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Barnwal R.P.,University of Washington | Van Voorhis W.C.,Seattle Structural Genomics Center for Infectious Disease | Van Voorhis W.C.,University of Washington | Varani G.,University of Washington
Acta Crystallographica Section F: Structural Biology and Crystallization Communications

Nearly complete resonance assignment and the high-resolution NMR structure of the acyl-carrier protein from Borrelia burgdorferi, a target of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) structure- determination pipeline, are reported. This protein was chosen as a potential target for drug-discovery efforts because of its involvement in fatty-acid biosynthesis, an essential metabolic pathway, in bacteria. It was possible to assign >98% of backbone resonances and >92% of side-chain resonances using multidimensional NMR spectroscopy. The NMR structure was determined to a backbone r.m.s.d. of 0.4 Å and contained four -helices and two 310-helices. A structure-homology search revealed that this protein is highly similar to the acyl-carrier protein from Aquifex aeolicus. Source

Abendroth J.,Emerald Biostructures | Abendroth J.,Seattle Structural Genomics Center for Infectious Disease | Gardberg A.S.,Emerald Biostructures | Gardberg A.S.,Seattle Structural Genomics Center for Infectious Disease | And 13 more authors.
Journal of Structural and Functional Genomics

The Seattle Structural Genomics Center for Infectious Disease (SSGCID) focuses on the structure elucidation of potential drug targets from class A, B, and C infectious disease organisms. Many SSGCID targets are selected because they have homologs in other organisms that are validated drug targets with known structures. Thus, many SSGCID targets are expected to be solved by molecular replacement (MR), and reflective of this, all proteins are expressed in native form. However, many community request targets do not have homologs with known structures and not all internally selected targets readily solve by MR, necessitating experimental phase determination. We have adopted the use of iodide ion soaks and single wavelength anomalous dispersion (SAD) experiments as our primary method for de novo phasing. This method uses existing native crystals and in house data collection, resulting in rapid, low cost structure determination. Iodide ions are non-toxic and soluble at molar concentrations, facilitating binding at numerous hydrophobic or positively charged sites. We have used this technique across a wide range of crystallization conditions with successful structure determination in 16 of 17 cases within the first year of use (94% success rate). Here we present a general overview of this method as well as several examples including SAD phasing of proteins with novel folds and the combined use of SAD and MR for targets with weak MR solutions. These cases highlight the straightforward and powerful method of iodide ion SAD phasing in a high-throughput structural genomics environment. © 2011 Springer Science+Business Media B.V. Source

Staker B.L.,Seattle Structural Genomics Center for Infectious Disease | Staker B.L.,Seattle Biomedical Research Institute | Buchko G.W.,Seattle Structural Genomics Center for Infectious Disease | Buchko G.W.,Pacific Northwest National Laboratory | And 3 more authors.
Current Opinion in Microbiology

According to a Pew Research study published in February 2015, there are 37 antibacterial programs currently in clinical trials in the United States. Protein structure-based methods for guiding small molecule design were used in at least 34 of these programs. Typically, this occurred at an early stage (drug discovery and/or lead optimization) prior to an Investigational New Drug (IND) application, although sometimes in retrospective studies to rationalize biological activity. Recognizing that structure-based methods are resource-intensive and often require specialized equipment and training, the NIAID has funded two Structural Genomics Centers to determine structures of infectious disease species proteins with the aim of supporting individual investigators' research programs with structural biology methods. © 2015. Source

Buchko G.W.,Pacific Northwest National Laboratory | Buchko G.W.,Seattle Structural Genomics Center for Infectious Disease | Abendroth J.,Seattle Structural Genomics Center for Infectious Disease | Robinson H.,Brookhaven National Laboratory | And 9 more authors.
Journal of Structural and Functional Genomics

Macrophage migration inhibitory factor (MIF) is a eukaryotic cytokine that affects a broad spectrum of immune responses and its activation/inactivation is associated with numerous diseases. During protozoan infections MIF is not only expressed by the host, but, has also been observed to be expressed by some parasites and released into the host. To better understand the biological role of parasitic MIF proteins, the crystal structure of the MIF protein from Giardia lamblia (Gl-MIF), the etiological agent responsible for giardiasis, has been determined at 2.30 Å resolution. The 114-residue protein adopts an α/β fold consisting of a four-stranded β-sheet with two anti-parallel α-helices packed against a face of the β-sheet. An additional short β-strand aligns anti-parallel to β4 of the β-sheet in the adjacent protein unit to help stabilize a trimer, the biologically relevant unit observed in all solved MIF crystal structures to date, and form a discontinuous β-barrel. The structure of Gl-MIF is compared to the MIF structures from humans (Hs-MIF) and three Plasmodium species (falciparum, berghei, and yoelii). The structure of all five MIF proteins are generally similar with the exception of a channel that runs through the center of each trimer complex. Relative to Hs-MIF, there are differences in solvent accessibility and electrostatic potential distribution in the channel of Gl-MIF and the Plasmodium-MIFs due primarily to two "gate-keeper" residues in the parasitic MIFs. For the Plasmodium MIFs the gate-keeper residues are at positions 44 (Y→R) and 100 (V→D) and for Gl-MIF it is at position 100 (V→R). If these gate-keeper residues have a biological function and contribute to the progression of parasitemia they may also form the basis for structure-based drug design targeting parasitic MIF proteins. © 2013 Springer Science+Business Media Dordrecht (Outside the USA). Source

Zhang Y.,Pacific Northwest National Laboratory | Zhang Y.,University of Texas Health Science Center at San Antonio | Gardberg A.S.,Emerald Biostructures | Gardberg A.S.,Seattle Structural Genomics Center for Infectious Disease | And 7 more authors.

Botulinum neurotoxin (BoNT), the causative agent of the deadly neuroparalytic disease botulism, is the most poisonous protein known for humans. Produced by different strains of the anaerobic bacterium Clostridium botulinum, BoNT effects cellular intoxication via a multistep mechanism executed by the three modules of the activated protein. Endocytosis, the first step of cellular intoxication, is triggered by the ∼50 kDa, heavy-chain receptor-binding domain (HCR) that is specific for a ganglioside and a protein receptor on neuronal cell surfaces. This dual receptor recognition mechanism between BoNT and the host cell's membrane is well documented and occurs via specific intermolecular interactions with the C-terminal sub-domain, Hcc, of BoNT-HCR. The N-terminal sub-domain of BoNT-HCR, Hcn, comprises ∼50% of BoNT-HCR and adopts a β-sheet jelly roll fold. While suspected in assisting cell surface recognition, no unambiguous function for the Hcn sub-domain in BoNT has been identified. To obtain insights into the potential function of the Hcn sub-domain in BoNT, the first crystal structure of a BoNT with an organic ligand bound to the Hcn sub-domain has been obtained. Here, we describe the crystal structure of BoNT/CD-HCR determined at 1.70 Å resolution with a tetraethylene glycol (PG4) moiety bound in a hydrophobic cleft between β-strands in the β-sheet jelly roll fold of the Hcn sub-domain. The PG4 moiety is completely engulfed in the cleft, making numerous hydrophilic (Y932, S959, W966, and D1042) and hydrophobic (S935, W977, L979, N1013, and I1066) contacts with the protein's side chain and backbone that may mimic in vivo interactions with the phospholipid membranes on neuronal cell surfaces. A sulfate ion was also observed bound to residues T1176, D1177, K1196, and R1243 in the Hcc sub-domain of BoNT/CD-HCR. In the crystal structure of a similar protein, BoNT/D-HCR, a sialic acid molecule was observed bound to the equivalent residues suggesting that residues T1176, D1177, K1196, and R1243 in BoNT/CD may play a role in ganglioside binding. © 2013 Elsevier Masson SAS. All rights reserved. Source

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