March 11, 2011
Complete title: Crystal structure of Toxoplasma gondii porphobilinogen synthase: insights on octameric structure and porphobilinogen formation
J Biol Chem (2011) [Epub ahead of print]. PMID:21383008.
Jaffe, E.K., Shanmugan, D., Gardberg, A., Dieterich, S., Sankaran, B., Stewart, L.J., Myler, P.J., and Roos, D.S.
ABSTRACT
Porphobilinogen synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product porphobilinogen. Unlike prior structures containing a substrate-derived heterocycle directly bound to an active site zinc ion, the product-bound TgPBGS active site contains neither zinc nor magnesium, placing in question the common notion that all PBGS enzymes require an active site metal ion. Unlike human PBGS, the TgPBGS octamer contains magnesium ions at the intersections between pro-octamer dimers, which are presumed to function in allosteric regulation. TgPBGS includes N- and C-terminal regions that differ considerably from previously solved crystal structures. In particular, the C-terminal extension found in all apicomplexan PBGS enzymes forms an intersubunit β-sheet, stabilizing a pro-octamer dimer and preventing formation of hexamers that can form in human PBGS. The TgPBGS structure suggests strategies for the development of parasite-selective PBGS inhibitors.
PHOTO: Paper models of structure created by Anna Gardberg.
March 11, 2011
J Struct Funct Genomics. 2011 Feb 26.
Begley DW, Hartley RC, Davies DR, Edwards TE, Leonard JT, Abendroth J, Burris CA, Bhandari J, Myler PJ, Staker BL, Stewart LJ.
ABSTRACT
As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.
February 4, 2011
Complete Title: Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer
Don Lorimer, Amy Raymond, John Walchli, Mark Mixon, Adrienne Barrow, Ellen Wallace, Rena Grice, Alex Burgin1, and Lance Stewart
BMC Biotechnology 2009, 9:37
Background
With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript.
Results
In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38α), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias.
Conclusion
The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.
February 4, 2011
Don Lorimer, Amy Raymond, John Walchli, Mark Mixon, Adrienne Barrow, Ellen Wallace, Rena Grice, Alex Burgin, and Lance Stewart
BMC Biotechnology, 2009 9:36
To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering.
Results
An interactive Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies.
Conclusion
We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis-match specific endonuclease error correction in combination with PIPE cloning. In a sister manuscript we present data on how Gene Composer designed genes and protein constructs can result in improved protein production for structural studies
December 22, 2010
Di Paolo JA, Huang T, Balazs M, Barbosa J, Barck KH, Bravo BJ, Carano RA, Darrow J, Davies DR, Deforge LE, Diehl L, Ferrando R, Gallion SL, Giannetti AM, Gribling P, Hurez V, Hymowitz SG, Jones R, Kropf JE, Lee WP, Maciejewski PM, Mitchell SA, Rong H, Staker BL, Whitney JA, Yeh S, Young WB, Yu C, Zhang J, Reif K, Currie KS.
Nature Chemical Biology (2010) Nov. 28 [Epub ahead of print]. PMID: 21113169
As part of a collaboration with CGI Pharmaceuticals, Inc., Emerald scientists contributed two X-ray crystal structures of Bruton's tyrosine kinase (Btk) bound to key small molecule inhibitors. Emerald's work was allowed CGI and their partners to gain important chemical insight into specificity factors between an novel Btk inhibitor, CGI1746, and the reference compound dasatinib.
December 17, 2010
Full title: Identifying regulators for EAG1 channels with a novel electrophysiololgy and tryptophan fluorescence based screen.
Brelidze TI, Carlson AE, Davies DR, Stewart LJ, Zagotta WN.
PLoS One. 2010 Sep 2;5(9). pii: e12523.
Scientists at Emerald BioStructures have co-authored a peer-reviewed article with University of Washington Professor William N. Zagotta that demonstrates an innovative use of the Emerald Fragments of Life™ ligand library to identify Regulators for EAG1 Channels with a Novel Electrophysiology and Tryptophan Fluorescence based screen,” was published today in PloS One, an open-access journal for the communication of peer-reviewed scientific and medical research.
December 17, 2010
Yamada S, Hatta M, Staker BL, Watanabe S, Imai M, Shinya K, Sakai-Tagawa Y, Ito M, Ozawa M, Watanabe T, Sakabe S, Li C, Kim JH, Myler PJ, Phan I, Raymond A, Smith E, Stacy R, Nidom CA, Lank SM, Wiseman RW, Bimber BN, O'Connor DH, Neumann G, Stewart LJ, Kawaoka Y.
PLoS Pathog. 2010 Aug 5;6(8). pii: e1001034.
Emerald scientists and members of the Seattle Structural Genomics Center for Infectious Disease (SSGCID), reported peer reviewed work from an international collaboration that describes how the H1N1 flu virus has used a new biochemical trick to spread efficiently in humans.
November 25, 2010
Davies et al. Crystal structure of a transition state mimic for Tdp1 assembled from vanadate, DNA, and a topoisomerase I-derived peptide.
(2003) Chem Biol. 10: 139–147
October 1, 2010
Ahn YM, Clare M, Ensinger CL, Hood MM, Lord JW, Lu WP, Miller DF, Patt WC, Smith BD, Vogeti L, Kaufman MD, Petillo PA, Wise SC, Abendroth J, Chun L, Clark R, Feese M, Kim H, Stewart L, Flynn DL.
Bioorg Med Chem Lett. 2010 Oct 1;20(19):5793-8. Epub 2010 Aug 3.
Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.