Biomedical subjects

Jacqueline M Matthews

Publications and source records attributed to Jacqueline M Matthews.

28 records · Page 2Linked to original sources

PubMed2003

Crystallization of FLINC4, an intramolecular LMO4-ldb1 complex.

LMO4 is the most recently discovered member of a small family of nuclear transcriptional regulators that are important for both normal development and disease processes. LMO4 is comprised primarily of two tandemly repeated LIM domains and interacts with the ubiquitous nuclear adaptor protein ldb1. This interaction is mediated via the LIM domains of LMO4 and the LIM-interaction domain (LID) of ldb1. An intramolecular complex, termed FLINC4, consisting of the two LIM domains from LMO4 linked to the LID domain of ldb1 via a flexible linker has been engineered, purified and crystallized. The trigonal crystals, which belong to space group P312 with unit-cell parameters a = 61.3, c = 93.2 A, diffract to 1.3 A resolution and contain one molecule of FLINC4 per asymmetric unit. Native and multiple-wavelength anomalous dispersion (MAD) data collected at the Zn X-ray absorption edge have been recorded to 1.3 and 1.7 A resolution, respectively. Anomalous Patterson maps calculated with data collected at the peak wavelength show strong peaks sufficient to determine the positions of four Zn atoms per asymmetric unit.

Adaptor Proteins, Signal Transducing↗

PubMed2003

CCHX zinc finger derivatives retain the ability to bind Zn(II) and mediate protein-DNA interactions.

Classical (CCHH) zinc fingers are among the most common protein domains found in eukaryotes. They function as molecular recognition elements that mediate specific contact with DNA, RNA, or other proteins and are composed of a betabetaalpha fold surrounding a single zinc ion that is ligated by two cysteine and two histidine residues. In a number of variant zinc fingers, the final histidine is not conserved, and in other unrelated zinc binding domains, residues such as aspartate can function as zinc ligands. To test whether the final histidine is required for normal folding and the DNA-binding function of classical zinc fingers, we focused on finger 3 of basic Krüppel-like factor. The structure of this domain was determined using NMR spectroscopy and found to constitute a typical classical zinc finger. We generated a panel of substitution mutants at the final histidine in this finger and found that several of the mutants retained some ability to fold in the presence of zinc. Consistent with this result, we showed that mutation of the final histidine had only a modest effect on DNA binding in the context of the full three-finger DNA-binding domain of basic Krüppel-like factor. Further, the zinc binding ability of one of the point mutants was tested and found to be indistinguishable from the wild-type domain. These results suggest that the final zinc chelating histidine is not an essential feature of classical zinc fingers and have implications for zinc finger evolution, regulation, and the design of experiments testing the functional roles of these domains.

Amino Acid Sequence↗

PubMed2003

Structural basis for the recognition of ldb1 by the N-terminal LIM domains of LMO2 and LMO4.

LMO2 and LMO4 are members of a small family of nuclear transcriptional regulators that are important for both normal development and disease processes. LMO2 is essential for hemopoiesis and angiogenesis, and inappropriate overexpression of this protein leads to T-cell leukemias. LMO4 is developmentally regulated in the mammary gland and has been implicated in breast oncogenesis. Both proteins comprise two tandemly repeated LIM domains. LMO2 and LMO4 interact with the ubiquitous nuclear adaptor protein ldb1/NLI/CLIM2, which associates with the LIM domains of LMO and LIM homeodomain proteins via its LIM interaction domain (ldb1-LID). We report the solution structures of two LMO:ldb1 complexes (PDB: 1M3V and 1J2O) and show that ldb1-LID binds to the N-terminal LIM domain (LIM1) of LMO2 and LMO4 in an extended conformation, contributing a third strand to a beta-hairpin in LIM1 domains. These findings constitute the first molecular definition of LIM-mediated protein-protein interactions and suggest a mechanism by which ldb1 can bind a variety of LIM domains that share low sequence homology.

Adaptor Proteins, Signal Transducing↗

PubMed2003

The LIM protein FHL3 binds basic Krüppel-like factor/Krüppel-like factor 3 and its co-repressor C-terminal-binding protein 2.

The ability of DNA-binding transcription factors to recruit specific cofactors is central to the mechanism by which they regulate gene expression. BKLF/KLF3, a member of the Krüppel-like factor family of zinc finger proteins, is a potent transcriptional repressor that recruits a CtBP co-repressor. We show here that BKLF also recruits the four and a half LIM domain protein FHL3. Different but closely linked regions of BKLF mediate contact with CtBP2 and FHL3. We present evidence that CtBP2 also interacts with FHL3 and demonstrate that the three proteins co-elute in gel filtration experiments. CtBP and FHL proteins have been implicated in both nuclear and cytoplasmic functions, but expression of BKLF promotes the nuclear accumulation of both FHL3 and CtBP2. FHL proteins have been shown to act predominantly as co-activators of transcription. However, we find FHL3 can repress transcription. We suggest that LIM proteins like FHL3 are important in assembling specific repression or activation complexes, depending on conditions such as cofactor availability and promoter context.

Animals↗

PubMed2003

Engineering a protein scaffold from a PHD finger.

The design of proteins with tailored functions remains a relatively elusive goal. Small size, a well-defined structure, and the ability to maintain structural integrity despite multiple mutations are all desirable properties for such designer proteins. Many zinc binding domains fit this description. We determined the structure of a PHD finger from the transcriptional cofactor Mi2beta and investigated the suitability of this domain as a scaffold for presenting selected binding functions. The two flexible loops in the structure were mutated extensively by either substitution or expansion, without affecting the overall fold of the domain. A binding site for the corepressor CtBP2 was also grafted onto the domain, creating a new PHD domain that can specifically bind CtBP2 both in vitro and in the context of a eukaryotic cell nucleus. These results represent a step toward designing new regulatory proteins for modulating aberrant gene expression in vivo.

Amino Acid Sequence↗

PubMed2003

LIM-domain-binding protein 1: a multifunctional cofactor that interacts with diverse proteins.

The ubiquitous nuclear adaptor protein LIM-domain-binding protein 1 (Ldb1) was originally identified as a cofactor for LIM-homeodomain and LIM-only (LMO) proteins that have fundamental roles in development. In parallel, Ldb1 has been shown to have essential functions in diverse biological processes in different organisms. The recent targeting of this gene in mice has revealed roles for Ldb1 in neural patterning and development that have been conserved throughout evolution. Furthermore, the elucidation of the three-dimensional structures of LIM-Ldb1 complexes has provided insight into the molecular basis for the ability of Ldb1 to contact diverse LIM-domain proteins. It has become evident that Ldb1 is a multi-adaptor protein that mediates interactions between different classes of transcription factors and their co-regulators and that the nature of these complexes determines cell fate and differentiation.

Amino Acid Sequence↗

PubMed2002

Characterization of the conserved interaction between GATA and FOG family proteins.

The N-terminal zinc finger (ZnF) from GATA transcription factors mediates interactions with FOG family proteins. In FOG proteins, the interacting domains are also ZnFs; these domains are related to classical CCHH fingers but have an His --> Cys substitution at the final zinc-ligating position. Here we demonstrate that different CCHC fingers in the FOG family protein U-shaped contact the N-terminal ZnF of GATA-1 in the same fashion although with different affinities. We also show that these interactions are of moderate affinity, which is interesting given the presumed low concentrations of these proteins in the nucleus. Furthermore, we demonstrate that the variant CCHC topology enhances binding affinity, although the His --> Cys change is not essential for the formation of a stably folded domain. To ascertain the structural basis for the contribution of the CCHC arrangement, we have determined the structure of a CCHH mutant of finger nine from U-shaped. The structure is very similar overall to the wild-type domain, with subtle differences at the C terminus that result in loss of the interaction in vivo. Taken together, these results suggest that the CCHC zinc binding topology is required for the integrity of GATA-FOG interactions and that weak interactions can play important roles in vivo.

Amino Acid Sequence↗

PubMed2002

Recombinant CLIC1 (NCC27) assembles in lipid bilayers via a pH-dependent two-state process to form chloride ion channels with identical characteristics to those observed in Chinese hamster ovary cells expressing CLIC1.

CLIC1 (NCC27) is an unusual, largely intracellular, ion channel that exists in both soluble and membrane-associated forms. The soluble recombinant protein can be expressed in Escherichia coli, a property that has made possible both detailed electrophysiological studies in lipid bilayers and an examination of the mechanism of membrane integration. Soluble E. coli-derived CLIC1 moves from solution into artificial bilayers and forms chloride-selective ion channels with essentially identical conductance, pharmacology, and opening and closing kinetics to those observed in CLIC1-transfected Chinese hamster ovary cells. The process of membrane integration of CLIC1 is pH-dependent. Following addition of protein to the trans solution, small conductance channels with slow kinetics (SCSK) appear in the bilayer. These SCSK modules then appear to undergo a transition to form a high conductance channel with fast kinetics. This has four times the conductance of the SCSK and fast kinetics that characterize the native channel. This suggests that the CLIC1 ion channel is likely to consist of a tetrameric assembly of subunits and indicates that despite its size and unusual properties, it is able to form a completely functional ion channel in the absence of any other ancillary proteins.

Animals↗

PubMed2002

A new zinc binding fold underlines the versatility of zinc binding modules in protein evolution.

Many different zinc binding modules have been identified. Their abundance and variety suggests that the formation of zinc binding folds might be relatively common. We have determined the structure of CH1(1), a 27-residue peptide derived from the first cysteine/histidine-rich region (CH1) of CREB binding protein (CBP). This peptide forms a highly ordered zinc-dependent fold that is distinct from known folds. The structure differs from a subsequently determined structure of a larger region from the CH3 region of CBP, and the CH1(1) fold probably represents a nonphysiologically active form. Despite this, the fold is thermostable and tolerant to both multiple alanine mutations and changes in the zinc-ligand spacing. Our data support the idea that zinc binding domains may arise frequently. Additionally, such structures may prove useful as scaffolds for protein design, given their stability and robustness.

Amino Acid Sequence↗

PubMed2002

Zinc fingers--folds for many occasions.

Zinc finger domains (ZnFs) are common, relatively small protein motifs that fold around one or more zinc ions. In addition to their role as a DNA-binding module, ZnFs have recently been shown to mediate protein:protein and protein:lipid interactions. This small zinc-ligating domain, often found in clusters containing fingers with different binding specificities, can facilitate multiple, often independent intermolecular interactions between nucleic acids and proteins. Classical ZnFs, typified by TFIIIA, ligate zinc via pairs of cysteine and histidine residues but there are at least 14 different classes of Zn fingers, which differ in the nature and arrangement of their zinc-binding residues. Some GATA-type ZnFs can bind to both DNA and a variety of other proteins. Thus proteins with multiple GATA-type fingers can play a complex role in regulating transcription through the interplay of these different binding selectivities and affinities. Other ZnFs have more specific functions, such as DNA-binding ZnFs in the nuclear hormone receptor proteins and small-molecule-binding ZnFs in protein kinase C. Some classes of ZnFs appear to act exclusively in protein-only interactions. These include the RING family of ZnFs that are involved in ubiquitination processes and in the assembly of large protein complexes, LIM, TAZ, and PHD domains. We review the similarities and differences in structure and functions of different ZnF classes and highlight the versatility of this fold.

Amino Acid Motifs↗