Summary Genetic studies have established anaplastic lymphoma kinase (ALK), a cell surface receptor tyrosine kinase, as a tractable molecular target in neuroblastoma. We describe comprehensive genomic, biochemical, and computational analyses of ALK mutations across 1596 diagnostic neuroblastoma samples. ALK tyrosine kinase domain mutations occurred in 8% of samples; at three hotspots plus 13 minor sites – and correlated significantly with poorer survival in high- and intermediate-risk neuroblastoma. Biochemical and computational studies distinguished oncogenic (constitutively activating) from non-oncogenic mutations and allowed robust computational prediction of their effects. We also established differential in vitro crizotinib sensitivity of mutated variants. Our studies identify ALK genomic status as a clinically important therapeutic stratification tool in neuroblastoma, and will allow tailoring of ALK-targeted therapy to specific mutations.

Molecular dynamics calculations have been used to determine the structure of phosphatidylinositol 4,5 bisphosphate (PIP2) at the quantum level and to quantify the propensity for PIP2 to bind two physiologically relevant divalent cations, Mg2+ and Ca2+. We performed a geometry optimization at the Hartree-Fock 6-31+G(d) level of theory in vacuum and with a polarized continuum dielectric to determine the conformation of the phospholipid headgroup in the presence of water and its partial charge distribution. The angle between the headgroup and the acyl chains is nearly perpendicular, suggesting that in the absence of other interactions, the inositol ring would lie flat along the cytoplasmic surface of the plasma membrane. Next, we employed hybrid quantum mechanics / molecular mechanics (QM/MM) simulations to investigate the protonation state of PIP2 and its interactions with magnesium or calcium. We test the hypothesis suggested by prior experiments that binding of magnesium to PIP2 is mediated by a water molecule that is absent when calcium binds. These results may explain the selective ability of calcium to induce the formation of PIP2 clusters and phase separation from other lipids.

Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9–11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes. These findings align with AID's competing requirements for specificity and flexibility to efficiently drive antibody maturation. Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function.

The cancer-predisposing Lynch Syndrome (LS) arises from germline mutations in DNA mismatch repair (MMR) genes, predominantly MLH1, MSH2, MSH6, and PMS2. A major challenge for clinical diagnosis of LS is the frequent identification of variants of uncertain significance (VUS) in these genes, as it is often difficult to determine variant pathogenicity, particularly for missense variants. Generic programs such as SIFT and PolyPhen-2, and MMR gene-specific programs such as PON-MMR and MAPP-MMR, are often used to predict deleterious or neutral effects of VUS in MMR genes. We evaluated the performance of multiple predictive programs in the context of functional biologic data for 15 VUS in MLH1, MSH2, and PMS2. Using cell line models, we characterized VUS predicted to range from neutral to pathogenic on mRNA and protein expression, basal cellular viability, viability following treatment with a panel of DNA-damaging agents, and functionality in DNA damage response (DDR) signaling, benchmarking to wild-type MMR proteins. Our results suggest that the MMR gene-specific classifiers do not always align with the experimental phenotypes related to DDR. Our study highlights the importance of complementary experimental and computational assessment to develop future predictors for the assessment of VUS.

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In CASP11, the organizers sought to bring the biological inferences from predicted structures to the fore. To accomplish this, we assessed the models for their ability to perform quantifiable tasks related to biological function. First, for 10 targets that were probable homodimers, we measured the accuracy of docking the models into homodimers as a function of GDT-TS of the monomers, which produced characteristic L-shaped plots. At low GDT-TS, none of the models could be docked correctly as homodimers. Above GDT-TS of~60%, some models formed correct homodimers in one of the largest docked clusters, while many other models at the same values of GDT-TS did not. Docking was more successful when many of the templates shared the same homodimer. Second, we docked a ligand from an experimental structure into each of the models of one of the targets. Docking to the models with two different programs produced poor ligand RMSDs with the experimental structure. Measures that evaluated similarity of contacts were reasonable for some of the models, although there was not a significant correlation with model accuracy. Finally, we assessed whether models would be useful in predicting the phenotypes of missense mutations in three human targets by comparing features calculated from the models with those calculated from the experimental structures. The models were successful in reproducing accessible surface areas but there was little correlation of model accuracy with calculation of FoldX evaluation of the change in free energy between the wild-type and the mutant.

GB3 and Ubiquitin, when bound to AuNPs. We find no significant changes in slow HDX rates (5-300 min), suggesting that AuNP-induced structural changes are small for these two proteins. Together, these results support a model where most of a protein's native contacts are preserved upon adsorption, although larger changes may occur over long timescales.

The cytochrome bc 1 complex (bc 1 ) is a widespread, dimeric redox-driven proton translocase. It oxidizes molecules of ubiquinol (UQH2) in the site Q P of the cytochrome b subunit, whereby the two released electrons are transferred, respectively, to the heme of cytochrome c 1 -via the mobile soluble domain of the Rieske protein (the ''head'' domain) -and to a ubiquinone (UQ) molecule in the Q N site of cytochrome b. Earlier kinetic studies have indicated that the movement of the Rieske ''head'' domain could be coupled with the quinone reduction in the Q N site. Thermodynamics considerations also indicate that the ability of the Rieske ''head'' domain in one monomer to move towards cytochrome c 1 might be coupled with the docking of the ''head'' domain to cytochrome b in the other monomer. However, the details of such mechanistic coupling within the bc 1 dimer remain elusive.In the present study, large-scale MD simulations were used to track the communications within the bc 1 of Rhodobacter sphaeroides. Different computational techniques (correlation analysis of interaction energies, motion cross-correlation, co-evolution of amino acid positions) have revealed possible pathways for transmembrane propagation of information about the redox state of UQ in the Q N site. Equilibrium and metadynamics simulations of the bc 1 revealed two distinct binding modes for semiquinone (SQ) and UQH2/UQ in the Q N site and the two positions of UQ in the Q P site which corresponded to these Q N modes, whereby a SQ in the Q N site favored tighter docking of the Rieske ''head'' domain and hindered its outward motion. Metadynamics simulations also showed that the Rieske ''head'' domain of one bc 1 monomer could easier move towards cytochrome c 1 when the ''head'' domain in the opposite monomer was docked to cytochrome b.