MICA is a major histocompatibility complex-like protein that undergoes a structural transition from disorder to order upon binding its immunoreceptor, NKG2D. We redesigned the disordered region of MICA with RosettaDesign to increase NKG2D binding. Mutations that stabilize this region were expected to increase association kinetics without changing dissociation kinetics, increase affinity of interaction, and reduce entropy loss upon binding. MICA mutants were stable in solution, and they were amenable to surface plasmon resonance evaluation of NKG2D binding kinetics and thermodynamics. Several MICA mutants bound NKG2D with enhanced affinity, kinetic changes were primarily observed during association, and thermodynamic changes in entropy were as expected. However, none of the 15 combinations of mutations predicted to stabilize the receptor-bound MICA conformation enhanced NKG2D affinity, whereas all 10 mutants predicted to be destabilized bound NKG2D with increased on-rates. Five of these had affinities enhanced by 0.9 -1.8 kcal/mol over wild type by one to three non-contacting substitutions. Therefore, in this case, mutations designed to mildly destabilize a protein enhanced association and affinity.NKG2D-ligand interactions play a central role in inducible NK cell and ␥␦ T cell activation, initiating cytotoxic responses to transformation and infection (1-3). All known NKG2D-binding proteins use a major histocompatibility complex platform-like tertiary structure as a scaffold to contact NKG2D, including two binding surface ␣ helices (␣1 and ␣2). In the crystal structure of the NKG2D ligand MICA, electron density was absent for a central portion of the ␣2 helix (residues 152-161), indicating that the helix in that region was disordered into a flexible loop (4). When bound to NKG2D, the residues were ordered beneath the receptor (5). This type of transition from disorder to order upon binding is similar to other immunoreceptor-ligand combinations (6 -9). The thermodynamics of four NKG2D-ligand interactions were compared and found to be driven by both enthalpy and entropy, distinct from the generally enthalpy-driven, entropy-hindered T-cell receptor-major histocompatibility complex interactions (10).The structural characterization of NKG2D-ligand interfaces allows computational optimization of the interactions. Algorithms for computational design of proteins have already been used for energetic dissection of NKG2D-ligand interactions, confirmed by experiment (11). Different rational design techniques applied to LFA-1 with ICAM-1 (intercellular adhesion molecule 1) (12) and a mature antibody-antigen complex (13), for example, have engineered interfaces toward increased affinities.We applied rational design to the MICA-NKG2D interface by attempting to stabilize MICA in its receptor-bound conformation. Loss of configurational freedom for the MICA ␣2 loop should pose an entropic barrier to MICA-NKG2D association, assuming protein entropy dominates. Using RosettaDesign, we used a two-stage design strategy to stabilize thi...

With more than 150,000 deaths per year in the US alone, lung cancer has the highest number of deaths for any cancer. These poor outcomes reflect a lack of treatment for the most common form of lung cancer, non-small cell lung carcinoma (NSCLC). Lung adenocarcinoma (ADC) is the most prevalent subtype of NSCLC, with the main oncogenic drivers being KRAS and epidermal growth factor receptor (EGFR). Whereas EGFR blockade has led to some success in lung ADC, effective KRAS inhibition is lacking. KRAS-mutant ADCs are characterized by high levels of gel-forming mucin expression, with the highest mucin levels corresponding to worse prognoses. Despite these well-recognized associations, little is known about roles for individual gel-forming mucins in ADC development causatively. We hypothesized that MUC5AC/Muc5ac, a mucin gene known to be commonly expressed in NSCLC, is crucial in KRAS/Kras-driven lung ADC. We found that MUC5AC was a significant determinant of poor prognosis, especially in patients with KRAS-mutant tumors. In addition, by using mice with lung ADC induced chemically with urethane or transgenically by mutant-Kras expression, we observed significantly reduced tumor development in animals lacking Muc5ac compared with controls. Collectively, these results provide strong support for MUC5AC as a potential therapeutic target for lung ADC, a disease with few effective treatments.

PCR amplification, a key step in next-generation sequencing (NGS) library construction, can generate an unlimited amount of product from limited input; however, it cannot create more information than was present in the original template. Thus, NGS libraries can be made from very little DNA, but reducing the input may compromise assay sensitivity in ways that are difficult to ascertain unless library complexity (ie, the number of unique DNA molecules represented in the library) and depth of coverage with unique sequence reads (those derived from input DNA molecules) versus duplicate sequence reads (those resulting from overamplification of particular molecules) are discretely measured. A series of experiments was performed to explore the impact of low DNA input on an amplicon-based NGS assay using unique molecular identifiers to track unique versus duplicate reads. At high sequencing depths, unique and total (unique plus duplicate) read coverage are not well correlated, so increasing the number of sequenced reads does not necessarily improve sensitivity. Unique coverage depth tends to improve with more input, but improvements are not consistent. Fluctuations in library complexity complicated variant detection using both standardized and clinical specimens, often resulting in technical replicates with vastly different estimates of variant allelic fraction. In conclusion, depth of coverage with unique reads must be tracked in clinical NGS to ensure that sensitivity and accuracy are maintained.

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