Genetic and structural basis of the human anti-α-galactosyl antibody response.

Humans lack the capacity to produce the Galα1-3Galß1-4GlcNAc (α-gal) glycan, and produce anti-α-gal antibodies upon exposure to the carbohydrate on a diverse set of immunogens, including commensal gut bacteria, malaria parasites, cetuximab, and tick proteins. Here we use X-ray crystallographic analysis of antibodies from α-gal knockout mice and humans in complex with the glycan to reveal a common binding motif, centered on a germline-encoded tryptophan residue at Kabat position 33 (W33) of the complementarity-determining region of the variable heavy chain (CDRH1). Immunoglobulin sequencing of anti-α-gal B cells in healthy humans and tick-induced mammalian meat anaphylaxis patients revealed preferential use of heavy chain germline IGHV3-7, encoding W33, among an otherwise highly polyclonal antibody response. Antigen binding was critically dependent on the presence of the germline-encoded W33 residue for all of the analyzed antibodies; moreover, introduction of the W33 motif into naive IGHV3-23 antibody phage libraries enabled the rapid selection of α-gal binders. Our results outline structural and genetic factors that shape the human anti-α-galactosyl antibody response, and provide a framework for future therapeutics development.

mRNA display reveals a class of high-affinity bromodomain-binding motifs that are not found in the human proteome.

Bromodomains (BDs) regulate gene expression by recognizing protein motifs containing acetyllysine. Although originally characterized as histone-binding proteins, it has since become clear that these domains interact with other acetylated proteins, perhaps most prominently transcription factors. The likely transient nature and low stoichiometry of such modifications, however, has made it challenging to fully define the interactome of any given BD. To begin to address this knowledge gap in an unbiased manner, we carried out mRNA display screens against a BD-the N-terminal BD of BRD3-using peptide libraries that contained either one or two acetyllysine residues. We discovered peptides with very strong consensus sequences and with affinities that are significantly higher than typical BD-peptide interactions. X-ray crystal structures also revealed modes of binding that have not been seen with natural ligands. Intriguingly, however, our sequences are not found in the human proteome, perhaps suggesting that strong binders to BDs might have been selected against during evolution.

Cyclic peptides can engage a single binding pocket through highly divergent modes.

Cyclic peptide library screening technologies show immense promise for identifying drug leads and chemical probes for challenging targets. However, the structural and functional diversity encoded within such libraries is largely undefined. We have systematically profiled the affinity, selectivity, and structural features of library-derived cyclic peptides selected to recognize three closely related targets: the acetyllysine-binding bromodomain proteins BRD2, -3, and -4. We report affinities as low as 100 pM and specificities of up to 106-fold. Crystal structures of 13 peptide-bromodomain complexes reveal remarkable diversity in both structure and binding mode, including both α-helical and ß-sheet structures as well as bivalent binding modes. The peptides can also exhibit a high degree of structural preorganization. Our data demonstrate the enormous potential within these libraries to provide diverse binding modes against a single target, which underpins their capacity to yield highly potent and selective ligands.

Correction: Mutations in MITF and PAX3 Cause "Splashed White" and Other White Spotting Phenotypes in Horses.

[This corrects the article DOI: 10.1371/journal.pgen.1002653.].

The characterization of protein interactions - what, how and how much?

Protein interactions underlie most molecular events in biology. Many methods have been developed to identify protein partners, to measure the affinity with which these biomolecules interact and to characterise the structures of the complexes. Each approach has its own advantages and limitations, and it can be difficult for the newcomer to determine which methodology would best suit their system. This review provides an overview of many of the techniques most widely used to identify protein partners, assess stoichiometry and binding affinity, and determine low-resolution models for complexes. Key methods covered include: yeast two-hybrid analysis, affinity purification mass spectrometry and proximity labelling to identify partners; size-exclusion chromatography, scattering methods, native mass spectrometry and analytical ultracentrifugation to estimate stoichiometry; isothermal titration calorimetry, biosensors and fluorometric methods (including microscale thermophoresis, anisotropy/polarisation, resonance energy transfer, AlphaScreen, and differential scanning fluorimetry) to measure binding affinity; and crosslinking and hydrogen-deuterium exchange mass spectrometry to probe the structure of complexes.

Disparate binding kinetics by an intrinsically disordered domain enables temporal regulation of transcriptional complex formation.

Intrinsically disordered regions are highly represented among mammalian transcription factors, where they often contribute to the formation of multiprotein complexes that regulate gene expression. An example of this occurs with LIM-homeodomain (LIM-HD) proteins in the developing spinal cord. The LIM-HD protein LHX3 and the LIM-HD cofactor LDB1 form a binary complex that gives rise to interneurons, whereas in adjacent cell populations, LHX3 and LDB1 form a rearranged ternary complex with the LIM-HD protein ISL1, resulting in motor neurons. The protein-protein interactions within these complexes are mediated by ordered LIM domains in the LIM-HD proteins and intrinsically disordered LIM interaction domains (LIDs) in LDB1 and ISL1; however, little is known about how the strength or rates of binding contribute to complex assemblies. We have measured the interactions of LIM:LID complexes using FRET-based protein-protein interaction studies and EMSAs and used these data to model population distributions of complexes. The protein-protein interactions within the ternary complexes are much weaker than those in the binary complex, yet surprisingly slow LDB1:ISL1 dissociation kinetics and a substantial increase in DNA binding affinity promote formation of the ternary complex over the binary complex in motor neurons. We have used mutational and protein engineering approaches to show that allostery and modular binding by tandem LIM domains contribute to the LDB1LID binding kinetics. The data indicate that a single intrinsically disordered region can achieve highly disparate binding kinetics, which may provide a mechanism to regulate the timing of transcriptional complex assembly.

Mutation in a flexible linker modulates binding affinity for modular complexes.

Tandem beta zippers are modular complexes formed between repeated linear motifs and tandemly arrayed domains of partner proteins in which ß-strands form upon binding. Studies of such complexes, formed by LIM domain proteins and linear motifs in their intrinsically disordered partners, revealed spacer regions between the linear motifs that are relatively flexible but may affect the overall orientation of the binding modules. We demonstrate that mutation of a solvent exposed side chain in the spacer region of an LHX4-ISL2 complex has no significant effect on the structure of the complex, but decreases binding affinity, apparently by increasing flexibility of the linker.

1D Self-Assembly and Ice Recrystallization Inhibition Activity of Antifreeze Glycopeptide-Functionalized Perylene Bisimides.

Antifreeze glycoproteins (AFGPs) are polymeric natural products that have drawn considerable interest in diverse research fields owing to their potent ice recrystallization inhibition (IRI) activity. Self-assembled materials have emerged as a promising class of biomimetic ice growth inhibitor, yet the development of AFGP-based supramolecular materials that emulate the aggregative behavior of AFGPs have not yet been reported. This work reports the first example of the 1D self-assembly and IRI activity of AFGP-functionalized perylene bisimides (AFGP-PBIs). Glycopeptide-functionalized PBIs underwent 1D self-assembly in water and showed modest IRI activity, which could be tuned through substitution of the PBI core. This work presents essential proof-of-principle for the development of novel IRIs as potential supramolecular cryoprotectants and glycoprotein mimics.

Discovery, characterization and in vivo activity of pyocin SD2, a protein antibiotic from Pseudomonas aeruginosa.

Increasing rates of antibiotic resistance among Gram-negative pathogens such as Pseudomonas aeruginosa means alternative approaches to antibiotic development are urgently required. Pyocins, produced by P. aeruginosa for intraspecies competition, are highly potent protein antibiotics known to actively translocate across the outer membrane of P. aeruginosa. Understanding and exploiting the mechanisms by which pyocins target, penetrate and kill P. aeruginosa is a promising approach to antibiotic development. In this work we show the therapeutic potential of a newly identified tRNase pyocin, pyocin SD2, by demonstrating its activity in vivo in a murine model of P. aeruginosa lung infection. In addition, we propose a mechanism of cell targeting and translocation for pyocin SD2 across the P. aeruginosa outer membrane. Pyocin SD2 is concentrated at the cell surface, via binding to the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide (LPS), from where it can efficiently locate its outer membrane receptor FpvAI. This strategy of utilizing both the CPA and a protein receptor for cell targeting is common among pyocins as we show that pyocins S2, S5 and SD3 also bind to the CPA. Additional data indicate a key role for an unstructured N-terminal region of pyocin SD2 in the subsequent translocation of the pyocin into the cell. These results greatly improve our understanding of how pyocins target and translocate across the outer membrane of P. aeruginosa. This knowledge could be useful for the development of novel anti-pseudomonal therapeutics and will also support the development of pyocin SD2 as a therapeutic in its own right.

Biochemical and biophysical characterisation of haloalkane dehalogenases DmrA and DmrB in Mycobacterium strain JS60 and their role in growth on haloalkanes.

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. The kinetic parameters of DmrA with 4-bromobutyronitrile as a substrate were Km = 1.9 ± 0.2 mM, kcat = 3.1 ± 0.2 s(-1) . DmrB showed the highest activity against 1-bromohexane. DmrA is monomeric, whereas DmrB is tetrameric. 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. We show that M. rhodesiae JS60 can utilise 1-iodopropane, 1-iodobutane and 1-bromobutane as sole carbon and energy sources. This ability appears to be conferred predominantly through DmrA, which shows significantly higher levels of upregulation in response to haloalkanes than DmrB.

The recently identified modifier of murine metastable epialleles, Rearranged L-Myc Fusion, is involved in maintaining epigenetic marks at CpG island shores and enhancers.

BACKGROUND: We recently identified a novel protein, Rearranged L-myc fusion (Rlf), that is required for DNA hypomethylation and transcriptional activity at two specific regions of the genome known to be sensitive to epigenetic gene silencing. To identify other loci affected by the absence of Rlf, we have now analysed 12 whole genome bisulphite sequencing datasets across three different embryonic tissues/stages from mice wild-type or null for Rlf. RESULTS: Here we show that the absence of Rlf results in an increase in DNA methylation at thousands of elements involved in transcriptional regulation and many of the changes occur at enhancers and CpG island shores. ChIP-seq for H3K4me1, a mark generally found at regulatory elements, revealed associated changes at many of the regions that are differentially methylated in the Rlf mutants. RNA-seq showed that the numerous effects of the absence of Rlf on the epigenome are associated with relatively subtle effects on the mRNA population. In vitro studies suggest that Rlf's zinc fingers have the capacity to bind DNA and that the protein interacts with other known epigenetic modifiers. CONCLUSION: This study provides the first evidence that the epigenetic modifier Rlf is involved in the maintenance of DNA methylation at enhancers and CGI shores across the genome.

A Quantitative Fluorescence-Based Assay for Assessing LIM Domain-Peptide Interactions.

We have developed Förster resonance energy transfer (FRET)-based experiments for measuring the binding affinity, off-rates, and inferred on-rates for interactions between a family of transcriptional regulators and their intrinsically disordered binding partners. It was difficult to evaluate these interactions previously, as the transcriptional regulators are obligate binding proteins that aggregate in the absence of a binding partner. The assays rely on fusion constructs where binding domains are linked by a flexible tether containing a specific protease site, with fluorescent proteins at either end that display FRET when the complex is formed. Loss of FRET is monitored after cutting the tether followed by dilution or competition with a non-fluorescent peptide. These methods allowed a wide range of binding affinities (10-9 -10-5 m) to be determined. Our data indicate that interactions of closely related proteins can have surprisingly different binding properties.

Analysis of disease-causing GATA1 mutations in murine gene complementation systems.

Missense mutations in transcription factor GATA1 underlie a spectrum of congenital red blood cell and platelet disorders. We investigated how these alterations cause distinct clinical phenotypes by combining structural, biochemical, and genomic approaches with gene complementation systems that examine GATA1 function in biologically relevant cellular contexts. Substitutions that disrupt FOG1 cofactor binding impair both gene activation and repression and are associated with pronounced clinical phenotypes. Moreover, clinical severity correlates with the degree of FOG1 disruption. Surprisingly, 2 mutations shown to impair DNA binding of GATA1 in vitro did not measurably affect in vivo target gene occupancy. Rather, one of these disrupted binding to the TAL1 complex, implicating it in diseases caused by GATA1 mutations. Diminished TAL1 complex recruitment mainly impairs transcriptional activation and is linked to relatively mild disease. Notably, different substitutions at the same amino acid can selectively inhibit TAL1 complex or FOG1 binding, producing distinct cellular and clinical phenotypes. The structure-function relationships elucidated here were not predicted by prior in vitro or computational studies. Thus, our findings uncover novel disease mechanisms underlying GATA1 mutations and highlight the power of gene complementation assays for elucidating the molecular basis of genetic diseases.

Mutations in MITF and PAX3 cause "splashed white" and other white spotting phenotypes in horses.

During fetal development neural-crest-derived melanoblasts migrate across the entire body surface and differentiate into melanocytes, the pigment-producing cells. Alterations in this precisely regulated process can lead to white spotting patterns. White spotting patterns in horses are a complex trait with a large phenotypic variance ranging from minimal white markings up to completely white horses. The "splashed white" pattern is primarily characterized by an extremely large blaze, often accompanied by extended white markings at the distal limbs and blue eyes. Some, but not all, splashed white horses are deaf. We analyzed a Quarter Horse family segregating for the splashed white coat color. Genome-wide linkage analysis in 31 horses gave a positive LOD score of 1.6 in a region on chromosome 6 containing the PAX3 gene. However, the linkage data were not in agreement with a monogenic inheritance of a single fully penetrant mutation. We sequenced the PAX3 gene and identified a missense mutation in some, but not all, splashed white Quarter Horses. Genome-wide association analysis indicated a potential second signal near MITF. We therefore sequenced the MITF gene and found a 10 bp insertion in the melanocyte-specific promoter. The MITF promoter variant was present in some splashed white Quarter Horses from the studied family, but also in splashed white horses from other horse breeds. Finally, we identified two additional non-synonymous mutations in the MITF gene in unrelated horses with white spotting phenotypes. Thus, several independent mutations in MITF and PAX3 together with known variants in the EDNRB and KIT genes explain a large proportion of horses with the more extreme white spotting phenotypes.

A structural basis for the regulation of the LIM-homeodomain protein islet 1 (Isl1) by intra- and intermolecular interactions.

Islet 1 (Isl1) is a transcription factor of the LIM-homeodomain (LIM-HD) protein family and is essential for many developmental processes. 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.

New insights into DNA recognition by zinc fingers revealed by structural analysis of the oncoprotein ZNF217.

Classical zinc fingers (ZFs) are one of the most abundant and best characterized DNA-binding domains. Typically, tandem arrays of three or more ZFs bind DNA target sequences with high affinity and specificity, and the mode of DNA recognition is sufficiently well understood that tailor-made ZF-based DNA-binding proteins can be engineered. 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 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-π 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. Finally, we investigated the sequence specificity of this two-ZF unit and discuss how ZNF217 might discriminate its target DNA sites in the cell.

A structural analysis of DNA binding by myelin transcription factor 1 double zinc fingers.

Myelin transcription factor 1 (MyT1/NZF2), a member of the neural zinc-finger (NZF) protein family, is a transcription factor that plays a central role in the developing central nervous system. It has also recently been shown that, in combination with two other transcription factors, the highly similar paralog MyT1L is able to direct the differentiation of murine and human stem cells into functional neurons. MyT1 contains seven zinc fingers (ZFs) that are highly conserved throughout the protein and throughout the NZF family. We recently presented a model for the interaction of the fifth ZF of MyT1 with a DNA sequence derived from the promoter of the retinoic acid receptor (RARE) gene. Here, we have used NMR spectroscopy, in combination with surface plasmon resonance and data-driven molecular docking, to delineate the mechanism of DNA binding for double ZF polypeptides derived from MyT1. Our data indicate that a two-ZF unit interacts with the major groove of the entire RARE motif and that both fingers bind in an identical manner and with overall two-fold rotational symmetry, consistent with the palindromic nature of the target DNA. Several key residues located in one of the irregular loops of the ZFs are utilized to achieve specific binding. Analysis of the human and mouse genomes based on our structural data reveals three putative MyT1 target genes involved in neuronal development.

Structural basis of simultaneous recruitment of the transcriptional regulators LMO2 and FOG1/ZFPM1 by the transcription factor GATA1.

The control of red blood cell and megakaryocyte development by the regulatory protein GATA1 is a paradigm for transcriptional regulation of gene expression in cell lineage differentiation and maturation. Most GATA1-regulated events require GATA1 to bind FOG1, and essentially all GATA1-activated genes are cooccupied by a TAL1/E2A/LMO2/LDB1 complex; however, it is not known whether FOG1 and TAL1/E2A/LMO2/LDB1 are simultaneously recruited by GATA1. Our structural data reveal that the FOG1-binding domain of GATA1, the N finger, can also directly contact LMO2 and show that, despite the small size (< 50 residues) of the GATA1 N finger, both FOG1 and LMO2 can simultaneously bind this domain. LMO2 in turn can simultaneously contact both GATA1 and the DNA-binding protein TAL1/E2A at bipartite E-box/WGATAR sites. Taken together, our data provide the first structural snapshot of multiprotein complex formation at GATA1-dependent genes and support a model in which FOG1 and TAL1/E2A/LMO2/LDB1 can cooccupy E-box/WGATAR sites to facilitate GATA1-mediated activation of gene activation.

Engineering specificity changes on a RanBP2 zinc finger that binds single-stranded RNA.

The realization that gene transcription is much more pervasive than previously thought and that many diverse RNA species exist in simple as well as complex organisms has triggered efforts to develop functionalized RNA-binding proteins (RBPs) that have the ability to probe and manipulate RNA function. Previously, we showed that the RanBP2-type zinc finger (ZF) domain is a good candidate for an addressable single-stranded-RNA (ssRNA) binding domain that can recognize ssRNA in a modular and specific manner. In the present study, we successfully engineered a sequence specificity change onto this ZF scaffold by using a combinatorial approach based on phage display. This work constitutes a foundation from which a set of RanBP2 ZFs might be developed that is able to recognize any given RNA sequence.

No evidence for cumulative effects in a Dnmt3b hypomorph across multiple generations.

Observations of inherited phenotypes that cannot be explained solely through genetic inheritance are increasing. Evidence points to transmission of non-DNA molecules in the gamete as mediators of the phenotypes. However, in most cases it is unclear what the molecules are, with DNA methylation, chromatin proteins, and small RNAs being the most prominent candidates. From a screen to generate novel mouse mutants of genes involved in epigenetic reprogramming, we produced a DNA methyltransferase 3b allele that is missing exon 13. Mice that are homozygous for the mutant allele have smaller stature and reduced viability, with particularly high levels of female post-natal death. Reduced DNA methylation was also detected at telocentric repeats and the X-linked Hprt gene. However, none of the abnormal phenotypes or DNA methylation changes worsened with multiple generations of homozygous mutant inbreeding. This suggests that in our model the abnormalities are reset each generation and the processes of transgenerational epigenetic reprogramming are effective in preventing their inheritance.