Colleen Ciccosanti

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.

Dimer Interface of the Effector Domain of Non-structural Protein 1 from Influenza A Virus AN INTERFACE WITH MULTIPLE FUNCTIONS

Non-structural protein 1 from influenza A virus, NS1A, is a key multifunctional virulence factor composed of two domains: an N-terminal double-stranded RNA (dsRNA)-binding domain and a C-terminal effector domain (ED). Isolated RNA-binding and effector domains of NS1A both exist as homodimers in solution. Despite recent crystal structures of isolated ED and full-length NS1A proteins from different influenza virus strains, controversy remains over the actual biologically relevant ED dimer interface. Here, we report the biophysical properties of the NS1A ED from H3N2 influenza A/Udorn/307/1972 (Ud) virus in solution. Several lines of evidence, including 15N NMR relaxation, NMR chemical shift perturbations, static light scattering, and analytical sedimentation equilibrium, demonstrate that Ud NS1A ED forms a relatively weak dimer in solution (Kd = 90 ± 2 μm), featuring a symmetric helix-helix dimer interface. Mutations within and near this interface completely abolish dimerization, whereas mutations consistent with other proposed ED dimer interfaces have no effect on dimer formation. In addition, the critical Trp-187 residue in this interface serves as a sensitive NMR spectroscopic marker for the concentration-dependent dimerization of NS1A ED in solution. Finally, dynamic light scattering and gel shift binding experiments demonstrate that the ED interface plays a role in both the oligomerization and the dsRNA binding properties of the full-length NS1A protein. In particular, mutation of the critical tryptophan in the ED interface substantially reduces the propensity of full-length NS1A from different strains to oligomerize and results in a reduction in dsRNA binding affinity for full-length NS1A.

Combining NMR and EPR Methods for Homodimer Protein Structure Determination

There is a general need to develop more powerful and more robust methods for structural characterization of homodimers, homo-oligomers, and multiprotein complexes using solution-state NMR methods. In recent years, there has been increasing emphasis on integrating distinct and complementary methodologies for structure determination of multiprotein complexes. One approach not yet widely used is to obtain intermediate and long-range distance constraints from paramagnetic relaxation enhancements (PRE) and electron paramagnetic resonance (EPR)-based techniques such as double electron electron resonance (DEER), which, when used together, can provide supplemental distance constraints spanning to 10-70 A. In this Communication, we describe integration of PRE and DEER data with conventional solution-state nuclear magnetic resonance (NMR) methods for structure determination of Dsy0195, a homodimer (62 amino acids per monomer) from Desulfitobacterium hafniense. Our results indicate that combination of conventional NMR restraints with only one or a few DEER distance constraints and a small number of PRE constraints is sufficient for the automatic NMR-based structure determination program CYANA to build a network of interchain nuclear Overhauser effect constraints that can be used to accurately define both the homodimer interface and the global homodimer structure. The use of DEER distances as a source of supplemental constraints as described here has virtually no upper molecular weight limit, and utilization of the PRE constraints is limited only by the ability to make accurate assignments of the protein amide proton and nitrogen chemical shifts.

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

Structural Basis of O6-Alkylguanine Recognition by a Bacterial Alkyltransferase-like DNA Repair Protein

Alkyltransferase-like proteins (ATLs) are a novel class of DNA repair proteins related to O6-alkylguanine-DNA alkyltransferases (AGTs) that tightly bind alkylated DNA and shunt the damaged DNA into the nucleotide excision repair pathway. Here, we present the first structure of a bacterial ATL, from Vibrio parahaemolyticus (vpAtl). We demonstrate that vpAtl adopts an AGT-like fold and that the protein is capable of tightly binding to O6-methylguanine-containing DNA and disrupting its repair by human AGT, a hallmark of ATLs. Mutation of highly conserved residues Tyr23 and Arg37 demonstrate their critical roles in a conserved mechanism of ATL binding to alkylated DNA. NMR relaxation data reveal a role for conformational plasticity in the guanine-lesion recognition cavity. Our results provide further evidence for the conserved role of ATLs in this primordial mechanism of DNA repair.

Solution NMR structure of Dsy0195 homodimer from Desulfitobacterium hafniense: first structure representative of the YabP domain family of proteins involved in spore coat assembly

Protein domain family YabP (PF07873) is a family of small protein domains that are conserved in a wide range of bacteria and involved in spore coat assembly during the process of sporulation. The 62-residue fragment of Dsy0195 from Desulfitobacterium hafniense, which belongs to the YabP family, exists as a homodimer in solution under the conditions used for structure determination using NMR spectroscopy. The structure of the Dsy0195 homodimer contains two identical 62-residue monomeric subunits, each consisting of five anti-parallel beta strands (β1, 23–29; β2, 31–38; β3, 41–46; β4, 49–59; β5, 69–80). The tertiary structure of the Dsy0195 monomer adopts a cylindrical fold composed of two beta sheets. The two monomer subunits fold into a homodimer about a single C2 symmetry axis, with the interface composed of two anti-parallel beta strands, β1–β1′ and β5b–β5b′, where β5b refers to the C-terminal half of the bent β5 strand, without any domain swapping. Potential functional regions of the Dsy0195 structure were predicted based on conserved sequence analysis. The Dsy0195 structure reported here is the first representative structure from the YabP family.