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…
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.
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…).
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.
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.
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…
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.
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.
Hydrophobins spontaneously self-assemble into functional amyloid monolayers at hydrophobic:hydrophilic interfaces. These amphipathic monolayers have amazing physicochemical properties and have been suggested for many different applications. Vanessa, under Margie’s supervision, determined the st ructure of DewA. While the pattern of four disulfide bonds that is a defining feature of hydrophobins is conserved, the arrangement and composition of secondary-structure elements in DewA are quite different to what has been observed in other hydrophobin structures. Her NMR data also showed that DewA populates two conformations in solution, both of which are assembly competent. One conformer forms a dimer at high concentrations, but this dimer is off-pathway to fibril formation and may represent an assembly control mechanism. These data highlight the structural differences between fibril-forming hydrophobins and those that form amorphous monolayers.
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.
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.
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.
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!).
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.
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.
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…
The Nucleosome Remodeling and Deacetylase (NuRD) complex is essential for the normal regulation of gene expression in a wide range of organisms (even plants!). We are slowly trying to build up a picture of how NuRD works at a mechanistic level. One of the NuRD components, CHD4, contains two PHD-type zinc-finger domains. In collaboration with Tanya Kutateladze from the University of Colorado at Denver, we have shown that one of these two PHD domains can recognize the N-terminal tail of histone H3 when specifically modified by trimethylation of K9 (a repressive mark). We have determined the structure of the PHD:H3 tail complex, showing how the methylated K9 is recognized (on the right of the pic).
The Nucleosome Remodeling and Deacetylase (NuRD) complex is essential for the normal regulation of gene expression in a wide range of organisms (even plants!). We are slowly trying to build up a picture of how NuRD works at a mechanistic level. One of the NuRD components, CHD4, contains two PHD-type zinc-finger domains. This picture shows the structure of the first PHD domains, which we have shown in collaboration with Tanya Kutateladze from the University of Colorado at Denver can recognize the N-terminal tail of histone H3.
FOG1 is a coregulator protein that assists the transcription factor GATA1 in the control of gene expression during erythroid development. Gerd Blobel, a collaborator of ours in Philadelphia, showed that at least part of the mechanism by which FOG1 regulates GATA1 expression is through its ability to recruit the multiprotein NuRD (Nucleosome Remodeling and Deacetylase) complex. We have determined the structure of a complex formed between the NuRD-binding region of FOG1 (residues 1-15) and the RbAp48 component of the NuRD complex. RbAp48 forms a beta-propellor and a basic sequence in the FOG1 peptide docks into a cavity at one end of the propellor.
The two zinc fingers of ZRANB2 (formerly known as ZNF265) can bind to single-stranded RNA with high sequence specificity. We have determined the structure of one of these fingers bound to its RNA target site (AGGUAA – determined by SELEX). The structure reveals a new class of RNA-binding domain. This ZnF forms a unique guanine-Trp-guanine aromatic stack, and the core nucleotides (GGU) are recognized by an extensive network of protein side-chain hydrogen bonds. Also notable are the two-headed hydrogen bonds that are formed between arginine side-chains and the two guanines – these interactions appear to provide strong selection for guanine in those positions.
MED-1 is a GATA-family transcription factor that is essential for development in Caenorhabditis elegans. It’s single GATA-type zinc finger was shown by Morris Maduro to bind to a long and slightly divergent DNA site (GTATACTTTT), compared to the well-known consensus for mammalian GATA-family zinc fingers (AGATAA). We used a combination of NMR spectroscopy, gel shifts and mutagenesis to demonstrate that the MED-1 zinc finger forms an additional C-terminal helix, which is induced only upon binding to DNA. This helix is inserted into the major groove of DNA to bind to the 3′ end of the recognition sequence. These data demonstrate the difficulties in predicting the structures of protein complexes from sequence data alone – the additional helix was not predicted by secondary structure prediction algorithms.
[ PDB file ]
The two zinc fingers of ZRANB2 (formerly known as ZNF265) can bind to single-stranded RNA with high sequence specificity. In addition to determining the structures of these two domains, we have used a combination of chemical shift mapping and mutagenesis to define the RNA-binding surface of these domains. Other zinc fingers in the same structural class (RanBP2-type zinc fingers) have been shown to mediate protein-protein interactions, another reminder of the versatility of small zinc-binding domains as recognition motifs.
We used intein technology to generate a circular protein complex between LMO4 and the LIM-binding domain of Ldb1 (Ldb1-LID). The proteins, which bind in a head-to-tail fashion are joined by a flexible linker at each end. The circular complex is more stable than a tethered complex where the C-terminus of LMO4 is linked to the N-terminus of Ldb1-LID, however, the crystal structure of this complex shows that the structures (in blue and gold), are identical. Curiously, when the order of a single tethering step is reversed (Ldb1-LID-to-LMO4) the resulting protein is just as stable as the circular complex.
In developing motor neurons Isl1 displaces Lhx3 as the binding partner of the essential LIM-HD cofactor protein Ldb1. Isl1 provides Lhx3 with a decoy binding domain from a region in the C-terminus of Isl1 that, despite very low sequence identity, binds Lhx3 is essentially the same way as Ldb1. The LIM domains from Lhx3 are shown as a grey surface. The Lhx3-binding domain from Isl1 is shown in green and the LIM-interaction domain from Ldb1 from the Lhx3-Ldb1 structure above is shown in gold.
We have used a variety of NMR, mutagenesis and binding data to build a model of the interaction between zinc finger 5 from the neuronal transcription factor MyT1 and its cognate DNA site. The model was built using the HADDOCK data-driven docking software (from Alexandre Bonvin) and shows that the domain fits snugly into the DNA major groove, making a number of base specific hydrogen bonds and hydrophobic contacts.
MyT1 is a zinc finger transcription factor that is involved in neuronal development, controlling genes that are important for myelin sheath formation. It contains 7 zinc fingers with an unusual consensus sequence, and these domains have been shown previously to be responsible for the DNA-binding properties of MyT1. We have determined the solution structure of one of these zinc fingers, as a preliminary step towards understanding how these domains recognize DNA. The structure is different from all other known classes of zinc fingers, and contains no elements of regular secondary structure. We have gone on to examine the binding of this domain to DNA (see below!).
The THAP (Thanatos-associated protein) domain is a recently discovered zinc-binding domain found in proteins involved in transcriptional regulation, cell-cycle control, apoptosis and chromatin modification. It contains a single zinc atom ligated by cysteine and histidine residues within a Cys-X(2-4)-Cys-X(35-53)-Cys-X(2)-His consensus. We determined the NMR solution structure of the THAP domain from Caenorhabditis elegans C-terminal binding protein (CtBP) and show that it adopts a fold containing a treble clef motif, with some similarity to the zinc finger-associated domain (ZAD) from Drosophila Grauzone. We have also shown using gel-shift data that CtBP-THAP is able to bind DNA. Other THAP domains have been reported to be involved in mediating protein interactions, suggesting that THAP domains might exhibit a functional diversity similar to that observed for classical and GATA-type zinc fingers.
Homeodomain-only protein (HOP) is an 8-kDa transcriptional corepressor that is essential for the normal development of the mammalian heart. A combination of sequence comparison and our structural data revealed that HOP consists entirely of a homeodomain, and it is the only human protein to have this topology. We have also shown that, unlike other classic homeodomain proteins, HOP does not appear to interact with DNA, and it appears that it instead functions as a bridge in the formation of HDAC-type repressive complexes on DNA. However, the mechanism by which this repression occurs is still only partially resolved. Our results demonstrate that the homeodomain fold has been co-opted during evolution for functions other than sequence-specific DNA binding.
p22HBP is a 22-kDa mammalian protein that is highly upregulated during erythroid development, and appears to be a target gene of GATA-1. Its function is currently unknown, although it has been reported to bind to a range of different porphyrins, suggesting a role in heme biosynthesis. We have determined the structure of p22HBP and used HSQC titration data to map its porphyrin binding site. Interestingly, our structure reveals that p22HBP has structural (but not sequence) homology to a bacterial multi-drug resistance protein BmrR that functions by binding to a variety of small hydrophobic drug molecules.
Hydrophobins are small fungal proteins that form polymeric fibrils, known as rodlets. These rodlets form a matted coating on the surface of aerial structures like air-dispersed spores. The coating is extremely amphipathic, with an outward facing hydrophobic surface that “water-proofs” the spores. The coating is also extremely robust and is being considered for material science applications. Our structure of the monomeric hydrophobin from Neurospora crassa shows that it is made up entirely of beta structure (top right), forming a 4-stranded beta-barrel. The structure contains two very flexible loops (seen top left), which may play a role in fibril formation. The electrostatic surface properties of the EAS structure are also consistent with the amphipathic nature of the rodlets (bottom), and have allowed us to create a model for fibril formation (see below).
This CHANCE peptide was one of two (+DFF5) designed to mimic the GATA-binding surface of the transcriptional repressor U-shaped. NMR structural work showed both that DFF7 was well-folded (right) and that the grafted residues occupied positions that were similar (although not identical) to those found in the native USF1 protein (left). Both DFF5 and DFF7 displayed measureable binding to GATA-1, as judged by NMR titration experiments, although the nature of the binding may not be as specific as intended.
This CHANCE peptide was designed to mimic the GATA-binding surface of the transcriptional repressor U-shaped. We have previously defined the surface involved in this interaction, and we attempted to transplant this surface onto the minimal CHANCE domain. NMR structural work showed both that DFF5 was well-folded and that the grafted residues occupied positions that were similar (although not identical) to those found in the native USF1 protein. In the picture, the grafted residues are shown overlayed with the same residues from USF1.
This is one of the first CHANCE peptides that was designed to emulate the binding function of another protein. The N-terminal nucleocapsid domain (NUC1, left) of the HIV-1 nucleocapsid protein is able to bind to a short oligonucleotide from the virus’ genome. We identified the binding residues from NUC1 and ‘grafted’ them onto our CHANCE domain, in an arrangement that appeared to mimic their spatial orientation in the NUC1 protein. The structure of DFF2 shows that the grafted residues do indeed form a surface that bears a reasonable resemblance to the template protein. Unfortunately, NMR titration data indicated that DFF2 was not actually able to specifically recognize the NUC1 target oligonucleotide. Back to the drawing board!
This is a second minimalised version of the CHANCE domain. This time, 13 of the 25 residues have been mutated (mostly to alanine), and the fold is retained. These two stripped down domains retain only the zinc-binding residues and the residues that make significant numbers of contacts.
This is a minimalised version of the CHANCE domain. 12 of the 25 residues have been mutated (mostly to alanine), but the fold is retained. This ‘stripping down’ of the surface of the domain is the first step in re-designing the domain to have new binding functionality. This structure contains ~50% Ala and has a very similar fold to the wild type sequence, indicating that zinc-ligands and a few key hydrophobic residues are all that is required for structure.
In order to get an idea of the conformation of SS3 at a sub-zero temperature, we determined the structure of SS3 at -5 degrees Celcius. Getting the solution down to that temperature without freezing was straighforward – slow cooling together with the salt present in the buffer (~50 mM phosphate) does the trick. No dramatic changes were observed – the structure comprised a large and a small helical region, connected by a short, slightly less well defined sequence.
Many animals that live in extremely cold climates produce proteins that act to prevent the formation of ice crystals in the bodily fluids of the organism. Antarctic fish produce a range of such proteins, as the water temperatures can drop below 0 degrees Celsius. We have determined the structure of the type I antifreeze protein (SS3) from the blood of the shorthorn sculpin at both 5 degrees and -5 degrees. The structure largely comprises a long alpha-helix that displays conserved Thr residues (red) along a single surface. These residues are important for preventing the growth of ice crystals, although the mechanism of action is not currently understood. An additional short helix exists at the N-terminus of the protein, although the conformational relationship between the two helices is not well defined. This is the first solution structure of a wild-type type I antifreeze protein.
[ PDB file ]
This structure shows a two-residue insertion mutant of BKLF F3 that possesses native function but not native structure. This zinc finger retains the ability to bind to GATA-1, even though it contains two additional bulky residues in a structurally important region.
Red blood cells contain an excess of the alpha-chain of hemoglobin over the beta-chain. It is known that excess alpha-globin is unstable, and its precipitation can lead to membrane damage and apoptosis. This phenomenon is the root of the beta-thalassemia phenotype, in which sufferers cannot make sufficient beta-globin. How is the normal excess of alpha-globin kept ‘safe’? The answer seems to lie in AHSP – a protein that appears to act as a specific chaperone of alpha-globin, binding to the globin and preventing its precipitation. We have determined the structure of AHSP and used NMR titration methods and mutagenesis to reveal which residues are involved in mediating this specific interaction. In addition, as part of a collaboration with Yigong Shi at Princeton, the structure of the AHSP:a-globin complex was determined by X-ray crystallography. This structure confirmed many of our predictions based on biophysical analysis of the interaction. Collaborative work is also ongoing with Peter Lay in the School of Chemistry at USyd to examine the properties of the metal-binding site in the complex.
FOG-1 is vital for the development of red blood cells and platelets. Originally identified as a Friend of GATA, GATA-independent functions are now emerging. Recently, we identified TACC3 as a protein partner of FOG-1. FOG-1 uses its third (of nine) ZnF to bind the coiled-coil domain of TACC3. The structure of FOG-1 finger 3 (left) shows that it is a typical classical CCHH finger (BMRB 6216). NMR and mutagenesis data identified the residues important for the interaction between FOG-1 (yellow) and TACC3 (blue) and this allowed us to use HADDOCK to calculate the structure of the FOG-TACC3 complex (right).
The transcriptional regulator LMO4 consists entirely of two LIM domains, and must bind to the partner protein ldb1 in order to be transported into the nucleus and carry out its role in the regulation of lymphogenesis. We have determined the structure of a complex formed betwen full-length LMO4 and the LMO4-binding region of ldb1 using X-ray crystallography. The interaction interface is completely extended, and the ldb1 peptide (yellow) forms additional beta-strands on beta-hairpins present in both of the LMO4 LIM domains. This tandem beta-zipper arrangement has been observed in one other recent structure – a bacterial pathogen that binds cellular fibronectin repeats (Potts et al., Nature 2003, 423, 177). This structure can now serve as a scaffold from which inhibitors of the LMO4:ldb1 interaction may be designed.
BKLF is a transcriptional repressor involved in erythropoiesis. It has been shown to interact with both DNA (CACCC boxes) and the C-terminal zinc finger of GATA-1. We have determined its solution structure as a first step towards characterizing this interaction. In addition, we have shown that variants of this domain in which the final zinc-ligating histidine is mutated to residues such as asparagine and alanine are still functional in binding both GATA-1 and, in the context of a 3-zinc-finger construct, DNA.
Shown is the structure of a mutant of Mi2-P2, with the second flexible loop (called L3) replaced by the corresponding region from another PHD domain from the protein WSTF. This structure shows that the PHD fold is indeed preserved in the mutant. The backbone of L3-WSTF overlays well with Mi2b-P2 except for some minor differences observed at residues 20-22 and 43-46, which correspond to the two flexible regions (shown in orange and purple).
We have used NMR methods to determine the solution structure of the second PHD finger from the transcriptional regulator Mi2b. This structure indicates that PHD fingers contain two relatively flexible loop regions (shown in orange and red) and a conserved stable core that ligates two zinc atoms (zinc ligation residues are shown in yellow and green). By using a strategy of mutagenic substitution and expansion, we have also shown that a wide variety of mutations in these loops can be tolerated without major disruption of the PHD fold. If you want to see for yourself, the solution structure of one of these mutants is shown in the next figure!
Bacterial superantigens are involved in overstimulating T cells. YPM has a structure that is unique amongst classical superantigens. Typical superantigens consist of 2 b-strand domains and a central α-helix. However YPM consists of 8 antiparallel β-strands that form a jelly roll motif. There are 2 main regions in the YPM structure, one that is quite rigid and another that undergoes intermediate exchange. The unique structure of YPM suggests that it may activate T cells through a mechanism that is distinct from other superantigens.
We have solved the structure of a complex of the transcription regulators LMO4 and ldb1 (PDB: 1M3V). To do this we engineered FLIN4, a fusion of the LID domain of ldb1 and the N-terminal LIM domain of LMO4. The structure of FLIN4 shows that ldb1-LID (yellow) binds to both Zn1 (cyan) and Zn2 (magenta) of LMO4-LIM1 in an extended fashion with ldb1-LID forming a short b-strand that extends a b-hairpin present in LMO4-LIM1.
FLIN2 (PDB: 1J2O) is an engineered intramolecular complex comprising a fusion of the LID domain of ldb1 and the N-terminal LIM domain of LMO2. The solution structure of FLIN2 shows that ldb1-LID (yellow ribbon) binds to the LMO2 LIM1 domain (surface representation) in an extended fashion. Half of the interaction interface involves predominantly backbone-backbone and side-chain hydrophobic interactions, the other involves mainly electrostatic interactions.
ZNF265 is a mammalian splicing factor, which contains an Arg-Ser rich domain and a putative double zinc finger motif that is capable of binding to RNA (putting it in the SR class of proteins). Shown here is the structure of one of the two zinc fingers. The structure comprises two beta-hairpins, crossing each other at an angle of ~80 degrees, and sandwiching a single zinc atom. The fold is unlike any known zinc-binding motifs and we have termed it the crossed finger. Remarkably, the positions of the residues shown in space-fill mimic the RNA-binding residues of the unrelated dsRBM (double-stranded RNA-binding module) domain, indicating the convergent evolution of a binding surface on different scaffolds.
This non-native structure corresponds to a fragment of the CH1 domain of CBP. Although the CH1 domain in its native form binds 3 zinc ions (Dames et al, PNAS 2002, 99, 5271-5276), this shorter domain binds only one zinc ion, through a different combination of zinc ligating residues. This novel zinc-binding fold is quite remarkable, given that a sequence, taken out of the context of a whole domain, is able to fold into a stable, yet different structure.
USF9 has the same zinc ligation topology as USF1 but the two fingers share only 14% sequence identity outside of the zinc ligands (blue). However, USF9 adopts an almost identical fold to USF1, with a short β-hairpin (orange) and an α-helix. Furthermore, USF9 is also able to interact with the N-terminal finger of GATA-1.
U-shaped is a member of the FOG family of transcription factors, which interact with GATA proteins to control gene expression in various tissues. U-shaped has nine zinc fingers. The structure of the first finger of U-shaped (USF1) is very similar to that of a classical zinc finger, consisting of a short β-hairpin (yellow) and an α-helix. The central zinc atom (green) is ligated by 1 His and 3 Cys residues (blue), in contrast to classical fingers, which have a CCHH zinc ligation topology. While many classical fingers bind to DNA, USF1 has been shown to interact with the N-terminal zinc finger of GATA-1.
The structure of GNF consists of two distorted β-hairpins and an α-helix, with a central zinc atom (green) ligated by four cysteine residues (red). GATA-1 interacts with transcription factors from the FOG family using this N-terminal zinc finger. This interaction is crucial for GATA-1’s ability to correctly regulate gene expression in hematopoetic cells.