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1.
J Mol Biol ; 425(7): 1101-10, 2013 Apr 12.
Article in English | MEDLINE | ID: mdl-23353824

ABSTRACT

LIM-only protein 4 (LMO4) is strongly linked to the progression of breast cancer. Although the mechanisms underlying this phenomenon are not well understood, a role is emerging for LMO4 in regulation of the cell cycle. We determined the solution structure of LMO4 in complex with CtIP (C-terminal binding protein interacting protein)/RBBP8, a tumour suppressor protein that is involved in cell cycle progression, DNA repair and transcriptional regulation. Our data reveal that CtIP and the essential LMO cofactor LDB1 (LIM-domain binding protein 1) bind to the same face on LMO4 and cannot simultaneously bind to LMO4. We hypothesise that overexpression of LMO4 may disrupt some of the normal tumour suppressor activities of CtIP, thereby contributing to breast cancer progression.


Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Carrier Proteins/chemistry , LIM Domain Proteins/chemistry , Nuclear Proteins/chemistry , Protein Structure, Tertiary , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Binding Sites/genetics , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Endodeoxyribonucleases , Female , Humans , LIM Domain Proteins/genetics , LIM Domain Proteins/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Sequence Homology, Amino Acid , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Two-Hybrid System Techniques
2.
Nucleic Acids Res ; 40(14): e105, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22492509

ABSTRACT

In recent years, evidence has emerged for the existence of many diverse types of RNA, which play roles in a wide range of biological processes in all kingdoms of life. These molecules generally do not, however, act in isolation, and identifying which proteins partner with RNA is a major challenge. Many methods, in vivo and in vitro, have been used to address this question, including combinatorial or high-throughput approaches, such as systematic evolution of ligands, cross-linking and immunoprecipitation and RNA immunoprecipitation combined with deep sequencing. However, most of these methods are not trivial to pursue and often require substantial optimization before results can be achieved. Here, we demonstrate a simple technique that allows one to screen proteins for RNA-binding properties in a gel-shift experiment and can be easily implemented in any laboratory. This assay should be a useful first-pass tool for assessing whether a protein has RNA- or DNA-binding properties, prior to committing resources to more complex procedures.


Subject(s)
Electrophoretic Mobility Shift Assay/methods , RNA-Binding Proteins/analysis , Heparin , RNA Probes/chemistry
3.
Curr Pharm Des ; 15(31): 3681-96, 2009.
Article in English | MEDLINE | ID: mdl-19925420

ABSTRACT

LIM (Lin-11, Isl-1, Mec-3), RING (Really interesting new gene), PHD (Plant homology domain) and MYND (myeloid, Nervy, DEAF-1) domains are all zinc-binding domains that ligate two zinc ions. Unlike the better known classical zinc fingers, these domains do not bind DNA, but instead mediate interactions with other proteins. LIM-domain containing proteins have diverse functions as regulators of gene expression, cell adhesion and motility and signal transduction. RING finger proteins are generally associated with ubiquitination; the presence of such a domain is the defining feature of a class of E3 ubiquitin protein ligases. PHD proteins have been associated with SUMOylation but most recently have emerged as a chromatin recognition motif that reads the methylation state of histones. The function of the MYND domain is less clear, but MYND domains are also found in proteins that have ubiquitin ligase and/or histone methyltransferase activity. Here we review the structure-function relationships for these domains and discuss strategies to modulate their activity.


Subject(s)
DNA-Binding Proteins/physiology , Homeodomain Proteins/physiology , Zinc Fingers/physiology , Animals , Binding Sites , DNA-Binding Proteins/chemistry , Homeodomain Proteins/chemistry , Humans , Protein Conformation , Protein Folding , RING Finger Domains/physiology , Sequence Homology, Amino Acid
4.
Proc Natl Acad Sci U S A ; 103(10): 3621-6, 2006 Mar 07.
Article in English | MEDLINE | ID: mdl-16537446

ABSTRACT

Class I hydrophobins are a unique family of fungal proteins that form a polymeric, water-repellent monolayer on the surface of structures such as spores and fruiting bodies. Similar monolayers are being discovered on an increasing range of important microorganisms. Hydrophobin monolayers are amphipathic and particularly robust, and they reverse the wettability of the surface on which they are formed. There are also significant similarities between these polymers and amyloid-like fibrils. However, structural information on these proteins and the rodlets they form has been elusive. Here, we describe the three-dimensional structure of the monomeric form of the class I hydrophobin EAS. EAS forms a beta-barrel structure punctuated by several disordered regions and displays a complete segregation of charged and hydrophobic residues on its surface. This structure is consistent with its ability to form an amphipathic polymer. By using this structure, together with data from mutagenesis and previous biophysical studies, we have been able to propose a model for the polymeric rodlet structure adopted by these proteins. X-ray fiber diffraction data from EAS rodlets are consistent with our model. Our data provide molecular insight into the nature of hydrophobin rodlet films and extend our understanding of the fibrillar beta-structures that continue to be discovered in the protein world.


Subject(s)
Fungal Proteins/chemistry , Amino Acid Sequence , Biophysical Phenomena , Biophysics , Fungal Proteins/classification , Fungal Proteins/genetics , Models, Molecular , Molecular Sequence Data , Neurospora crassa/chemistry , Neurospora crassa/genetics , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sequence Deletion , Static Electricity , X-Ray Diffraction
5.
J Biol Chem ; 276(43): 40306-12, 2001 Oct 26.
Article in English | MEDLINE | ID: mdl-11522785

ABSTRACT

We have isolated a novel family of insect-selective neurotoxins that appear to be the most potent blockers of insect voltage-gated calcium channels reported to date. These toxins display exceptional phylogenetic specificity, with at least a 10,000-fold preference for insect versus vertebrate calcium channels. The structure of one of the toxins reveals a highly structured, disulfide-rich core and a structurally disordered C-terminal extension that is essential for channel blocking activity. Weak structural/functional homology with omega-agatoxin-IVA/B, the prototypic inhibitor of vertebrate P-type calcium channels, suggests that these two toxin families might share a similar mechanism of action despite their vastly different phylogenetic specificities.


Subject(s)
Calcium Channel Blockers/pharmacology , Insecticides/pharmacology , Neurotoxins/pharmacology , Spider Venoms/pharmacology , Amino Acid Sequence , Animals , Bees/cytology , Insect Control/methods , Models, Molecular , Molecular Sequence Data , Neurons/drug effects , Neurotoxins/genetics , Nuclear Magnetic Resonance, Biomolecular , Patch-Clamp Techniques , Protein Precursors/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Spider Venoms/chemistry , Spider Venoms/genetics , Spiders , omega-Agatoxin IVA/chemistry
6.
Protein Eng ; 14(7): 493-9, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11522923

ABSTRACT

The nuclear LIM-only (LMO) transcription factors LMO2 and LMO4 play important roles in both normal and leukemic T-cell development. LIM domains are cysteine/histidine-rich domains that contain two structural zinc ions and that function as protein-protein adaptors; members of the LMO family each contain two closely spaced LIM domains. These LMO proteins all bind with high affinity to the nuclear protein LIM domain binding protein 1 (ldb1). The LMO-ldb1 interaction is mediated through the N-terminal LIM domain (LIM1) of LMO proteins and a 38-residue region towards the C-terminus of ldb1 [ldb1(LID)]. Unfortunately, recombinant forms of LMO2 and LMO4 have limited solubility and stability, effectively preventing structural analysis. Therefore, we have designed and constructed a fusion protein in which ldb1(LID) and LIM1 of LMO2 can form an intramolecular complex. The engineered protein, FLIN2 (fusion of the LIM interacting domain of ldb1 and the N-terminal LIM domain of LMO2) has been expressed and purified in milligram quantities. FLIN2 is monomeric, contains significant levels of secondary structure and yields a sharp and well-dispersed one-dimensional (1)H NMR spectrum. The analogous LMO4 protein, FLIN4, has almost identical properties. These data suggest that we will be able to obtain high-resolution structural information about the LMO-ldb1 interactions.


Subject(s)
DNA-Binding Proteins/chemistry , Homeodomain Proteins/chemistry , Metalloproteins/chemistry , Protein Engineering , Transcription Factors/chemistry , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Binding Sites , Cloning, Molecular , DNA-Binding Proteins/isolation & purification , Escherichia coli/genetics , Homeodomain Proteins/isolation & purification , LIM Domain Proteins , Magnetic Resonance Spectroscopy , Metalloproteins/isolation & purification , Mice , Nuclear Proteins/chemistry , Protein Binding , Protein Folding , Protein Structure, Secondary , Recombinant Fusion Proteins , Transcription Factors/isolation & purification , Zinc/chemistry
7.
Biochemistry ; 40(15): 4867-78, 2001 Apr 17.
Article in English | MEDLINE | ID: mdl-11294655

ABSTRACT

The molecular recognition of oligonucleotides by chiral ruthenium complexes has been probed by NMR spectroscopy using the template Delta-cis-alpha- and Delta-cis-beta-[Ru(RR-picchxnMe(2)) (bidentate)](2+), where the bidentate ligand is one of phen (1,10-phenanthroline), dpq (dipyrido[3,2-f:2',3'-h]quinoxaline), or phi (9,10-phenanthrenequinone diimine) and picchxnMe(2)() is N,N'-dimethyl-N,N'-di(2-picolyl)-1,2-diaminocyclohexane. By varying only the bidentate ligand in a series of complexes, it was shown that the bidentate alone can alter binding modes. DNA binding studies of the Delta-cis-alpha-[Ru(RR-picchxnMe(2))(phen)](2+) complex indicate fast exchange kinetics on the chemical shift time scale and a "partial intercalation" mode of binding. This complex binds to [d(CGCGATCGCG)](2) and [d(ATATCGATAT)](2) at AT, TA, and GA sites from the minor groove, as well as to the ends of the oligonucleotide at low temperature. Studies of the Delta-cis-beta-[Ru(RR-picchxnMe(2))(phen)](2+) complex with [d(CGCGATCGCG)](2) showed that the complex binds only weakly to the ends of the oligonucleotide. The interaction of Delta-cis-alpha-[Ru(RR-picchxnMe(2))(dpq)](2+) with [d(CGCGATCGCG)](2) showed intermediate exchange kinetics and evidence of minor groove intercalation at the GA base step. In contrast to the phen and dpq complexes, Delta-cis-alpha- and Delta-cis-beta-[Ru(RR-picchxnMe(2))(phi)](2+) showed evidence of major groove binding independent of the metal ion configuration. DNA stabilization induced by complex binding to [d(CGCGATCGCG)](2) (measured as DeltaT(m)) increases in the order phen < dpq and DNA affinity in the order phen < dpq < phi. The groove binding preferences exhibited by the different bidentate ligands is explained with the aid of molecular modeling experiments.


Subject(s)
Cobalt/chemistry , DNA/chemistry , Intercalating Agents/chemistry , Models, Molecular , Organometallic Compounds/chemistry , Picolines/chemistry , Ruthenium/chemistry , Binding Sites , DNA Adducts/chemistry , Hot Temperature , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Denaturation , Oligodeoxyribonucleotides/chemistry , Phenanthrolines/chemistry , Pyridines/chemistry , Quinoxalines/chemistry , Stereoisomerism , Thermodynamics
8.
Structure ; 9(2): 83-91, 2001 Feb 07.
Article in English | MEDLINE | ID: mdl-11250193

ABSTRACT

BACKGROUND: Fungal hydrophobin proteins have the remarkable ability to self-assemble into polymeric, amphipathic monolayers on the surface of aerial structures such as spores and fruiting bodies. These monolayers are extremely resistant to degradation and as such offer the possibility of a range of biotechnological applications involving the reversal of surface polarity. The molecular details underlying the formation of these monolayers, however, have been elusive. We have studied EAS, the hydrophobin from the ascomycete Neurospora crassa, in an effort to understand the structural aspects of hydrophobin polymerization. RESULTS: We have purified both wild-type and uniformly 15N-labeled EAS from N. crassa conidia, and used a range of physical methods including multidimensional NMR spectroscopy to provide the first high resolution structural information on a member of the hydrophobin family. We have found that EAS is monomeric but mostly unstructured in solution, except for a small region of antiparallel beta sheet that is probably stabilized by four intramolecular disulfide bonds. Polymerised EAS appears to contain substantially higher amounts of beta sheet structure, and shares many properties with amyloid fibers, including a characteristic gold-green birefringence under polarized light in the presence of the dye Congo Red. CONCLUSIONS: EAS joins an increasing number of proteins that undergo a disorder-->order transition in carrying out their normal function. This report is one of the few examples where an amyloid-like state represents the wild-type functional form. Thus the mechanism of amyloid formation, now thought to be a general property of polypeptide chains, has actually been applied in nature to form these remarkable structures.


Subject(s)
Amyloid/chemistry , Fungal Proteins/chemistry , Amino Acid Sequence , Circular Dichroism , Coloring Agents , Congo Red , Fungal Proteins/isolation & purification , Molecular Sequence Data , Neurospora crassa/chemistry , Nuclear Magnetic Resonance, Biomolecular , Protein Isoforms/chemistry , Protein Isoforms/isolation & purification , Protein Structure, Secondary , Solutions
10.
Structure ; 8(11): 1157-66, 2000 Nov 15.
Article in English | MEDLINE | ID: mdl-11080638

ABSTRACT

BACKGROUND: Zinc finger domains have traditionally been regarded as sequence-specific DNA binding motifs. However, recent evidence indicates that many zinc fingers mediate specific protein-protein interactions. For instance, several zinc fingers from FOG family proteins have been shown to interact with the N-terminal zinc finger of GATA-1. RESULTS: We have used NMR spectroscopy to determine the first structures of two FOG family zinc fingers that are involved in protein-protein interactions: fingers 1 and 9 from U-shaped. These fingers resemble classical TFIIIA-like zinc fingers, with the exception of an unusual extended portion of the polypeptide backbone prior to the fourth zinc ligand. [15N,(1)H]-HSQC titrations have been used to define the GATA binding surface of USH-F1, and comparison with other FOG family proteins indicates that the recognition mechanism is conserved across species. The surface of FOG-type fingers that interacts with GATA-1 overlaps substantially with the surface through which classical fingers typically recognize DNA. This suggests that these fingers could not contact both GATA and DNA simultaneously. In addition, results from NMR, gel filtration, and sedimentation equilibrium experiments suggest that the interactions are of moderate affinity. CONCLUSIONS: Our results demonstrate unequivocally that zinc fingers comprising the classical betabetaalpha fold are capable of mediating specific contacts between proteins. The existence of this alternative function has implications for the prediction of protein function from sequence data and for the evolution of protein function.


Subject(s)
Drosophila Proteins , Insect Proteins/chemistry , Transcription Factors/chemistry , Zinc Fingers , Amino Acid Sequence , Animals , Carrier Proteins/chemistry , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Drosophila melanogaster/chemistry , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , Insect Proteins/classification , Insect Proteins/genetics , Insect Proteins/metabolism , Magnetic Resonance Spectroscopy , Mice , Models, Molecular , Molecular Sequence Data , Multigene Family , Nuclear Proteins/chemistry , Protein Binding , Protein Conformation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Transcription Factors/classification , Transcription Factors/genetics , Transcription Factors/metabolism
11.
J Biol Chem ; 275(48): 37454-61, 2000 Dec 01.
Article in English | MEDLINE | ID: mdl-10978321

ABSTRACT

The transcription factor Max is the obligate dimerization partner of the Myc oncoprotein. The pivotal role of Max within the Myc regulatory network is dependent upon its ability to dimerize via the helix-loop-helix leucine zipper domain. The Max homodimer contains a tetrad of polar residues at the interface of the leucine zipper domain. A conserved interfacial Asn residue at an equivalent position in two other leucine zipper proteins has been shown to decrease homodimer stability. The unusual arrangement of this Gln-Asn/Gln'-Asn' tetrad prompted us to investigate whether Asn(92) plays a similar role in destabilizing the Max homodimer. This residue was sequentially replaced with aliphatic and charged residues. Thermal denaturation, redox time course and analytical ultracentrifugation studies show that the N92V mutation does not increase homodimer stability. Replacing this residue with negatively charged side chains in N92D and N92E destabilizes the mutant homodimer. Further replacement of Gln(91) indicated that H bonding between Gln(91) and Asn(92) residues is not significant to the stability of the native protein. These data collectively demonstrate the central role of Asn(92) in homodimer interactions. Molecular modelling studies illustrate the favorable packing of the native Asn residue at the dimer interface compared with that of the mutant Max peptides.


Subject(s)
Amides/chemistry , Asparagine/chemistry , Helix-Loop-Helix Motifs , Leucine Zippers , Base Sequence , Circular Dichroism , DNA Primers , Dimerization , Leucine Zippers/genetics , Models, Molecular , Mutagenesis , Oxidation-Reduction , Protein Denaturation
12.
J Virol ; 74(13): 5911-20, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10846072

ABSTRACT

Entry into the host cell by enveloped viruses is mediated by fusion (F) or transmembrane glycoproteins. Many of these proteins share a fold comprising a trimer of antiparallel coiled-coil heterodimers, where the heterodimers are formed by two discontinuous heptad repeat motifs within the proteolytically processed chain. The F protein of human respiratory syncytial virus (RSV; the major cause of lower respiratory tract infections in infants) contains two corresponding regions that are predicted to form coiled coils (HR1 and HR2), together with a third predicted heptad repeat (HR3) located in a nonhomologous position. In order to probe the structures of these three domains and ascertain the nature of the interactions between them, we have studied the isolated HR1, HR2, and HR3 domains of RSV F by using a range of biophysical techniques, including circular dichroism, nuclear magnetic resonance spectroscopy, and sedimentation equilibrium. HR1 forms a symmetrical, trimeric coiled coil in solution (K(3) approximately 2.2 x 10(11) M(-2)) which interacts with HR2 to form a 3:3 hexamer. The HR1-HR2 interaction domains have been mapped using limited proteolysis, reversed-phase high-performance liquid chromatography, and electrospray-mass spectrometry. HR2 in isolation exists as a largely unstructured monomer, although it exhibits a tendency to form aggregates with beta-sheet-like characteristics. Only a small increase in alpha-helical content was observed upon the formation of the hexamer. This suggests that the RSV F glycoprotein contains a domain that closely resembles the core structure of the simian parainfluenza virus 5 fusion protein (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309-319, 1999). Finally, HR3 forms weak alpha-helical homodimers that do not appear to interact with HR1, HR2, or the HR1-HR2 complex. The results of these studies support the idea that viral fusion proteins have a common core architecture.


Subject(s)
HN Protein , Respiratory Syncytial Virus, Human/chemistry , Viral Envelope Proteins/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , Circular Dichroism , Humans , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular/methods , Oligopeptides/chemistry , Peptide Biosynthesis , Protein Structure, Secondary , Protein Structure, Tertiary , Viral Envelope Proteins/biosynthesis , Viral Envelope Proteins/metabolism , Viral Proteins/biosynthesis , Viral Proteins/metabolism
13.
J Biol Chem ; 275(20): 15128-34, 2000 May 19.
Article in English | MEDLINE | ID: mdl-10748221

ABSTRACT

cAMP-response element-binding protein-binding protein (CBP) is a transcriptional coactivator that interacts with a number of DNA-binding proteins and cofactor proteins involved in the regulation of transcription. Relatively little is known about the structure of CBP, but it has been noted that it contains three domains that are rich in cysteine and histidine (CH1, CH2, and CH3). The sequence of CH2 conforms to that of a leukemia-associated protein domain (PHD finger), and it has been postulated that this and both CH1 and CH3 may be zinc finger domains. This has not, however, been demonstrated experimentally. We have studied CH1 and show that it is composed of two novel zinc-binding modules, which we term "zinc bundles." Each bundle contains the sequence Cys-X(4)-Cys-X(8)-His-X(3)-Cys, and we show that a synthetic peptide comprising one zinc bundle from CH1 can fold in a zinc-dependent manner. CH3 also appears to contain two zinc bundles, one with the variant sequence Cys-X(2)-Cys-X(9)-His-X(3)-Cys, and we demonstrate that this variant motif also undergoes Zn(II)-induced folding. CH1 acts as a transcriptional activation domain in cellular assays. We show that mutations in any of the four zinc-chelating residues in either zinc bundle of CH1 significantly impair this activity and that these mutations also interfere with certain protein-protein interactions mediated by CH1. Our results indicate that CBP is a genuine zinc-binding protein and introduce zinc bundles as novel protein interaction domains.


Subject(s)
Nuclear Proteins/chemistry , Trans-Activators/chemistry , Amino Acid Sequence , Animals , Arabidopsis , Binding Sites , CREB-Binding Protein , Caenorhabditis elegans , Circular Dichroism , Cysteine , Drosophila melanogaster , Histidine , Humans , Mice , Molecular Sequence Data , Nuclear Proteins/metabolism , Plants, Toxic , Protein Folding , Sequence Alignment , Sequence Homology, Amino Acid , Nicotiana , Trans-Activators/metabolism , Transcriptional Activation , Zinc/metabolism , Zinc Fingers
14.
Eur J Biochem ; 267(4): 1030-8, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10672011

ABSTRACT

Zinc fingers (ZnFs) are extremely common protein domains. Several classes of ZnFs are distinguished by the nature and spacing of their zinc-coordinating residues. While the structure and function of some ZnFs are well characterized, many others have been identified only through their amino acid sequence. A number of proteins contain a conserved C-X2-C-X12-H-X1-5-C sequence, which is similar to the spacing observed for the 'classic' CCHH ZnFs. Although these domains have been implicated in protein-protein (and not protein-nucleic acid) interactions, nothing is known about their structure or function at a molecular level. Here, we address this problem through the expression and biophysical characterization of several CCHC-type zinc fingers from the erythroid transcription factor FOG and the related Drosophila protein U-shaped. Each of these domains does indeed fold in a zinc-dependent fashion, coordinating the metal in a tetrahedral manner through the sidechains of one histidine and three cysteine residues, and forming extremely thermostable structures. Analysis of CD spectra suggests an overall fold similar to that of the CCHH fingers, and indeed a point mutant of FOG-F1 in which the final cysteine residue is replaced by histidine remains capable of folding. However, the CCHC (as opposed to CCHH) motif is a prerequisite for GATA-1 binding activity, demonstrating that CCHC and CCHH topologies are not interchangeable. This demonstration that members of a structurally distinct subclass of genuine zinc finger domains are involved in the mediation of protein-protein interactions has implications for the prediction of protein function from nucleotide sequences.


Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Drosophila Proteins , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Zinc Fingers/physiology , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Carrier Proteins/isolation & purification , Cysteine/genetics , Cysteine/metabolism , DNA-Binding Proteins/metabolism , Drosophila melanogaster , Erythroid-Specific DNA-Binding Factors , Histidine/genetics , Histidine/metabolism , Hydrogen-Ion Concentration , Insect Proteins/chemistry , Insect Proteins/genetics , Insect Proteins/isolation & purification , Insect Proteins/metabolism , Molecular Sequence Data , Mutation/genetics , Nuclear Proteins/genetics , Nuclear Proteins/isolation & purification , Protein Binding , Protein Folding , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Spectrum Analysis , Temperature , Thermodynamics , Transcription Factors/genetics , Transcription Factors/isolation & purification , Two-Hybrid System Techniques , Zinc/metabolism , Zinc Fingers/genetics
15.
Protein Sci ; 8(6): 1320-31, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10386882

ABSTRACT

The N-terminal 17 residues of ubiquitin have been shown by 1H NMR to fold autonomously into a beta-hairpin structure in aqueous solution. This structure has a specific, native-like register, though side-chain contacts differ in detail from those observed in the intact protein. An autonomously folding hairpin has previously been identified in the case of streptococcal protein G, which is structurally homologous with ubiquitin, but remarkably, the two are not in topologically equivalent positions in the fold. This suggests that the organization of folding may be quite different for proteins sharing similar tertiary structures. Two smaller peptides have also been studied, corresponding to the isolated arms of the N-terminal hairpin of ubiquitin, and significant differences from simple random coil predictions observed in the spectra of these subfragments, suggestive of significant limitation of the backbone conformational space sampled, presumably as a consequence of the strongly beta-structure favoring composition of the sequences. This illustrates the ability of local sequence elements to express a propensity for beta-structure even in the absence of actual sheet formation. Attempts were made to estimate the population of the folded state of the hairpin, in terms of a simple two-state folding model. Using published "random coil" values to model the unfolded state, and values derived from native ubiquitin for the putative unique, folded state, it was found that the apparent population varied widely for different residues and with different NMR parameters. Use of the spectra of the subfragment peptides to provide a more realistic model of the unfolded state led to better agreement in the estimates that could be obtained from chemical shift and coupling constant measurements, while making it clear that some other approaches to population estimation could not give meaningful results, because of the tendency to populate the beta-region of conformational space even in the absence of the hairpin structure.


Subject(s)
Peptide Fragments/chemistry , Protein Folding , Ubiquitins/chemistry , Magnetic Resonance Spectroscopy , Protein Conformation
16.
J Biomol NMR ; 13(3): 249-62, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10212985

ABSTRACT

Zinc fingers (ZnFs) are generally regarded as DNA-binding motifs. However, a number of recent reports have implicated particular ZnFs in the mediation of protein-protein interactions. The N-terminal ZnF of GATA-1 (NF) is one such finger, having been shown to interact with a number of other proteins, including the recently discovered transcriptional co-factor FOG. Here we solve the three-dimensional structure of the NF in solution using multidimensional 1H/15N NMR spectroscopy, and we use 1H/15N spin relaxation measurements to investigate its backbone dynamics. The structure consists of two distorted beta-hairpins and a single alpha-helix, and is similar to that of the C-terminal ZnF of chicken GATA-1. Comparisons of the NF structure with those of other C4-type zinc binding motifs, including hormone receptor and LIM domains, also reveal substantial structural homology. Finally, we use the structure to map the spatial locations of NF residues shown by mutagenesis to be essential for FOG binding, and demonstrate that these residues all lie on a single face of the NF. Notably, this face is well removed from the putative DNA-binding face of the NF, an observation which is suggestive of simultaneous roles for the NF; that is, stabilisation of GATA-1 DNA complexes and recruitment of FOG to GATA-1-controlled promoter regions.


Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Binding Sites , Computer Simulation , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , Mice , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Software , Zinc Fingers
17.
Mol Microbiol ; 31(4): 1161-9, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10096083

ABSTRACT

Correct placement of the division septum in Escherichia coli requires the co-ordinated action of three proteins, MinC, MinD and MinE. MinC and MinD interact to form a non-specific division inhibitor that blocks septation at all potential division sites. MinE is able to antagonize MinCD in a topologically sensitive manner, as it restricts MinCD activity to the unwanted division sites at the cell poles. Here, we show that the topological specificity function of MinE residues in a structurally autonomous, trypsin-resistant domain comprising residues 31-88. Nuclear magnetic resonance (NMR) and circular dichroic spectroscopy indicate that this domain includes both alpha and beta secondary structure, while analytical ultracentrifugation reveals that it also contains a region responsible for MinE homodimerization. While trypsin digestion indicates that the anti-MinCD domain of MinE (residues 1-22) does not form a tightly folded structural domain, NMR analysis of a peptide corresponding to MinE1-22 indicates that this region forms a nascent helix in which the peptide rapidly interconverts between disordered (random coil) and alpha-helical conformations. This suggests that the N-terminal region of MinE may be poised to adopt an alpha-helical conformation when it interacts with the target of its anti-MinCD activity, presumably MinD.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/chemistry , Escherichia coli/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Cell Cycle Proteins , Circular Dichroism , Dimerization , Escherichia coli/growth & development , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Sequence Data , Protein Structure, Secondary , Structure-Activity Relationship , Substrate Specificity , Ultracentrifugation
18.
Mol Cell ; 3(2): 219-28, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10078204

ABSTRACT

GATA-1 and FOG (Friend of GATA-1) are each essential for erythroid and megakaryocyte development. FOG, a zinc finger protein, interacts with the amino (N) finger of GATA-1 and cooperates with GATA-1 to promote differentiation. To determine whether this interaction is critical for GATA-1 action, we selected GATA-1 mutants in yeast that fail to interact with FOG but retain normal DNA binding, as well a compensatory FOG mutant that restores interaction. These novel GATA-1 mutants do not promote erythroid differentiation of GATA-1- erythroid cells. Differentiation is rescued by the second-site FOG mutant. Thus, interaction of FOG with GATA-1 is essential for the function of GATA-1 in erythroid differentiation. These findings provide a paradigm for dissecting protein-protein associations involved in mammalian development.


Subject(s)
Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Erythropoiesis/physiology , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Cell Differentiation , DNA/metabolism , DNA-Binding Proteins/genetics , Erythroid Precursor Cells/metabolism , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , Gene Expression Regulation , Genetic Complementation Test , Macromolecular Substances , Mice , Molecular Sequence Data , Nuclear Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Structure-Activity Relationship , Transcription Factors/genetics , Transfection , Zinc Fingers
19.
J Biol Chem ; 273(50): 33595-603, 1998 Dec 11.
Article in English | MEDLINE | ID: mdl-9837943

ABSTRACT

Protein-protein interactions play significant roles in the control of gene expression. These interactions often occur between small, discrete domains within different transcription factors. In particular, zinc fingers, usually regarded as DNA-binding domains, are now also known to be involved in mediating contacts between proteins. We have investigated the interaction between the erythroid transcription factor GATA-1 and its partner, the 9 zinc finger protein, FOG (Friend Of GATA). We demonstrate that this interaction represents a genuine finger-finger contact, which is dependent on zinc-coordinating residues within each protein. We map the contact domains to the core of the N-terminal zinc finger of GATA-1 and the 6th zinc finger of FOG. Using a scanning substitution strategy we identify key residues within the GATA-1 N-finger which are required for FOG binding. These residues are conserved in the N-fingers of all GATA proteins known to bind FOG, but are not found in the respective C-fingers. This observation may, therefore, account for the particular specificity of FOG for N-fingers. Interestingly, the key N-finger residues are seen to form a contiguous surface, when mapped onto the structure of the N-finger of GATA-1.


Subject(s)
Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Zinc Fingers , 3T3 Cells , Amino Acid Sequence , Animals , Base Sequence , Conserved Sequence , DNA Primers , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Promoter Regions, Genetic , Protein Binding , Protein Conformation , Sequence Homology, Amino Acid , Transcription Factors/chemistry , Transcription Factors/genetics
20.
Biochemistry ; 37(47): 16663-70, 1998 Nov 24.
Article in English | MEDLINE | ID: mdl-9843434

ABSTRACT

The motor protein kinesin is a heterotetramer composed of two heavy chains of approximately 120 kDa and two light chains of approximately 65 kDa protein. Kinesin motor activity is dependent on the presence of ATP and microtubules. The kinesin light chain-binding site in human kinesin heavy chain was determined by reconstituting in vitro a complex of recombinant heavy and light chains. The proteins expressed in bacteria included oligohistidine-tagged fragments of human ubiquitous kinesin heavy chain, spanning most of the stalk and all of the tail domain (amino acids 555-963); and untagged, essentially full-length human kinesin light chain (4-569) along with N-terminal (4-363) and C-terminal (364-569) light chain fragments. Heavy chain fragments were attached to Ni2+-charged beads and incubated with untagged light chain fragments. Analysis of eluted complexes by SDS-PAGE and immunoblotting mapped the light chain-binding site in heavy chain to amino acids 771-813, a region close to the C-terminal end of the heavy chain stalk domain. In addition, only the full-length and N-terminal kinesin light chain fragments bound to this heavy chain region. Within this heavy chain region are four highly conserved contiguous heptad repeats (775-802) which are predicted to form a tight alpha-helical coiled-coil interaction with the heptad repeat-containing N-terminus of the light chain, in particular region 106-152 of human light chain. This predicted hydrophobic, alpha-helical coiled-coil interaction is supported by both circular dichroism spectroscopy of the recombinant kinesin heavy chain fragment 771-963, which displays an alpha-helical content of 70%, and the resistance of the heavy/light chain interaction to high salt (0.5 M).


Subject(s)
Kinesins/metabolism , Microtubule-Associated Proteins/metabolism , Peptide Fragments/metabolism , Amino Acid Sequence , Animals , Binding Sites/genetics , Cattle , Decapodiformes , Drosophila , Humans , Kinesins/chemistry , Kinesins/genetics , Mice , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/genetics , Molecular Sequence Data , Neurons/chemistry , Neurons/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Sea Urchins
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