[ PDB file ] [ PubMed link ]

Chu Wai knocked this structure off working together with Ann. The structure highlights how many partners can interact with these LMO proteins and how they do so through these very short sequence motifs (that aren’t very well conserved). It makes you realize that these vast unstructured tracts found in most transcription factors could be full of function…

[ PDB file ] [ PubMed link ]

Our collaborator (and former PhD student in Jacqui’s lab) Daniel Ryan identified a conserved region in CHD1 that hadn’t previously been appreciated. It turned out to be nicely structured and Biswa and Ana together were able to work it up to solve its structure by NMR. Steph helped out by looking to see whether it was a DNA-binding domain – seems to have some DNA-binding activity that is non-specific – which might well be functionally relevant.

[ PDB file ] [ PubMed link ]

Ana identified and determined the structure (using NMR – rather than her favoured crystallographic method…) of a small domain from the N-terminal region of the chromatin remodeller CHD4 (a member of our favourite chromatin remodelling complex). The domain resembles HMG-type domains that are often involved in non-specific DNA binding. Binding experiments suggest that this domain might have a penchant for poly-ADPribose – a branched chain nucleic acid that is found at sites of DNA damage (where NuRD is also found…).

[ PDB file ] [ PubMed link ]

Haloalkane dehalogenases (HLDs) catalyse the hydrolysis of haloalkanes to alcohols, offering a biological solution for toxic haloalkane industrial wastes. Hundreds of putative HLD genes have been identified in bacterial genomes, but relatively few enzymes have been characterised. We identified two novel HLDs in the genome of Mycobacterium rhodesiae strain JS60, an isolate from an organochlorine-contaminated site: DmrA and DmrB. Both recombinant enzymes were active against C2-C6 haloalkanes, with a preference for brominated linear substrates. However, DmrA had higher activity against a wider range of substrates, such as 4-bromobutyronitrile. We determined the crystal structure of selenomethionyl DmrA to 1.7 Å resolution. A spacious active site and alternate conformations of a methionine side-chain in the slot access tunnel may contribute to the broad substrate activity of DmrA. M. rhodesiae JS60 can utilise 1-iodopropane, 1-iodobutane and 1-bromobutane as sole carbon and energy sources, and this ability appears to be conferred predominantly through DmrA, which shows significantly higher levels of upregulation in response to haloalkanes than DmrB.

[ PDB file ] [ PubMed link ]

Nina Ripin, a German Masters student, was able to determine this structure with help from David Jacques and Mitchell Guss. analysis of the structure indicates that although the N-terminal ZF (NF) can modulate GATA1 DNA binding, the NF binds DNA so poorly under physiological conditionsthat it cannot play a direct role in DNA looping (a suggestion made recently). Rather, the ability of the NF to stabilise transcriptional complexes through protein-protein interactions, and thereby recruit looping factors such as Ldb1, seems a more likely model for GATA-mediated looping.

[ PDB file ] [ PubMed link ]

Many years ago, Gerd and I spotted a section of FOG1 outside the ZFs that looked like it might have been ordered. After only 12 years or so, Joel finally finished solving the structure of the domain (which is only ~110 residues, so who knows *why* it took him so long!). It turns out to be a PR domain – a fold that is essentially the same as the SET domains that act as methyltransferases – mostly adding methyl groups tolysines at the N-terminal tails of histones. This means that FOG1 is *potentially* an enzyme, although we were unable to demonstrate methyltransferase activity (via a collaboration with Masoud Vedadi in Toronto). It also makes FOG1 a member of a family of 16 other human proteins that contain this domain – some of which *have* been demonstrated to be enzymes. So, we shall see…

[ PDB file ] [ PubMed link ]

A *third* structure of the RbAp48-MTA1 subcomplex – just for good measure. No extra interactions this time though…

[ PDB file ] [ PubMed link ]

A second structure of the RbAp48-MTA1 subcomplex. A little bit more of MTA1 gave a substantially higher affinity, which can be rationalized in the structure by the formation of an additional hydrophobic cluster.

[ PDB file ] [ PubMed link ]

RbAP48 and MTA1 are components of the Nucleosome Remodeling and Deacetylase (NuRD) complex. This structure provides a small step towards understanding how the complex is put together. Still plenty more to be done though!

[ PDB file ] [ PubMed link ]

Islet 1 (Isl1) is a transcription factor of the LIM-homeodomain (LIM-HD) protein family. LIM-HD proteins all contain two protein-interacting LIM domains, a DNA-binding homeodomain (HD), and a C-terminal region. In Isl1, the C-terminal region also contains the LIM homeobox 3 (Lhx3)-binding domain (LBD), which interacts with the LIM domains of Lhx3. The LIM domains of Isl1 have been implicated in inhibition of DNA binding potentially through an intramolecular interaction with or close to the HD. Here we investigate the LBD as a candidate intramolecular interaction domain. Competitive yeast-two hybrid experiments indicate that the LIM domains and LBD from Isl1 can interact with apparently low affinity, consistent with no detection of an intermolecular interaction in the same system. Nuclear magnetic resonance studies show that the interaction is specific, whereas substitution of the LBD with peptides of the same amino acid composition but different sequence is not specific. We solved the crystal structure of a similar but higher affinity complex between the LIM domains of Isl1 and the LIM interaction domain from the LIM-HD cofactor protein LIM domain-binding protein 1 (Ldb1) and used these coordinates to generate a homology model of the intramolecular interaction that indicates poorer complementarity for the weak intramolecular interaction. The intramolecular interaction in Isl1 may provide protection against aggregation, minimize unproductive DNA binding, and facilitate cofactor exchange within the cell.

[ PDB file ] [ PubMed link ]

Another step in the story of understanding how Staph aureus steals iron from host red blood cells.

[ PDB file ] [ PubMed link ]

This structure shows that the second NEAT domain of IsdH is involved in recognizing hemoglobin rather than extracting heme/iron. That task falls to the third NEAT domains (as elegantly shown from Claire Dickson’s structure in the same paper).

[ PDB file ] [ PubMed link ]

We have shown previously that a two-zinc finger unit found in the transcriptional coregulator ZNF217 recognizes DNA but with an affinity and specificity that is lower than other classical ZF arrays. To investigate the basis for these differences, we determined the structure of a ZNF217-DNA complex. We show that although the overall position of the ZFs on the DNA closely resembles that observed for other ZFs, the side-chain interaction pattern differs substantially from the canonical model. The structure also reveals the presence of two methyl-p interactions, each featuring a tyrosine contacting a thymine methyl group. To our knowledge, interactions of this type have not previously been described in classical ZF-DNA complexes. We speculate that relatively low affinity/specificity interactions of this type might be important for gene regulation.

[ PDB file ] [ PubMed link ]

In developing neuronal tissue expression of the key specification factor Lhx3 is supplemented by the redundant protein Lhx4. In motor neurons the balance between Lhx3 and Isl1 is critical for proper cell fate determination. In order to achieve the correct stoichiometric balance between Lhx3/4 and Isl1, Isl2 is additionally expressed to supplement Isl1 protein levels. The structure of the Lhx4 LIM domains complexed with the Isl2 LIM-interaction domain illustrates strong structural conservation in the interactions made between Lhx3/4 and Isl1/2 (when compared with our Lhx3/Isl1 structure). This emphasises the need to so strictly preserve these redundant interactions for the correct developmental outcome.

[ PDB file ] [ PubMed link ]

Fungi make a hydrophobic coating on their spores by assembling an amphipathic surface monolayer made from small proteins termed hydrophobins. In an effort to try to understand the structural basis for the non-covalent assembly process (in which long, thin, amyloid-like structures known as rodlets are formed), Ingrid Macindoe in Margie’s lab determined the structure of a point mutant – F72G of the Neurospora hydrophobin EAS. This mutant takes much longer to form rodlets – although the rodlets that it finally forms closely resemble wildtype ones. Surprisingly the structure of F72G was indistinguishable from the wild-type protein. On the other hand, a small but measureable increase in flexibility was observed for the mutated region, suggesting that this increased dynamics is responsible for the longer lag time in rodlet formation. This work starts to give us an idea of which parts of EAS are important for rodlet formation.

[ PDB file ] [ PubMed link ]

Pathogens must steal iron from their hosts to establish infection. In mammals, hemoglobin (Hb) represents the largest reservoir of iron, and pathogens express Hb-binding proteins to access this source. Here, we show how one of the commonest and most significant human pathogens, Staphylococcus aureus, captures Hb as the first step of an iron-scavenging pathway. The x-ray crystal structure of Hb bound to a domain from the Isd (iron-regulated surface determinant) protein, IsdH, is the first structure of a Hb capture complex to be determined. Surface mutations in Hb that reduce binding to the Hb-receptor limit the capacity of S. aureus to utilize Hb as an iron source, suggesting that Hb sequence is a factor in host susceptibility to infection. The demonstration that pathogens make highly specific recognition complexes with Hb raises the possibility of developing inhibitors of Hb binding as antibacterial agents.

[ PDB file ] [ PubMed link ]

All GATA1-activated genes are co-occupied by LMO2, LDB1 and a number of other proteins. As part of a project to understand how these proteins come together at such gene promoters, we have determined the solution structure of the C-terminal LIM domain of LMO2 bound to its cognate target peptide in ldb1. This domain is able to bind to the N-terminal zinc finger of GATA1 – and we have used NMR data to build a model of this complex (see above!).

[ PDB file ] [ PubMed link ]

All GATA1-activated genes are co-occupied by LMO2, LDB1 and a number of other proteins. Here, we have used a range of NMR and mutagenesis data to create a model of the complex formed by LMO2, LDB1 and the GATA1 N-finger. Surprisingly, the model suggests that GATA1 N-finger will also be able to contact FOG1 at the same time as LMO2 and gives us more insight into the molecular details surrounding gene activation by multi-protein transcriptional assemblies.

[ PDB file ] [ PubMed link ]

It has been known for some time that the sidechains of several lysine residues in the transcription factor GATA1 can be modified by acetylation, but the function of these modifications has not been so clear. Our collaborator Gerd Blobel from the Children’s Hospital in Philadelphia has shown that, although acetylation doesn’t reduce the in vitro binding of GATA1 to DNA, acetylation of these lysines is essential for the localization of GATA1 to a chromatinized template. He more recently showed that acetylated GATA1 is recognized by Brd3. Together with Gerd, we have shown that the Brd3 bromodomain recognized a *doubly* acetylated GATA1 motif, and we have determined the structure of Brd3-bromodomain 1 bound to a GATA1 peptide containing two acetyllysine residues.

[ PDB file ] [ PubMed link ]

The (�a)8 barrel is one of the most common protein folds and it is thought it was one of the very first protein folds to emerge. Our collaborator Wayne Patrick previously isolated a three-quarter barrel that is soluble and almost as thermostable as full-length protein (PRAI). Our NMR structure of this three-quarter barrel shows that, despite missing a quarter of the normal foled, the subdomain is monomeric, well ordered and adopts a native-like structure in solution. These proteins – they’re crazy…