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Jacqueline M Matthews

Publications and source records attributed to Jacqueline M Matthews.

At least 19 recordsLinked to original sources
PubMed2007

Dimerization of CtIP may stabilize in vivo interactions with the Retinoblastoma-pocket domain.

CtIP is a tumor suppressor that interacts with Retinoblastoma protein (Rb) to regulate the G1/S-phase transition of the cell cycle. Despite its large size (897 residues) CtIP has few known structured regions. Rather it contains several linear motifs that interact with known binding partners, including an LXCXE motif that binds the pocket domain of Rb-family proteins. This LXCXE motif lies at the C-terminus of the only known structured domain, an N-terminal coiled-coil dimerization domain (DD; residues 45-160). Yeast two-hybrid (Y2H) and GST-pulldown analyses showed that CtIP requires the LXCXE motif to bind the Rb-pocket. Although isothermal titration calorimetry data indicates that the LXCXE motif is the sole determinant of binding affinity for the Rb-pocket domain (K(A) approximately 10(6)M(-1)), Y2H data indicates that the DD is required to stabilize the interaction in vivo. Thus dimerization may increase the apparent stability of the proteins and/or the lifetime of the complexes.

Amino Acid Motifs↗
PubMed2006

Mechanism of allosteric regulation of transglutaminase 2 by GTP.

Allosteric regulation is a fundamental mechanism of biological control. Here, we investigated the allosteric mechanism by which GTP inhibits cross-linking activity of transglutaminase 2 (TG2), a multifunctional protein, with postulated roles in receptor signaling, extracellular matrix assembly, and apoptosis. Our findings indicate that at least two components are involved in functionally coupling the allosteric site and active center of TG2, namely (i) GTP binding to mask a conformationally destabilizing switch residue, Arg-579, and to facilitate interdomain interactions that promote adoption of a compact, catalytically inactive conformation and (ii) stabilization of the inactive conformation by an uncommon H bond between a cysteine (Cys-277, an active center residue) and a tyrosine (Tyr-516, a residue located on a loop of the beta-barrel 1 domain that harbors the GTP-binding site). Although not essential for GTP-mediated inhibition of cross-linking, this H bond enhances the rate of formation of the inactive conformer.

Allosteric Regulation↗
PubMed2006

Stabilization of a binary protein complex by intein-mediated cyclization.

The study of protein-protein interactions can be hampered by the instability of one or more of the protein complex components. In this study, we showed that intein-mediated cyclization can be used to engineer an artificial intramolecular cyclic protein complex between two interacting proteins: the largely unstable LIM-only protein 4 (LMO4) and an unstructured domain of LIM domain binding protein 1 (ldb1). The X-ray structure of the cyclic complex is identical to noncyclized versions of the complex. Chemical and thermal denaturation assays using intrinsic tryptophan fluorescence and dynamic light scattering were used to compare the relative stabilities of the cyclized complex, the intermolecular (or free) complex, and two linear versions of the intramolecular complex (in which the interacting domains of LMO4 and ldb1 were fused, via a flexible linker, in either orientation). In terms of resistance to denaturation, the cyclic complex is the most stable variant and the intermolecular complex is the least stable; however, the two linear intramolecular variants show significant differences in stability. These differences appear to be related to the relative contact order (the average distance in sequence between residues that make contacts within a structure) of key binding residues at the interface of the two proteins. Thus, the restriction of the more stable component of a complex may enhance stability to a greater extent than restraining less stable components.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Disparate thermodynamics governing T cell receptor-MHC-I interactions implicate extrinsic factors in guiding MHC restriction.

The underlying basis of major histocompatibility complex (MHC) restriction is unclear. Nevertheless, current data suggest that a common thermodynamic signature dictates alphabeta T cell receptor (TcR) ligation. To evaluate whether this thermodynamic signature defines MHC restriction, we have examined the thermodynamic basis of a highly characterized immunodominant TcR interacting with its cognate peptide-MHC-I ligand. Surprisingly, we observed this interaction to be governed by favorable enthalpic and entropic forces, which is in contrast to the prevailing generality, namely, enthalpically driven interactions combined with markedly unfavorable entropic forces. We conclude that extrinsic molecular factors, such as coreceptor ligation, conformational adjustments involved in TcR signaling, or constraints dictated by higher-order arrangement of ligated TcRs, might play a greater role in guiding MHC restriction than appreciated previously.

Amino Acid Sequence↗
PubMed2006

Identification of the key LMO2-binding determinants on Ldb1.

The overexpression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations, retroviral insertion during gene therapy, or in transgenic mice models, leads to the onset of T-cell leukemias. LMO2 comprises two protein-binding LIM domains that allow LMO2 to interact with multiple protein partners, including LIM domain-binding protein 1 (Ldb1, also known as CLIM2 and NLI), an essential cofactor for LMO proteins. Sequestration of Ldb1 by LMO2 in T-cells may prevent it binding other key partners, such as LMO4. Here, we show using protein engineering and enzyme-linked immunosorbent assay (ELISA) methodologies that LMO2 binds Ldb1 with a twofold lower affinity than does LMO4. Thus, excess LMO2 rather than an intrinsically higher binding affinity would lead to sequestration of Ldb1. Both LIM domains of LMO2 are required for high-affinity binding to Ldb1 (K(D) = 2.0 x 10(-8) M). However, the first LIM domain of LMO2 is primarily responsible for binding to Ldb1 (K(D) = 2.3 x 10(-7) M), whereas the second LIM domain increases binding by an order of magnitude. We used mutagenesis in combination with yeast two-hybrid analysis, and phage display selection to identify LMO2-binding "hot spots" within Ldb1 that locate to the LIM1-binding region. The delineation of this region reveals some specific differences when compared to the equivalent LMO4:Ldb1 interaction that hold promise for the development of reagents to specifically bind LMO2 in the treatment of leukemia.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Mutation of a critical arginine in the GTP-binding site of transglutaminase 2 disinhibits intracellular cross-linking activity.

Transglutaminase type 2 (TG2; also known as G(h)) is a multifunctional protein involved in diverse cellular processes. It has two well characterized enzyme activities: receptor-stimulated signaling that requires GTP binding and calcium-activated transamidation or cross-linking that is inhibited by GTP. In addition to the GDP binding residues identified from the human TG2 crystal structure (Liu, S., Cerione, R. A., and Clardy, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 2743-2747), we have previously implicated Ser171 in GTP binding, as binding is lost with glutamate substitution (Iismaa, S. E., Wu, M.-J., Nanda, N., Church, W. B., and Graham, R. M. (2000) J. Biol. Chem. 275, 18259-18265). Here, we have shown that alanine substitution of homologous residues in rat TG2 (Phe174 in the core domain or Arg476, Arg478, or Arg579 in barrel 1) does not affect TG activity but reduces or abolishes GTP binding and GTPgammaS inhibition of TG activity in vitro, indicating that these residues are important in GTP binding. Alanine substitution of Ser171 does not impair GTP binding, indicating this residue does not interact directly with GTP. Arg579 is particularly important for GTP binding, as isothermal titration calorimetry demonstrated a 100-fold reduction in GTP binding affinity by the R579A mutant. Unlike wild-type TG2 or its S171E or F174A mutants, which are sensitive to both trypsin and mu-calpain digestion, R579A is inherently more resistant to mu-calpain, but not trypsin, digestion, indicating reduced accessibility and/or flexibility of this mutant in the region of the calpain cleavage site(s). Basal TG activity of intact R579A stable SH-SY5Y neuroblastoma cell transfectants was slightly increased relative to wild-type transfectants and, in contrast to the TG activity of the latter, was further stimulated by muscarinic receptor-activated calcium mobilization. Thus, loss of GTP binding sensitizes TG2 to intracellular calcium concentrations. These findings are consistent with the notion that intracellularly, under physiological conditions, TG2 is maintained largely as a latent enzyme, its calcium-activated cross-linking activity being suppressed allosterically by guanine nucleotide binding.

Animals↗
PubMed2005

Stabilization of native protein fold by intein-mediated covalent cyclization.

A mutant version of the N-terminal domain of Escherichia coli DnaB helicase was used as a model system to assess the stabilization against unfolding gained by covalent cyclization. Cyclization was achieved in vivo by formation of an amide bond between the N and C termini with the help of a split mini-intein. Linear and circular proteins were constructed to be identical in amino acid sequence. Mutagenesis of Phe102 to Glu rendered the protein monomeric even at high concentration. A difference in free energy of unfolding, DeltaDeltaG, between circular and linear protein of 2.3(+/-0.5) kcal mol(-1) was measured at 10 degrees C by circular dichroism. A theoretical estimate of the difference in conformational entropy of linear and circular random chains in a three-dimensional cubic lattice model predicted DeltaDeltaG=2.3 kcal mol(-1), suggesting that stabilization by protein cyclization is driven by the reduced conformational entropy of the unfolded state. Amide-proton exchange rates measured by NMR spectroscopy and mass spectrometry showed a uniform, approximately tenfold decrease of the exchange rates of the most slowly exchanging amide protons, demonstrating that cyclization globally decreases the unfolding rate of the protein. The amide proton exchange was found to follow EX1 kinetics at near-neutral pH, in agreement with an unusually slow refolding rate of less than 4 min(-1) measured by stopped-flow circular dichroism. The linear and circular proteins differed more in their unfolding than in their folding rates. Global unfolding of the N-terminal domain of E.coli DnaB is thus promoted strongly by spatial separation of the N and C termini, whereas their proximity is much less important for folding.

Adenosine Triphosphatases↗
PubMed2005

A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis.

Two dimers of the replication terminator protein (RTP) of Bacillus subtilis bind to a chromosomal DNA terminator site to effect polar replication fork arrest. Cooperative binding of the dimers to overlapping half-sites within the terminator is essential for arrest. It was suggested previously that polarity of fork arrest is the result of the RTP dimer at the blocking (proximal) side within the complex binding very tightly and the permissive-side RTP dimer binding relatively weakly. In order to investigate this "differential binding affinity" model, we have constructed a series of mutant terminators that contain half-sites of widely different RTP binding affinities in various combinations. Although there appeared to be a correlation between binding affinity at the proximal half-site and fork arrest efficiency in vivo for some terminators, several deviated significantly from this correlation. Some terminators exhibited greatly reduced binding cooperativity (and therefore have reduced affinity at each half-site) but were highly efficient in fork arrest, whereas one terminator had normal affinity over the proximal half-site, yet had low fork arrest efficiency. The results show clearly that there is no direct correlation between the RTP binding affinity (either within the full complex or at the proximal half-site within the full complex) and the efficiency of replication fork arrest in vivo. Thus, the differential binding affinity over the proximal and distal half-sites cannot be solely responsible for functional polarity of fork arrest. Furthermore, efficient fork arrest relies on features in addition to the tight binding of RTP to terminator DNA.

Bacillus subtilis↗
PubMed2005

Zinc fingers as protein recognition motifs: structural basis for the GATA-1/friend of GATA interaction.

GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1/FOG interaction, we have determined the three-dimensional structure of a complex comprising the interaction domains of these proteins. The structure reveals how zinc fingers can act as protein recognition motifs. Details of the architecture of the contact domains and their physical properties provide a molecular explanation for how the GATA-1 mutations contribute to distinct but related genetic diseases.

Binding Sites↗
PubMed2005

Protein-protein interactions in human disease.

Many human diseases are the result of abnormal protein-protein interactions involving endogenous proteins, proteins from pathogens or both. The inhibition of these aberrant associations is of obvious clinical significance. Because of the diverse nature of protein-protein interactions, however, the successful design of therapeutics requires detailed knowledge of each system at a molecular and atomic level. Several recent studies have identified and/or characterised specific interactions from various disease systems, including cervical cancer, bacterial infection, leukaemia and neurodegenerative disease. A range of approaches are being developed to generate inhibitors of protein-protein interactions that may form useful therapeutics for human disease.

Disease↗
PubMed2005

Assessment of the robustness of a serendipitous zinc binding fold: mutagenesis and protein grafting.

Zinc binding motifs have received much attention in the area of protein design. Here, we have tested the suitability of a recently discovered nonnative zinc binding structure as a protein design scaffold. A series of multiple alanine mutants was created to investigate the minimal requirements for folding, and solution structures of these mutants showed that the original fold was maintained, despite changes in approximately 50% of the sequence. We next attempted to transplant binding faces from chosen bimolecular interactions onto one of these mutants, and many of the resulting "chimeras" were shown to adopt a native-like fold. These results both highlight the robust nature of small zinc binding domains and underscore the complexity of designing functional proteins, even using such small, highly ordered scaffolds as templates.

Alanine↗
PubMed2004

Structural studies on a protein-binding zinc-finger domain of Eos reveal both similarities and differences to classical zinc fingers.

The oligomerization domain that is present at the C terminus of Ikaros-family proteins and the protein Trps-1 is important for the proper regulation of developmental processes such as hematopoiesis. Remarkably, this domain is predicted to contain two classical zinc fingers (ZnFs), domains normally associated with the recognition of nucleic acids. The preference for protein binding by these predicted ZnFs is not well-understood. We have used a range of methods to gain insight into the structure of this domain. Circular dichroism, UV-vis, and NMR experiments carried out on the C-terminal domain of Eos (EosC) revealed that the two putative ZnFs (C1 and C2) are separable, i.e., capable of folding independently in the presence of Zn(II). We next determined the structure of EosC2 using NMR spectroscopy, revealing that, although the overall fold of EosC2 is similar to other classical ZnFs, a number of differences exist. For example, the conformation of the C terminus of EosC2 appears to be flexible and may result in a major rearrangement of the zinc ligands. Finally, alanine-scanning mutagenesis was used to identify the residues that are involved in the homo- and hetero-oligomerization of Eos, and these results are discussed in the context of the structure of EosC. These studies provide the first structural insights into how EosC mediates protein-protein interactions and contributes to our understanding of why it does not exhibit high-affinity DNA binding.

Amino Acid Sequence↗
PubMed2004

Tandem LIM domains provide synergistic binding in the LMO4:Ldb1 complex.

Nuclear LIM-only (LMO) and LIM-homeodomain (LIM-HD) proteins have important roles in cell fate determination, organ development and oncogenesis. These proteins contain tandemly arrayed LIM domains that bind the LIM interaction domain (LID) of the nuclear adaptor protein LIM domain-binding protein-1 (Ldb1). We have determined a high-resolution X-ray crystal structure of LMO4, a putative breast oncoprotein, in complex with Ldb1-LID, providing the first example of a tandem LIM:Ldb1-LID complex and the first structure of a type-B LIM domain. The complex possesses a highly modular structure with Ldb1-LID binding in an extended manner across both LIM domains of LMO4. The interface contains extensive hydrophobic and electrostatic interactions and multiple backbone-backbone hydrogen bonds. A mutagenic screen of Ldb1-LID, assessed by yeast two-hybrid and competition ELISA analysis, identified key features at the interface and revealed that the interaction is tolerant to mutation. These combined properties provide a mechanism for the binding of Ldb1 to numerous LMO and LIM-HD proteins. Furthermore, the modular extended interface may form a general mode of binding to tandem LIM domains.

Adaptor Proteins, Signal Transducing↗
PubMed2004

A classic zinc finger from friend of GATA mediates an interaction with the coiled-coil of transforming acidic coiled-coil 3.

Classic zinc finger domains (cZFs) consist of a beta-hairpin followed by an alpha-helix. They are among the most abundant of all protein domains and are often found in tandem arrays in DNA-binding proteins, with each finger contributing an alpha-helix to effect sequence-specific DNA recognition. Lone cZFs, not found in tandem arrays, have been postulated to function in protein interactions. We have studied the transcriptional co-regulator Friend of GATA (FOG), which contains nine zinc fingers. We have discovered that the third cZF of FOG contacts a coiled-coil domain in the centrosomal protein transforming acidic coiled-coil 3 (TACC3). Although FOG-ZF3 exhibited low solubility, we have used a combination of mutational mapping and protein engineering to generate a derivative that was suitable for in vitro and structural analysis. We report that the alpha-helix of FOG-ZF3 recognizes a C-terminal portion of the TACC3 coiled-coil. Remarkably, the alpha-helical surface utilized by FOG-ZF3 is the same surface responsible for the well established sequence-specific DNA-binding properties of many other cZFs. Our data demonstrate the versatility of cZFs and have implications for the analysis of many as yet uncharacterized cZF proteins.

Amino Acid Sequence↗
PubMed2004

TC-1 is a novel tumorigenic and natively disordered protein associated with thyroid cancer.

A novel gene, thyroid cancer 1 (TC-1), was found recently to be overexpressed in thyroid cancer. TC-1 shows no homology to any of the known thyroid cancer-associated genes. We have produced stable transformants of normal thyroid cells that express the TC-1 gene, and these cells show increased proliferation rates and anchorage-independent growth in soft agar. Apoptosis rates also are decreased in the transformed cells. We also have expressed recombinant TC-1 protein and have undertaken a structural and functional characterization of the protein. The protein is monomeric and predominantly unstructured under conditions of physiologic salt and pH. This places it in the category of natively disordered proteins, a rapidly expanding group of proteins, many members of which play critical roles in cell regulation processes. We show that the protein can be phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C, and the activity of both of these kinases is up-regulated when cells are stably transfected with TC-1. These results suggest that overexpression of TC-1 may be important in thyroid carcinogenesis.

Apoptosis↗
PubMed2004

Dimerization of CtIP, a BRCA1- and CtBP-interacting protein, is mediated by an N-terminal coiled-coil motif.

CtIP is a transcriptional co-regulator that binds a number of proteins involved in cell cycle control and cell development, such as CtBP (C terminus-binding protein), BRCA1 (breast cancer-associated protein-1), and LMO4 (LIM-only protein-4). The only recognizable structural motifs within CtIP are two putative coiled-coil domains located near the N and C termini of the protein. We now show that the N-terminal coiled coil (residues 45-160), but not the C-terminal coiled coil, mediates homodimerization of CtIP in mammalian 293T cells. The N-terminal coiled coil did not facilitate binding to LMO4 and BRCA1 proteins in these cells. A protease-resistant domain (residues 27-168) that minimally encompasses the putative N-terminal coiled coil was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This region is predicted to contain two smaller coiled-coil regions. The CtIP-(45-160) dimerization domain is helical and dimeric, indicating that the domain does form a coiled coil. The two smaller domains, CtIP-(45-92) and CtIP-(93-160), also formed dimers of lower binding affinity, but with less helical content than the longer peptide. The hydrodynamic radius of CtIP-(45-160) is the same as those of CtIP-(45-92) and CtIP-(93-160), implying that CtIP-(45-160) does not form a single long coiled coil, but a more compact structure involving homodimerization of the two smaller coiled coils, which fold back as a four-helix bundle or other compact structure. These results suggest a specific model for CtIP homodimerization via its N terminus and contribute to an improved understanding of how this protein might assemble other factors required for its role as a transcriptional corepressor.

Adaptor Proteins, Signal Transducing↗
PubMed2004

The power of two: protein dimerization in biology.

The self-association of proteins to form dimers and higher-order oligomers is a very common phenomenon. Recent structural and biophysical studies show that protein dimerization or oligomerization is a key factor in the regulation of proteins such as enzymes, ion channels, receptors and transcription factors. In addition, self-association can help to minimize genome size, while maintaining the advantages of modular complex formation. Oligomerization, however, can also have deleterious consequences when nonnative oligomers associated with pathogenic states are generated. Specific protein dimerization is integral to biological function, structure and control, and must be under substantial selection pressure to be maintained with such frequency throughout biology.

Allosteric Regulation↗