Dongyan Wang

The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium

We describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods. In most cases, the 6X-His-tagged proteins are sufficiently purified (>97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open reading frames of over 16,000 different targeted proteins (or domains) have been cloned as>26,000 constructs. Over the past 10 years, more than 16,000 of these expressed protein, and more than 4400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank (PDB). The methods described here are effective in producing eukaryotic and prokaryotic protein samples in E. coli. This paper summarizes some of the updates made to the protein production pipeline in the last 5 years, corresponding to phase 2 of the NIGMS Protein Structure Initiative (PSI-2) project. The NESG Protein Production Platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities.

Preparation of protein samples for NMR structure, function, and small-molecule screening studies.

In this chapter, we concentrate on the production of high-quality protein samples for nuclear magnetic resonance (NMR) studies. In particular, we provide an in-depth description of recent advances in the production of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein production platform of the Northeast Structural Genomics Consortium and outline our high-throughput strategies for producing high-quality protein samples for NMR studies. Our strategy is based on the cloning, expression, and purification of 6×-His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and analytical expression systems, parallel preparative scale fermentation, and high-throughput purification protocols. The 6×-His affinity tag allows for a similar two-step purification procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (>97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5000 of these proteins have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html), resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this chapter describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening, and structural genomics research.

Solution NMR structure of photosystem II reaction center protein Psb28 from Synechocystis sp. Strain PCC 6803

Oxygenic photosynthesis is initiated by photosystem II (PSII) in the thylakoid membranes of plants, algae and cyanobacteria. PSII is a multi-subunit pigment-protein complex responsible for splitting water into oxygen gas, hydrogen ions and electrons transferred to electron acceptors during photosynthesis.1 Two homologous membrane-spanning proteins D1 (PsbA) and D2 (PsbD) form the PSII complex core.1 Peripherally, two chlorophyll (Chl)-binding inner antenna proteins CP47 (PsbB) and CP43 (PsbC) are bound to the D1-D2 PSII complex core.1 These four large proteins are surrounded by a large number of smaller membrane proteins.2 Most of these small proteins have been observed in the crystal structures of the PSII complex from cyanobacteria.3,4 However, one small protein, Psb28, previously detected as a nonstoichiometric component of PSII,5 was not observed in the crystal structures indicating that Psb28 might not be a true PSII subunit. Recent studies revealed that Psb28 was preferentially bound to PSII core complex lacking CP43 (RC47) and involved in the biogenesis of CP47.6 Understanding the association of Psb28 with the PSII core complex should provide additional insight into its role in PSII-mediated function. However, the structure of Psb28 has remained unknown up until now. In this note, we report the solution NMR structure of Psb28 protein encoded by gene sll1398 [gi|952386] of Synechocystis sp. strain PCC 6803 (SWISS-PROT ID: PSB28_SYNY3, NESG target ID: SgR171).7 This protein, also named Psb13 or ycf79, belongs to the Psb28 protein family (Pfam ID: PF03912), which is currently made up of ~48 protein sequences (E score less than 0.001 using PSI-BLAST, Table S1). Both PSI-BLAST sequence similarity and Dali8 structure similarity searches indicate that this is the first atomic resolution structure available for the Psb28 family. ConSurf9 was used to identify conserved surface residues potentially involved in binding to the PSII core complex.10

Solution NMR structure of the ARID domain of human AT-rich interactive domain-containing protein 3A: a human cancer protein interaction network target.

The AT-rich interactive domain (ARID) of human AT-rich interactive domain-containing protein 3A (ARID3A) has been selected for structural characterization by Northeast Structural Genomics Consortium (residues 218-351 NESG ID HR4394C) as part of our Human Cancer Protein Interaction Network (HCPIN) project. Protein ARID3A belongs to the ARID family DNA-binding protein and is known to play important roles in embryonic patterning, cell lineage gene regulation, and cell cycle control, chromatin remodeling and transcriptional regulations. The solution NMR structure of ARID3A ARID domain consists of eight α-helices α0-α7 and a short β hairpin. Helix α0 and α1 form a V shape, helix α2-α4 and helix α5-α7 form two U shapes, and the V and two U shapes packed orthogonal to each other. The NMR structure of the ARID domain of human ARID3A reported here provides a structural basis for elucidating the regulatory mechanisms of ARID3A function, and the molecular mechanism of ARID3A interactions with DNA. It also has potential value in future drug discovery and design.

NMR structure of F-actin-binding domain of Arg/Abl2 from Homo sapiens.

The Northeast Structural Genomics Consortium has used bioinformatics methods to construct a Human Cancer Pathway Interaction Network (HCPIN),1 a comprehensive 3D structure-function database of human-cancer-associated proteins and protein complexes in the context of their interaction networks. The FABD domain of Arg (Abl-related gene; Abl2) was selected as NESG HCPIN target HR5537. Arg and Abl (Abelson tyrosine kinase; Abl1), the Abl non-receptor tyrosine kinases, link diverse cell surface receptors to the regulation of cytoskeletal dynamics and regulate cytoskeletal reorganization, cell proliferation, survival, adhesion, migration and stress responses in multiple cells types.2–7 Abl and Arg kinases are multi-domain proteins with highly conserved Src kinase homology 3 (SH3), SH2, kinase (SH1) domains in the N-terminal half. The C-terminal halves of these kinases are more divergent, however, the functions encoded by the C-terminus are critical for the overall functions of these proteins.4 Although Abl and Arg exhibit overlapping expression in many tissues, Arg is most highly expressed in brain, thymus, spleen, and muscle.8 Differences in regulation of cell migration by Abl and Arg have also been reported.7 Consistent with the nuclear and cytoplasmic localization of Abl and the predominant cytoplasmic localization of Arg, three nuclear localization signals (NLS), one nuclear export signal NES motifs and a DNA-binding domain are found in Abl but not in Arg.4 Abl and Arg share a C-terminal calponin homology F-actin-binding domain (FABD) with ~44% sequence identity, which distinguishes Abl family kinases from other non-receptor tyrosine kinases.4,9,10 Preceding this shared FABD, Arg has a microtubule-binding (MT) domain and a second talin-like F-actin-binding domain that is characterized by an I/LWEQ sequence while Abl kinase has a globular (G)-actin binding domain.4,9,10,11 Arg uses its FABD to anchoring actin and other cytoskeletal partner for signal transfer and other biological functions.4 Both human Abl FABD and Arg FABD belong to the F_actin_bind protein domain family (Pfam12 entry PF08919) comprised of 21 sequences. The NMR structure of the human Abl FABD, the only available structure in F_actin_bind family, has been reported recently13 ; it forms a compact left-handed four helix bundle in solution. The Arg FABD was selected as HCPIN target by NESG for structure determination.1 The NMR structure of human Arg FABD reported here can serve as a structural basis for elucidating the molecular mechanism of Arg pathway, for studies of protein complex formation, and potentially in small molecule drug design.