Structure-Based Rational Design of a Toll-like Receptor 4 (TLR4) Decoy Receptor with High Binding Affinity for a Target Protein

Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4) decoy receptor composed of leucine-rich repeat (LRR) modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2). Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (KD) one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities

AA mismatched DNAs with a single base difference exhibit a large structural change and a propensity for the parallel-stranded conformation

AA mismatches in DNA with different nearest-neighbor sequences were studied to understand the structural changes that accompany base-pair mismatches and the associated thermodynamics. Two synthesized duplexes, , 5' -d(CGACAATTGACG) (called AA1) and 5' -d(CGAGAATTCACG) (called AA2) as a palindrome sequences, had different nearest-neighbor sequences to the AA mismatches. This study focused on elucidating the structural and thermodynamic differences between these two molecules. A hydrogen bond between the mismatched adenines in AA1 was found, while no hydrogen bond in AA2. Both of the mismatched adenines in AA1 were stacked in the helix, while the mismatched adenine in AA2 surrounded by guanines was partially out of the helix and the other mismatched adenine surrounded by cytosines was stacked in the helix. Thermodynamically, AA1 was more stable than AA2. The melting temperature of the internal bases of AA1 was about 7 degree higher than that of AA2. The standard Gibbs free energy change for the duplex formation of AA1 was 1.30 Kcal/mol smaller than that of AA2. These thermal properties could be ascribed to the formation of the hydrogen bond. The conformational changes of these molecules at low pH were also investigated and compared. AA1 unambiguously assumed a parallel-stranded duplex at pH 4, while AA2 existed as a mixture of anti-parallel and parallel duplexes below pH 5

Rapid preparation of RNA samples for NMR spectroscopy and X-ray crystallography

Knowledge of the three-dimensional structures of RNA and its complexes is important for understanding the molecular mechanism of RNA recognition by proteins or ligands. Enzymatic synthesis using T7 bacteriophage RNA polymerase is used to prepare samples for NMR spectroscopy and X-ray crystallography. However, this run-off transcription method results in heterogeneity at the RNA 3-terminus. For structural studies, RNA purification requires a single nucleotide resolution. Usually PAGE purification is used, but it is tedious, time-consuming and cost ineffective. To overcome these problems in high-throughput RNA synthesis, we devised a method of RNA preparation that uses trans-acting DNAzyme and sequence-specific affinity column chromatography. A tag sequence is added at the 3′ end of RNA, and the tagged RNA is picked out using an affinity column that contains the complementary DNA sequence. The 3′ end tag is then removed by sequence-specific cleavage using trans-acting DNAzyme, the arm lengths of which are optimized for turnover number. This purification method is simpler and faster than the conventional method

Magnetic control of self-assembly and disassembly in organic materials

Abstract Because organic molecules and materials are generally insensitive or weakly sensitive to magnetic fields, a certain means to enhance their magnetic responsiveness needs to be exploited. Here we show a strategy to amplify the magnetic responsiveness of self-assembled peptide nanostructures by synergistically combining the concepts of perfect α-helix and rod-coil supramolecular building blocks. Firstly, we develop a monomeric, nonpolar, and perfect α-helix (MNP-helix). Then, we employ the MNP-helix as the rod block of rod-coil amphiphiles (rod-coils) because rod-coils are well-suited for fabricating responsive assemblies. We show that the self-assembly processes of the designed rod-coils and disassembly of rod-coil/DNA complexes can be controlled in a magnetically responsive manner using the relatively weak magnetic field provided by the ordinary neodymium magnet [0.07 ~ 0.25 Tesla (T)]. These results demonstrate that magnetically responsive organic assemblies usable under practical conditions can be realized by using rod-coil supramolecular building blocks containing constructively organized diamagnetic moieties

Solution Structure and Rpn1 Interaction of the UBL Domain of Human RNA Polymerase II C-Terminal Domain Phosphatase

<div><p>The ubiquitin-like modifier (UBL) domain of ubiquitin-like domain proteins (UDPs) interacts specifically with subunits of the 26 S proteasome. A novel UDP, ubiquitin-like domain-containing C-terminal domain phosphatase (UBLCP1), has been identified as an interacting partner of the 26 S proteasome. We determined the high-resolution solution structure of the UBL domain of human UBLCP1 by nuclear magnetic resonance spectroscopy. The UBL domain of hUBLCP1 has a unique β-strand (β3) and β3-α2 loop, instead of the canonical β4 observed in other UBL domains. The molecular topology and secondary structures are different from those of known UBL domains including that of fly UBLCP1. Data from backbone dynamics shows that the β3-α2 loop is relatively rigid although it might have intrinsic dynamic profile. The positively charged residues of the β3-α2 loop are involved in interacting with the C-terminal leucine-rich repeat-like domain of Rpn1.</p></div