes to a close this month, we take a look back at all the biological advances made by the PSI Network.


The active site of NDM-1, in which many of the residues responsible for substrate binding and catalysis are on flexible loops. Image courtesy of Jim Sacchettini.

The Protein Structure Initiative has solved 6,900 protein structures in 15 years as a result of newly developed high-throughput (HTP) technologies. As the PSI comes to a close this month, we highlight some of the biological advances made by the PSI:Biology Network. For a broader review of PSI results, we invite you to revisit the ∼500 research and technical reviews written by Nature Publishing Group, and the Featured Systems illustrated by David Goodsell on the SBKB's new homepage beginning in July 2015.

The Protein Structure Initiative was created in 2000 to extend the results of the Human Genome Project by determining the three-dimensional structures of key proteins in an analogous high-throughput (HTP) manner. The pilot phase (2000–2005) initiated the creation of pipelines for protein production and structure determination to quickly isolate thousands of constructs and obtain more than 1,000 structures as a proof of concept. During PSI-2 (2005–2010), the dials were turned up as the PSI aimed to cover more structural space, adding 5,000 more structures to the PDB consisting of key proteins, domains and novel representative structures. During the third and final phase of the PSI:Biology program (2010–2015), HTP efforts were directed towards more difficult biological and biomedical problems, such as membrane proteins, complexes and networks.

The PSI:Biology HTP centers selected targets from a wide variety of biological subject areas, from basic cell biology to infectious diseases to drug discovery. For example, researchers at the PSI JCSG characterized many novel proteins and domains of unknown function from human gut flora to help determine their role in maintaining human health 1, 2, 3 . They also used their HTP pipelines to study viral-host interactions. After ten years and thousands of constructs, JCSG researchers captured the structure of HIV-1 envelope proteins in complex with antibodies from HIV-infected individuals 4, 5 , providing insights into the very elongated and specific interaction interface. This knowledge should be useful to direct structure-based vaccine development.

Researchers at PSI MCSG focused on proteins related to microbial pathogenesis. One example from their numerous achievements was the structure of Klebsiella pneumoniae New Delhi metalloproteinase-1 (NDM-1), an enzyme responsible for inactivating nearly all available β-lactam antibiotics 6, 7 . Through the use of X-ray crystallography and computational techniques, the PSI MCSG researchers and their biological partners at PSI MTBI were able to solve the first structure of NDM-1 and then model modes for substrate recognition and the catalytic mechanism, explaining how the active site could utilize zinc and other metal cofactors depending on reaction conditions. This study will enable the development of better NDM-1 inhibitors.

PSI NESG researchers continued covering the human cancer proteome 8, 9 , and also explored the rules of protein folding and design with community collaborator David Baker 10 . To accomplish this, hundreds of engineered sequences were modeled, and samples were then created and structurally determined. The researchers found that the sequence wasn't the sole determining factor driving folding, but the length of the sequences and loops that connect different secondary structure elements could favor a particular fold.

The PSI NYSGRC, partnering with PSI IFN, aimed to cover the secreted proteome that modulates adaptive and innate immunity, in the hopes of developing new therapeutics against chronic infections or cancer. This project faced many challenges regarding eukaryotic protein production and cocrystallization, but ultimately produced a half-dozen structures of wild-type and mutant cytokine LIGHT 11 in complex with its inhibitor, the soluble TNF decoy 3 receptor (DcR3), and its TNF modulation receptors HVEM and LT?R. These studies allowed NYSGRC investigators to determine the basis for substrate promiscuity for both LIGHT and DcR3, and will open the door for the development of new mechanistic probes.

The PSI:Biology program also set its sights on empowering the membrane protein community, since there were relatively few such structures in the PDB. Nine membrane protein centers focused on GPCRs, transporters and other representatives of the membrane proteome. The PSI GPCR Network determined 16 unique GPCR superfamily structures, many of which were cocrystallized with antagonists to facilitate further drug discovery. One particular milestone was the first structure of the human glucagon receptor 12 , determined in collaboration with the PSI JCSG. This structure offered the first look at Class B (secretin-like) GPCRs, and revealed a much wider active site than that observed in Class A receptors. In addition, the structure of the delta-opioid receptor 13 was selected as one of Science Signaling's Signaling Breakthroughs of 2014 14 . PSI NYCOMPS researchers solved over 30 structures of human membrane proteins or their bacterial orthologs to elucidate the mechanisms of key biological functions. The 2.3-MDa rabbit ryanodine receptor mediates muscle action through the release of calcium from intracellular stores to the cytosol, and had evaded structural determination for a long time due to its large size; this target was finally tackled by PSI NYCOMPS using cryo-electron microscopy 15 .

The 13 HTP-enabled Biology Partners benefited from the expertise of the HTP centers, as they were able to obtain hundreds of protein samples and use the centers' structure determination pipelines to advance research on T-cell biology, chemokines, stem cells, cell-cell and cell-matrix interactions, tight junctions, the mitochondrion, epigenetics, tuberculosis inhibitors and many more fields. Their outcomes are too numerous to report here, but can be surveyed in the 200+ research articles published by the Partners. We hope that these, and all PSI biological advances, will enable research for years to come.

References

  1. D. Das et al. Structure and function of the DUF2233 domain in bacteria and in the human mannose 6-phosphate uncovering enzyme.
    J Biol Chem. 288, 16789-16799 (2013). doi:10.1074/jbc.M112.434977

  2. N. M. Fleischman et al. Molecular characterization of novel pyridoxal-5′-phosphate-dependent enzymes from the human microbiome.
    Protein Sci. 23, 1060-1076 (2014). doi:10.1002/pro.2493

  3. P. Natarajan et al. Structure and sequence analyses of Bacteroides proteins BVU_4064 and BF1687 reveal presence of two novel predominantly-beta domains, predicted to be involved in lipid and cell surface interactions.
    BMC Bioinformatics. 16, 7 (2015). doi:10.1186/s12859-014-0434-7

  4. J. P. Julien et al. Crystal structure of a soluble cleaved HIV-1 envelope trimer.
    Science. 342, 1477-1483 (2013). doi:10.1126/science.1245625

  5. L. Kong et al. Supersite of immune vulnerability on the glycosylated face of HIV-1 envelope glycoprotein gp120.
    Nat Struct Mol Biol. 20, 796-803 (2013). doi:10.1038/nsmb.2594

  6. Y. Kim et al. Structure of apo- and monometalated forms of NDM-1-a highly potent carbapenem-hydrolyzing metallo-beta-lactamase.
    PLoS One. 6, e24621 (2011). doi:10.1371/journal.pone.0024621

  7. Y. Kim et al. NDM-1, the ultimate promiscuous enzyme: substrate recognition and catalytic mechanism.
    FASEB J. 27, 1917-1927 (2013). doi:10.1096/fj.12-224014

  8. S. V. Pulavarti et al. Solution NMR structures of immunoglobulin-like domains 7 and 12 from obscurin-like protein 1 contribute to the structural coverage of the human cancer protein interaction network.
    J Struct Funct Genomics. 15, 209-214 (2014). doi:10.1007/s10969-014-9185-y

  9. X. Xu et al. Solution NMR structures of homeodomains from human proteins ALX4, ZHX1, and CASP8AP2 contribute to the structural coverage of the Human Cancer Protein Interaction Network.
    J Struct Funct Genomics. 15, 201-207 (2014). doi:10.1007/s10969-014-9184-z

  10. N. Koga et al. Principles for designing ideal protein structures.
    Nature. 491, 222-227 (2012). doi:10.1038/nature11600

  11. W. Liu et al. Mechanistic basis for functional promiscuity in the TNF and TNF receptor superfamilies: structure of the LIGHT:DcR3 assembly.
    Structure. 22, 1252-1262 (2014). doi:10.1016/j.str.2014.06.013

  12. F. Y. Siu et al. Structure of the human glucagon class B G-protein-coupled receptor.
    Nature. 499, 444-449 (2013). doi:10.1038/nature12393

  13. G. Fenalti et al. Molecular control of delta-opioid receptor signalling.
    Nature. 506, 191-196 (2014). doi:10.1038/nature12944

  14. J. D. Berndt & W. Wong 2014: signaling breakthroughs of the year.
    Sci Signal. 8, eg1 (2015). doi:10.1126/scisignal.aaa4696

  15. R. Zalk et al. Structure of a mammalian ryanodine receptor.
    Nature. 517, 44-49 (2015). doi:10.1038/nature13950

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