Recognition by the Thyroid Hormone Receptor of Canonical DNA Response Elements

To shed more light on the molecular requirements for recognition of thyroid response elements (TRES) by thyroid receptors (TRs), we compared the specific aspects of DNA TRE recognition by different TR constructs. Using fluorescence anisotropy, we performed a detailed and hierarchical study of TR-TRE binding. This wits done by comparing the binding affinities of three different TR constructs for four different TRE DNA elements, including palindromic sequences and direct repeats (F2, PAL, DR-1, and DR-4) as well as their interactions with nonspecific DNA sequences. The effect of MgCl(2) on suppressing of nonselective DNA binding to TR was also investigated. Furthermore, we determined the dissociation constants of the hTR beta DBD (DNA binding domain) and hTR beta DBD-LBD (DNA binding and ligand binding domains) for specific TRES. We found that a minimum DNA recognition peptide derived from DBD (H1TR) is sufficient for recognition and interaction with TREs, whereas scrambled DNA sequences were unrecognized. Additionally, we determined that the TR DBD binds to F2, PAL, and DR-4 with high affinity and similar K(d) values. The TR DBD-LBD recognizes all the tested TRES but binds preferentially to F2, with even higher affinity. Finally, our results demonstrate the important role played by LBDs in modulating TR-DNA binding.

On the Denaturation Mechanisms of the Ligand Binding Domain of Thyroid Hormone Receptors

The ligand binding domain (LBD) of nuclear hormone receptors adopts a very compact, mostly alpha-helical structure that binds specific ligands with very high affinity. We use circular dichroism spectroscopy and high-temperature molecular dynamics Simulations to investigate unfolding of the LBDs of thyroid hormone receptors (TRs). A molecular description of the denaturation mechanisms is obtained by molecular dynamics Simulations of the TR alpha and TR beta LBDs in the absence and in the presence of the natural ligand Triac. The Simulations Show that the thermal unfolding of the LBD starts with the loss of native contacts and secondary Structure elements, while the Structure remains essentially compact, resembling a molten globule state. This differs From most protein denaturation simulations reported to date and suggests that the folding mechanism may start with the hydrophobic collapse of the TR LBDs. Our results reveal that the stabilities of the LBDs of the TR alpha and TR beta Subtypes are affected to different degrees by the binding of the isoform selective ligand Triac and that ligand binding confers protection against thermal denaturation and unfolding in a subtype specific manner. Our Simulations indicate two mechanisms by which the ligand stabilizes the LBD: (1) by enhancing the interactions between H8 and H 11, and the interaction of the region between H I and the Omega-loop with the core of the LBD, and (2) by shielding the hydrophobic H6 from hydration.

Gaining ligand selectivity in thyroid hormone receptors via entropy

Nuclear receptors are important targets for pharmaceuticals, but similarities between family members cause difficulties in obtaining highly selective compounds. Synthetic ligands that are selective for thyroid hormone (TH) receptor beta (TR beta) vs. TR alpha reduce cholesterol and fat without effects on heart rate; thus, it is important to understand TR beta-selective binding. Binding of 3 selective ligands (GC-1, KB141, and GC-24) is characterized at the atomic level; preferential binding depends on a nonconserved residue (Asn-331 beta) in the TR beta ligand-binding cavity (LBC), and GC-24 gains extra selectivity from insertion of a bulky side group into an extension of the LBC that only opens up with this ligand. Here we report that the natural TH 3,5,3`-triodothyroacetic acid (Triac) exhibits a previously unrecognized mechanism of TR beta selectivity. TR x-ray structures reveal better fit of ligand with the TR alpha LBC. The TR beta LBC, however, expands relative to TR alpha in the presence of Triac (549 angstrom(3) vs. 461 angstrom(3)), and molecular dynamics simulations reveal that water occupies the extra space. Increased solvation compensates for weaker interactions of ligand with TR beta and permits greater flexibility of the Triac carboxylate group in TR beta than in TR alpha. We propose that this effect results in lower entropic restraint and decreases free energy of interactions between Triac and TR beta, explaining subtype-selective binding. Similar effects could potentially be exploited in nuclear receptor drug design.

Geodesics and Almost Geodesic Cycles in Random Regular Graphs

A geodesic in a graph G is a shortest path between two vertices of G. For a specific function e(n) of n, we define an almost geodesic cycle C in G to be a cycle in which for every two vertices u and v in C, the distance d(G)(u, v) is at least d(C)(u, v) - e(n). Let omega(n) be any function tending to infinity with n. We consider a random d-regular graph on n vertices. We show that almost all pairs of vertices belong to an almost geodesic cycle C with e(n)= log(d-1)log(d-1) n+omega(n) and vertical bar C vertical bar =2 log(d-1) n+O(omega(n)). Along the way, we obtain results on near-geodesic paths. We also give the limiting distribution of the number of geodesics between two random vertices in this random graph. (C) 2010 Wiley Periodicals, Inc. J Graph Theory 66: 115-136, 2011

The 3-colored Ramsey number of even cycles

Denote by R(L, L, L) the minimum integer N such that any 3-coloring of the edges of the complete graph on N vertices contains a monochromatic copy of a graph L. Bondy and Erdos conjectured that when L is the cycle C(n) on n vertices, R(C(n), C(n), C(n)) = 4n - 3 for every odd n > 3. Luczak proved that if n is odd, then R(C(n), C(n), C(n)) = 4n + o(n), as n -> infinity, and Kohayakawa, Simonovits and Skokan confirmed the Bondy-Erdos conjecture for all sufficiently large values of n. Figaj and Luczak determined an asymptotic result for the `complementary` case where the cycles are even: they showed that for even n, we have R(C(n), C(n), C(n)) = 2n + o(n), as n -> infinity. In this paper, we prove that there exists n I such that for every even n >= n(1), R(C(n), C(n), C(n)) = 2n. (C) 2009 Elsevier Inc. All rights reserved.