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

Abstract

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.

Interfering with the high-affinity interaction between wheat amylase trypsin inhibitor CM3 and toll-like receptor 4: in silico and biosensor-based studies

“…The resulting 1:1 ATI-TLR4 complex was characterized by K D in the nanomolar range. ATI binding to TLR4 occurred with fully comparable affinity to that of TLR4 physiological partner MD2 32 , and similar to other ATI targets, such as digestive serine proteases and amylases 23 . Additionally, wheat ATI CM3 preserved part of the native TLR4-binding ability upon in vitro enzymatic digestion, confirming the role of intramolecular disulphide bonds as key determinants of wheat ATI capacity to survive gastro-intestinal transit and trigger TLR4 signalling 25 .…”

Dynamics on human Toll‐like receptor 4 complexation to MD‐2: The coreceptor stabilizing function

“…To trace the B‐factor pattern in TLRs family dependent on MD‐2, we performed a screening on crystallographical B‐factor data avaiable for different crystal structures of TLRs. We select eighteen structures of TLRs in the absence of MD‐2 and five structures that do possessed MD‐2 (see Supporting Information Table S2) and performed multiple sequence alignment with these two groups of structures (whether or not porting MD‐2) and matched the correspondent residues in both groups. Once aligned, we calculated the average B‐factor values and standard deviations by residue and compared both groups thus characterizing the influence of presence or absence of MD‐2 on residues fluctuation in TLRs family.…”

Increasing Affinity of Interferon-γReceptor 1 to Interferon-γby Computer-Aided Design

“…Similarly, five amino acid substitutions increased affinity between integrin antigen LFA-1 and its ligand about twentyfold [46]. Single amino acid substitutions in decoy receptor TLR4 constructed of leucine-rich repeats increased affinity to myeloid differentiation protein 2 about tenfold [47]. Interestingly, this study reports high cooperativity among the single mutations as the affinity of double mutants has been reported up to a thousand-times higher compared to WT.…”