Allostery mediates ligand binding to Grb2 adaptor in a mutually exclusive manner

Allostery plays a key role in dictating the stoichiometry and thermodynamics of multi‐protein complexes driving a plethora of cellular processes central to health and disease. Herein, using various biophysical tools, we demonstrate that although Sos1 nucleotide exchange factor and Gab1 docking protein recognize two non‐overlapping sites within the Grb2 adaptor, allostery promotes the formation of two distinct pools of Grb2–Sos1 and Grb2–Gab1 binary signaling complexes in concert in lieu of a composite Sos1–Grb2–Gab1 ternary complex. Of particular interest is the observation that the binding of Sos1 to the nSH3 domain within Grb2 sterically blocks the binding of Gab1 to the cSH3 domain and vice versa in a mutually exclusive manner. Importantly, the formation of both the Grb2–Sos1 and Grb2–Gab1 binary complexes is governed by a stoichiometry of 2:1, whereby the respective SH3 domains within Grb2 homodimer bind to Sos1 and Gab1 via multivalent interactions. Collectively, our study sheds new light on the role of allostery in mediating cellular signaling machinery. Copyright © 2013 John Wiley & Sons, Ltd.     

Discrete elements within the SV40 enhancer region display different cell-specific enhancer activities.

The SV40 enhancer contains three genetically defined elements, called A, B and C, that can functionally compensate for one another. By using short, synthetic DNA oligonucleotides, we show that each of these elements can act autonomously as an enhancer when present as multiple tandem copies. Analysis of a progressive series of B element oligomers shows a single element is ineffective as an enhancer and that the activity of two or more elements increases with copy number. Assay in five different cell lines of two separate enhancers containing six tandem copies of either the B or C element shows that these elements possess different cell-specific activities. Parallel oligomer enhancer constructs containing closely spaced double point mutations display no enhancer activity in any of the cell lines tested, indicating that these elements represent single units of enhancer function. These elements contain either a 'core' or 'octamer' consensus sequence but these consensus sequences alone are not sufficient for enhancer activity. The different cell-specific activities of the B and C elements are consistent with functional interactions with different trans-acting factors. We discuss how tandem duplication of such dissimilar elements, as in the wild-type SV40 72-bp repeats, can serve to expand the conditions under which an enhancer can function.     

Multiple nuclear factors interact with the immunoglobulin enhancer sequences

To characterize proteins that bind to the immunoglobulin (Ig) heavy chain and the kappa light chain enhancers, an electrophoretic mobility shift assay with end-labeled DNA fragments was used. Three binding proteins have been found. One is NF-A, a factor found in all tested cell types that binds to the octamer sequence found upstream of all Ig variable region gene segments and to the same octamer in the heavy chain enhancer. The second, also ubiquitous, protein binds to a sequence in both the heavy chain and the kappa enhancers that was previously shown to be protected from methylation in vivo. Other closely related sites do not compete for this binding, implying a restriction enzyme-like binding specificity. The third protein binds to a sequence in the kappa enhancer (and to an identical sequence in the SV40 enhancer) and is restricted in its occurrence to B cells.     

Determination of the DNA conformation of the simian virus 40 (SV40) enhancer in SV40 minichromosomes

The simian virus 40 (SV40) enhancer contains three 8-bp purine-pyrimidine alternating sequences which are known to adopt the left-handed Z-DNA conformation in vitro. In this paper, we have undertaken the determination of the DNA conformation adopted by these Z-motifs in the SV40 minichromosome. We have analyzed the presence of Z-DNA through the change in linkage which should accompany formation of this left-handed conformation. Our results indicate that, regardless of the precise moment of the viral lytic cycle at which minichromosomes are harvested and the condition of the transfected DNA, either relaxed or negatively supercoiled, none of the three Z motifs of the SV40 enhancer exist to a significant extent as Z-DNA in SV40 minichromosomes. The SV40 enhancer adopts predominantly a right-handed B-DNA conformation in vivo.     

The CLIP-170 N-terminal domain binds directly to both F-actin and microtubules in a mutually exclusive manner

The cooperation between the actin and microtubule (MT) cytoskeletons is important for cellular processes such as cell migration and muscle cell development. However, a full understanding of how this cooperation occurs has yet to be sufficiently developed. The MT plus-end tracking protein CLIP-170 has been implicated in this actin–MT coordination by associating with the actin-binding signaling protein IQGAP1 and by promoting actin polymerization through binding with formins. Thus far, the interactions of CLIP-170 with actin were assumed to be indirect. Here, we demonstrate using high-speed cosedimentation assays that CLIP-170 can bind to filamentous actin (F-actin) directly. We found that the affinity of this binding is relatively weak but strong enough to be significant in the actin-rich cortex, where actin concentrations can be extremely high. Using CLIP-170 fragments and mutants, we show that the direct CLIP-170–F-actin interaction is independent of the FEED domain, the region that mediates formin-dependent actin polymerization, and that the CLIP-170 F-actin-binding region overlaps with the MT-binding region. Consistent with these observations, in vitro competition assays indicate that CLIP-170–F-actin and CLIP-170–MT interactions are mutually exclusive. Taken together, these observations lead us to speculate that direct CLIP-170–F-actin interactions may function to reduce the stability of MTs in actin-rich regions of the cell, as previously proposed for MT end-binding protein 1.     

Prediction of 'hot spots' in SV40 enhancer and relation with experimental data

A theoretical method for prediction of the points in the sequence which are relevant for its biological function, the so-called 'hot spots', is presented. This method is based on significant correlation between the spectrum of numerical presentation of any genetic sequence and its biological function. One number corresponds to the particular nucleotide, thus forming a numerical sequence. The 'hot spot' prediction has been tested on the SV40 enhancer as a model system. The SV40 enhancer was chosen because of existing detailed data about activities of systematically obtained mutants. These results have been compared with results obtained theoretically.