PublicationsHart Team
ORF-selector ESPRIT: A second generation library screen for soluble protein expression employing precise open reading frame selection.
An, Y., Yumerefendi, H., Mas, P.J., Chesneau, A. & Hart, D.J.
J Struct Biol. 2011 Apr 16.
Here we present ORF-selector ESPRIT, a 9-fold enhanced version of our technology for screening incremental truncation libraries to identify soluble high yielding constructs of challenging proteins. Gene fragments are truncated at both termini to access internal domains and the resulting reading frame problem is addressed by an unbiased, intein-based open reading frame selection yielding only in-frame DNA inserts. This enriched library is then subcloned into a standard high-level expression plasmid where tens of thousands of constructs can be assayed in a two-step process using colony- and liquid-handling robots to isolate rare highly expressing clones useful for production of multi milligram quantities of purifiable proteins. The p85alpha protein was used to benchmark the system resulting in isolation of all known domains, either alone or in tandem. The human kinase IKK1 was then screened resulting in purification of a predicted internal domain. This strategy provides an integrated, facile route to produce soluble proteins from challenging and poorly understood target genes at quantities compatible with structural biology, screening applications and immunisation studies. The high genetic diversity that can be sampled opens the way to study more diverse systems including multisubunit complexes.
PubMed
Interaction of the influenza A virus polymerase PB2 C-terminal region with importin {alpha} isoforms provides insights into host adaptation and polymerase assembly.
Boivin, S. & Hart, D.J.
J Biol Chem. 2011 Jan 7.
In the adaptation of avian viruses to mammalian hosts, mutations in the viral polymerase, notably in the PB2 subunit, play an important role. A PB2 C-terminal domain rich in putative host adaptation residues has been shown to bind importin alpha nuclear import receptors. Adaptation has been proposed to involve binding of PB2 to importins of the new host. To date PB2-importin complexes have been characterised semi-quantitatively with no precise measurement of binding parameters. To investigate the effects of adaptive mutations on importin interaction and selectivity, surface plasmon resonance was used to compare the binding rate constants and affinities of avian H5N1 and human H3N2 PB2 C-terminal variants with importin isoforms human alpha 1, 3, 5 and 7, and avian alpha 1. Using purified proteins eliminates host environment effects and permits measurement of intrinsic affinities and rates of complex formation and dissociation. Two effects were observed: firstly adaptive mutations D701N, R702K and S714R in the NLS domain increased 2-4 fold the association rates with avian and human importins; secondly, measurement of different structural forms of the PB2 C-terminus demonstrated that the upstream 627 domain reduced binding affinity, consistent with a steric clash predicted from crystal structures. From these kinetic data, structural analyses and the data of others, a model is proposed in which an increase in charged surface residues during host adaptation increases the association rate of PB2 to cytoplasmic importins, and where the C-terminal 627-NLS domain may reorganise upon importin binding, consistent with a role in active polymerase assembly.
PubMed
CoESPRIT: a library-based construct screening method for identification and expression of soluble protein complexes.
An, Y., Meresse, P., Mas, P.J. & Hart, D.J.
PLoS One. 2011 Feb 22;6(2):e16261.
Structural and biophysical studies of protein complexes require multi-milligram quantities of soluble material. Subunits are often unstable when expressed separately so co-expression strategies are commonly employed since in vivo complex formation can provide stabilising effects. Defining constructs for subunit co-expression experiments is difficult if the proteins are poorly understood. Even more problematic is when subunit polypeptide chains co-fold since individually they do not have predictable domains. We have developed CoESPRIT, a modified version of the ESPRIT random library construct screen used previously on single proteins, to express soluble protein complexes. A random library of target constructs is screened against a fixed bait protein to identify stable complexes. In a proof-of-principle study, C-terminal fragments of the influenza polymerase PB2 subunit containing folded domains were isolated using importin alpha as bait. Separately, a C-terminal fragment of the PB1 subunit was used as bait to trap N-terminal fragments of PB2 resulting in co-folded complexes. Subsequent expression of the target protein without the bait indicates whether the target is independently stable, or co-folds with its partner. This highly automated method provides an efficient strategy for obtaining recombinant protein complexes at yields compatible with structural, biophysical and functional studies.
PubMed
Influenza A virus polymerase: structural insights into replication and host adaptation mechanisms.
Boivin, S., Cusack, S., Ruigrok, R.W. & Hart, D.J.
J Biol Chem. 2010 Sep 10;285(37):28411-7. doi: 10.1074/jbc.R110.117531. Epub 2010Jun 10.
The heterotrimeric RNA-dependent RNA polymerase of influenza viruses catalyzes RNA replication and transcription activities in infected cell nuclei. The nucleotide polymerization activity is common to both replication and transcription processes, with an additional cap-snatching function being employed during transcription to steal short 5'-capped RNA primers from host mRNAs. Cap-binding, endonuclease, and polymerase activities have long been studied biochemically, but structural studies on the polymerase and its subunits have been hindered by difficulties in producing sufficient quantities of material. Recently, because of heightened effort and advances in expression and crystallization technologies, a series of high resolution structures of individual domains have been determined. These shed light on intrinsic activities of the polymerase, including cap snatching, subunit association, and nucleocytoplasmic transport, and open up the possibility of structure-guided development of new polymerase inhibitors. Furthermore, the activity of influenza polymerase is highly host- and cell type-specific, being dependent on the identity of a few key amino acid positions in the different subunits, especially in the C-terminal region of PB2. New structures demonstrate the surface exposure of these residues, consistent with ideas that they might modulate interactions with host-specific factors that enhance or restrict activity. Recent proteomic and genome-wide interactome and RNA interference screens have suggested the identities of some of these potential regulators of polymerase function.
PubMed