Glycine Insertion in the Hinge Region of Lactose Repressor Protein Alters DNA Binding

Falcon, Catherine M.; Matthews, Kathleen S.

doi:10.1074/jbc.274.43.30849

Cited by 44 publications

(66 citation statements)

References 72 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It was shown that the mutated subunit formed a heterodimer that did not bind to the receptor in vitro. The uncoupling of a quaternary event and target binding are also supported by recent mutation studies of the Escherichia coli lactose repressor protein (LacI), where it was demonstrated that although assembly and folding of the two functional domains were not significantly affected, their affinity for the DNA target sequence was reduced dramatically (33). These results are consistent with our earlier studies that gonadotropin variants composed of more than 2 subunit domains bind and activate the receptor (21).…”

Section: Biosynthesis Of Hcg Tethersupporting

confidence: 82%

The Position of the α and β Subunits in a Single Chain Variant of Human Chorionic Gonadotropin Affects the Heterodimeric Interaction of the Subunits and Receptor-binding Epitopes

Ben-Menahem

Jablonka‐Shariff

Hyde

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

The glycoprotein hormone family represents a class of heterodimers, which include the placental hormone human chorionic gonadotropin (CG) and the anterior pituitary hormones follitropin, lutropin, and thyrotropin. They are composed of common ␣ subunit and a hormone-specific ␤ subunit. Based on the CG crystal structure, it was suggested that the quaternary subunit interactions are crucial for biological activity. However, recent observations using single chain glycoprotein hormone analogs, where the ␤ and ␣ subunits are linked (NH 2 -CG␤-␣; CG␤␣ orientation), implied that the heterodimeric-like quaternary configuration is not a prerequisite for receptor binding/signal transduction. To study the heterodimeric alignment of the two subunit domains in a single chain and its role in the intracellular behavior and biological action of the hormone, a single chain CG variant was constructed in which the carboxyl terminus of ␣ was fused to the CG␤ amino terminus (NH 2 -␣-CG␤; ␣CG␤ orientation). The secretion rate of ␣CG␤ from transfected Chinese hamster ovary cells was less than that seen for CG␤␣. The ␣CG␤ tether was not recognized by dimer-specific monoclonal antibodies and did not bind to lutropin/CG receptor. To define if one or both subunit domains were modified in ␣CG␤, it was co-transfected with a monomeric ␣ or CG␤ gene. In each case, ␣CG␤/␣ and ␣CG␤/CG␤ complexes were formed indicating that CG dimer-specific epitopes were established. The ␣CG␤/␣ complex bound to receptor indicating that the ␤ domain in the ␣CG␤ tether was still functional. In contrast, no significant receptor binding of ␣CG␤/CG␤ was observed indicating a major perturbation in the ␣ domain. These results suggest that although dimeric-like determinants are present in both ␣CG␤/␣ and ␣CG␤/CG␤ complexes, the receptor binding determinants in the ␣ domain of the tether are absent. These results show that generating heterodimeric determinants do not necessarily result in a bioactive molecule. Our data also indicate that the determinants for biological activity are distinct from those associated with intracellular behavior.The glycoprotein hormones lutropin (LH), 1 follitropin (FSH), thyrotropin, and choriogonadotropin (CG) are heterodimers, which consist of a common ␣ subunit and a unique ␤ subunit that confer the receptor specificity of the ligand. The subunits combine non-covalently early in the secretory pathway, and formation of the heterodimer is crucial for binding to the gonadal and thyroid receptors. Although it is well established that both subunits contain residues critical for bioactivity, recent evidence indicates that the quaternary interactions between the two subunits are essential for intracellular behavior but not for in vitro bioactivity (1-3). This conclusion was based on the single chain gonadotropin model where the amino end of the common ␣ subunit was genetically fused to the carboxyl end of CG␤ subunit (designated CG␤␣; Fig. 1) (4). This analog was secreted efficiently and was active in in vitro and in vivo bioassays. The CG␤␣ ori...

show abstract

Section: Biosynthesis Of Hcg Tethersupporting

confidence: 82%

The Position of the α and β Subunits in a Single Chain Variant of Human Chorionic Gonadotropin Affects the Heterodimeric Interaction of the Subunits and Receptor-binding Epitopes

Ben-Menahem

Jablonka‐Shariff

Hyde

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…We next tested a set of LacI variants expected to have weak affinity for their binding sequences based on previous studies (Falcon and Matthews 1999). Notably, one of these variants, LacI** (see the Materials and Methods), enabled the detection of a lacO array in vivo without interfering with the expression of a neighboring silencer-flanked reporter gene ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Tight protein–DNA interactions favor gene silencing

Dubarry¹,

Loïodice²,

Chen³

et al. 2011

Genes Dev.

View full text Add to dashboard Cite

The heterochromatin-like structure formed by the yeast silent information regulator complex (SIR) represses transcription at the silent mating type loci and telomeres. Here, we report that tight protein-DNA complexes induce ectopic recruitment of the SIR complex, promoting gene silencing and changes in subnuclear localization when cis-acting elements are nearby. Importantly, lack of the replication fork-associated helicase Rrm3 enhances this induced gene repression. Additionally, Sir3 and Sir4 are enriched genome-wide at natural replication pause sites, including tRNA genes. Consistently, inserting a tRNA gene promotes SIR-mediated silencing of a nearby gene. These results reveal that replication stress arising from tight DNAprotein interactions favors heterochromatin formation.

show abstract

“…To further examine the effects of substitutions on the allosteric transition of this protein, operator release experiments were conducted. Nitrocellulose filter binding was used to monitor decreased levels of bound DNA as a function of inducer concentration (30,31,38,39). Results are presented in Table 3 and Figure 4.…”

Section: Inducibility Of V52 Mutantsmentioning

confidence: 99%

Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

2006

View full text Add to dashboard Cite

A significant number of eukaryotic regulatory proteins are predicted to have disordered regions. Many of these proteins bind DNA, which may serve as a template for protein folding. Similar behavior is seen in the prokaryotic LacI/GalR family of proteins that couple hinge-helix folding with DNA binding. These hinge regions form short α-helices when bound to DNA, but appear to be disordered in other states. An intriguing question is whether and to what degree intrinsic helix propensity contributes to the function of these proteins. In addition to its interaction with operator DNA, the LacI hinge helix interacts with the hinge helix of the homodimer partner as well as to the surface of the inducer-binding domain. To explore the hierarchy of these interactions, we made a series of substitutions in the LacI hinge helix at position 52, the only site in the helix that does not interact with DNA and/or the inducer-binding domain. The substitutions at V52 have significant effects on operator binding affinity and specificity, and several substitutions also impair functional communication with the inducer-binding domain. Results suggest that helical propensity of amino acids in the hinge region alone does not dominate function; helix-helix packing interactions appear to also contribute. Further, the data demonstrate that variation in operator sequence can overcome side chain effects on hinge helix folding and/or hinge•hinge interactions. Thus, this system provides a direct example whereby an extrinsic interaction (DNA binding) guides internal events that influence folding and functionality. KeywordsLacI; helical propensity; allostery; DNA binding Protein folding has long been a fundamental issue in biological sciences. Beyond the general question of how a polypeptide sequence adopts a three-dimensional structure, coupled folding and binding have been identified as a key feature of partially unstructured proteins in multiple regulatory processes (e.g., transcription regulation, signal transduction, membrane transport and signaling (6-9)). Indeed, more and more intrinsically disordered proteins are found to fold upon binding to their target partners, ligands, or even small ions (10-13). In the case of transcription regulation, mutual cooperative folding of both protein and DNA has been † This work was supported by NIH Grant GM22441 and Robert A. Welch Grant C-576 to Kathleen Shive Matthews. Liskin Swint-Kruse is supported by NIH Grant P20 RR17708 from the Institutional Development Award program of the National Center for Research Resources.*To whom correspondence should be addressed. Telephone: 713−348−4871; Fax: 713−348−6149; Email: E-mail: ksm@bioc.rice.edu.. observed (6,11,14,15). In this paper, coupled folding is explored in greater detail for the hinge helix of the lactose repressor protein (LacI) 1 . NIH Public AccessLacI is a premier model for the regulation of gene expression and allosteric transition in biological systems (2) 2 . This 150-kDa homotetramer is a dimer of functionally independent dimers, with ...

show abstract

Glycine Insertion in the Hinge Region of Lactose Repressor Protein Alters DNA Binding

Cited by 44 publications

References 72 publications

The Position of the α and β Subunits in a Single Chain Variant of Human Chorionic Gonadotropin Affects the Heterodimeric Interaction of the Subunits and Receptor-binding Epitopes

The Position of the α and β Subunits in a Single Chain Variant of Human Chorionic Gonadotropin Affects the Heterodimeric Interaction of the Subunits and Receptor-binding Epitopes

Tight protein–DNA interactions favor gene silencing

Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

Contact Info

Product

Resources

About